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Learning Objectives
• Explain why algae are included within the discipline of microbiology
• Describe the unique characteristics of algae
• Identify examples of toxin-producing algae
• Compare the major groups of algae in this chapter, and give examples of each
• Classify algal organisms according to major groups
The algae are autotrophic protists that can be unicellular or multicellular. These organisms are found in the supergroups Chromalveolata (dinoflagellates, diatoms, golden algae, and brown algae) and Archaeplastida (red algae and green algae). They are important ecologically and environmentally because they are responsible for the production of approximately 70% of the oxygen and organic matter in aquatic environments. Some types of algae, even those that are microscopic, are regularly eaten by humans and other animals. Additionally, algae are the source for agar, agarose, and carrageenan, solidifying agents used in laboratories and in food production. Although algae are typically not pathogenic, some produce toxins. Harmful algal blooms, which occur when algae grow quickly and produce dense populations, can produce high concentrations of toxins that impair liver and nervous-system function in aquatic animals and humans.
Like protozoans, algae often have complex cell structures. For instance, algal cells can have one or more chloroplasts that contain structures called pyrenoids to synthesize and store starch. The chloroplasts themselves differ in their number of membranes, indicative of secondary or rare tertiary endosymbiotic events. Primary chloroplasts have two membranes—one from the original cyanobacteria that the ancestral eukaryotic cell engulfed, and one from the plasma membrane of the engulfing cell. Chloroplasts in some lineages appear to have resulted from secondary endosymbiosis, in which another cell engulfed a green or red algal cell that already had a primary chloroplast within it. The engulfing cell destroyed everything except the chloroplast and possibly the cell membrane of its original cell, leaving three or four membranes around the chloroplast. Different algal groups have different pigments, which are reflected in common names such as red algae, brown algae, and green algae.
Some algae, the seaweeds, are macroscopic and may be confused with plants. Seaweeds can be red, brown, or green, depending on their photosynthetic pigments. Green algae, in particular, share some important similarities with land plants; however, there are also important distinctions. For example, seaweeds do not have true tissues or organs like plants do. Additionally, seaweeds do not have a waxy cuticle to prevent desiccation. Algae can also be confused with cyanobacteria, photosynthetic bacteria that bear a resemblance to algae; however, cyanobacteria are prokaryotes (see Nonproteobacteria Gram-negative Bacteria and Phototrophic Bacteria).
Algae have a variety of life cycles. Reproduction may be asexual by mitosis or sexual using gametes.
Algal Diversity
Although the algae and protozoa were formerly separated taxonomically, they are now mixed into supergroups. The algae are classified within the Chromalveolata and the Archaeplastida. Although the Euglenozoa (within the supergroup Excavata) include photosynthetic organisms, these are not considered algae because they feed and are motile.
The dinoflagellates and stramenopiles fall within the Chromalveolata. The dinoflagellates are mostly marine organisms and are an important component of plankton. They have a variety of nutritional types and may be phototrophic, heterotrophic, or mixotrophic. Those that are photosynthetic use chlorophyll a, chlorophyll c2, and other photosynthetic pigments (Figure \(1\)). They generally have two flagella, causing them to whirl (in fact, the name dinoflagellate comes from the Greek word for “whirl”: dini). Some have cellulose plates forming a hard outer covering, or theca, as armor. Additionally, some dinoflagellates produce neurotoxins that can cause paralysis in humans or fish. Exposure can occur through contact with water containing the dinoflagellate toxins or by feeding on organisms that have eaten dinoflagellates.
When a population of dinoflagellates becomes particularly dense, a red tide (a type of harmful algal bloom) can occur. Red tides cause harm to marine life and to humans who consume contaminated marine life. Major toxin producers include Gonyaulax and Alexandrium, both of which cause paralytic shellfish poisoning. Another species, Pfiesteria piscicida, is known as a fish killer because, at certain parts of its life cycle, it can produce toxins harmful to fish and it appears to be responsible for a suite of symptoms, including memory loss and confusion, in humans exposed to water containing the species.
The stramenopiles include the golden algae (Chrysophyta), the brown algae (Phaeophyta), and the diatoms(Bacillariophyta). Stramenopiles have chlorophyll a, chlorophyll c1/c2, and fucoxanthin as photosynthetic pigments. Their storage carbohydrate is chrysolaminarin. While some lack cell walls, others have scales. Diatoms have flagella and frustules, which are outer cell walls of crystallized silica; their fossilized remains are used to produce diatomaceous earth, which has a range of uses such as filtration and insulation. Additionally, diatoms can reproduce sexually or asexually. One diatom genus, Pseudo-nitzschia, is known to be associated with harmful algal blooms.
Brown algae (Phaeophyta) are multicellular marine seaweeds. Some can be extremely large, such as the giant kelp (Laminaria). They have leaf-like blades, stalks, and structures called holdfasts that are used to attach to substrate. However, these are not true leaves, stems, or roots (Figure \(2\)). Their photosynthetic pigments are chlorophyll a, chlorophyll c, β-carotene, and fucoxanthine. They use laminarin as a storage carbohydrate.
The Archaeplastids include the green algae (Chlorophyta), the red algae (Rhodophyta), another group of green algae (Charophyta), and the land plants. The Charaphyta are the most similar to land plants because they share a mechanism of cell division and an important biochemical pathway, among other traits that the other groups do not have. Like land plants, the Charophyta and Chlorophyta have chlorophyll a and chlorophyll b as photosynthetic pigments, cellulose cell walls, and starch as a carbohydrate storage molecule. Chlamydomonas is a green alga that has a single large chloroplast, two flagella, and a stigma (eyespot); it is important in molecular biology research (Figure \(3\)).
Chlorella is a nonmotile, large, unicellular alga, and Acetabularia is an even larger unicellular green alga. The size of these organisms challenges the idea that all cells are small, and they have been used in genetics research since Joachim Hämmerling (1901–1980) began to work with them in 1943. Volvox is a colonial, unicellular alga (Figure \(3\)). A larger, multicellular green alga is Ulva, also known as the sea lettuce because of its large, edible, green blades. The range of life forms within the Chlorophyta—from unicellular to various levels of coloniality to multicellular forms—has been a useful research model for understanding the evolution of multicellularity. The red algae are mainly multicellular but include some unicellular forms. They have rigid cell walls containing agar or carrageenan, which are useful as food solidifying agents and as a solidifier added to growth media for microbes.
Exercise \(1\)
Which groups of algae are associated with harmful algal blooms?
Key Concepts and Summary
• Algae are a diverse group of photosynthetic eukaryotic protists.
• Algae may be unicellular or multicellular.
• Large, multicellular algae are called seaweeds but are not plants and lack plant-like tissues and organs.
• Although algae have little pathogenicity, they may be associated with toxic algal blooms that can harm aquatic wildlife and contaminate seafood with toxins that cause paralysis.
• Algae are important for producing agar, which is used as a solidifying agent in microbiological media, and carrageenan, which is used as a solidifying agent. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/05%3A_The_Eukaryotes_of_Microbiology/5.04%3A_Algae.txt |
Learning Objectives
• Explain why lichens are included in the study of microbiology
• Describe the unique characteristics of a lichen and the role of each partner in the symbiotic relationship of a lichen
• Describe ways in which lichens are beneficial to the environment
No one has to worry about getting sick from a lichen infection, but lichens are interesting from a microbiological perspective and they are an important component of most terrestrial ecosystems. Lichens provide opportunities for study of close relationships between unrelated microorganisms. Lichens contribute to soil production by breaking down rock, and they are early colonizers in soilless environments such as lava flows. The cyanobacteria in some lichens can fix nitrogen and act as a nitrogen source in some environments. Lichens are also important soil stabilizers in some desert environments and they are an important winter food source for caribou and reindeer. Finally, lichens produce compounds that have antibacterial effects, and further research may discover compounds that are medically useful to humans.
Characteristics
A lichen is a combination of two organisms, a green alga or cyanobacterium and an ascomycete fungus, living in a symbiotic relationship. Whereas algae normally grow only in aquatic or extremely moist environments, lichens can potentially be found on almost any surface (especially rocks) or as epiphytes (meaning that they grow on other plants).
In some ways, the symbiotic relationship between lichens and algae seems like a mutualism (a relationship in which both organisms benefit). The fungus can obtain photosynthates from the algae or cyanobacterium and the algae or cyanobacterium can grow in a drier environment than it could otherwise tolerate. However, most scientists consider this symbiotic relationship to be a controlled parasitism (a relationship in which one organism benefits and the other is harmed) because the photosynthetic organism grows less well than it would without the fungus. It is important to note that such symbiotic interactions fall along a continuum between conflict and cooperation.
Lichens are slow growing and can live for centuries. They have been used in foods and to extract chemicals as dyes or antimicrobial substances. Some are very sensitive to pollution and have been used as environmental indicators.
Lichens have a body called a thallus, an outer, tightly packed fungal layer called a cortex, and an inner, loosely packed fungal layer called a medulla (Figure \(1\)). Lichens use hyphal bundles called rhizines to attach to the substrate.
Lichen Diversity
Lichens are classified as fungi and the fungal partners belong to the Ascomycota and Basidiomycota. Lichens can also be grouped into types based on their morphology. There are three major types of lichens, although other types exist as well. Lichens that are tightly attached to the substrate, giving them a crusty appearance, are called crustose lichens. Those that have leaf-like lobes are foliose lichens; they may only be attached at one point in the growth form, and they also have a second cortex below the medulla. Finally, fruticose lichens have rounded structures and an overall branched appearance. Figure \(2\) shows an example of each of the forms of lichens.
Exercise \(1\)
1. What types of organisms are found in lichens?
2. What are the three growth forms of lichens?
Clinical Focus: Resolution
Sarah’s mother asks the doctor what she should do if the cream prescribed for Sarah’s ringworm does not work. The doctor explains that ringworm is a general term for a condition caused by multiple species. The first step is to take a scraping for examination under the microscope, which the doctor has already done. He explains that he has identified the infection as a fungus, and that the antifungal cream works against the most common fungi associated with ringworm. However, the cream may not work against some species of fungus. If the cream is not working after a couple of weeks, Sarah should come in for another visit, at which time the doctor will take steps to identify the species of the fungus.
Positive identification of dermatophytes requires culturing. For this purpose, Sabouraud’s agar may be used. In the case of Sarah’s infection, which cleared up within 2 weeks of treatment, the culture would have a granular texture and would appear pale pink on top and red underneath. These features suggest that the fungus is Trichophyton rubrum, a common cause of ringworm.
Key Concepts and Summary
• Lichens are a symbiotic association between a fungus and an algae or a cyanobacterium
• The symbiotic association found in lichens is currently considered to be a controlled parasitism, in which the fungus benefits and the algae or cyanobacterium is harmed
• Lichens are slow growing and can live for centuries in a variety of habitats
• Lichens are environmentally important, helping to create soil, providing food, and acting as indicators of air pollution | textbooks/bio/Microbiology/Microbiology_(OpenStax)/05%3A_The_Eukaryotes_of_Microbiology/5.05%3A_Lichens.txt |
5.1: Unicellular Eukaryotic Microorganisms
Protists are a diverse, polyphyletic group of eukaryotic organisms. Protists may be unicellular or multicellular. They vary in how they get their nutrition, morphology, method of locomotion, and mode of reproduction. Important structures of protists include contractile vacuoles, cilia, flagella, pellicles, and pseudopodia; some lack organelles such as mitochondria. Taxonomy of protists is changing rapidly as relationships are reassessed using newer techniques.
Multiple Choice
Which genus includes the causative agent for malaria?
1. Euglena
2. Paramecium
3. Plasmodium
4. Trypanosoma
Answer
C
Which protist is a concern because of its ability to contaminate water supplies and cause diarrheal illness?
1. Plasmodium vivax
2. Toxoplasma gondii
3. Giardia lamblia
4. Trichomonas vaginalis
Answer
C
Fill in the Blank
The plasma membrane of a protist is called the __________.
Answer
plasmalemma
Animals belong to the same supergroup as the kingdom __________.
Answer
Fungi
Short Answer
What are kinetoplastids?
Aside from a risk of birth defects, what other effect might a toxoplasmosis infection have?
What is the function of the ciliate macronucleus?
Critical Thinking
The protist shown has which of the following?
1. pseudopodia
2. flagella
3. a shell
4. cilia
(credit: modification of work by Richard Robinson)
Protist taxonomy has changed greatly in recent years as relationships have been re-examined using newer approaches. How do newer approaches differ from older approaches?
What characteristics might make you think a protist could be pathogenic? Are certain nutritional characteristics, methods of locomotion, or morphological differences likely to be associated with the ability to cause disease?
5.2: Parasitic Helminths
Helminth parasites are included within the study of microbiology because they are often identified by looking for microscopic eggs and larvae. The two major groups of helminth parasites are the roundworms (Nematoda) and the flatworms (Platyhelminthes). Nematodes are common intestinal parasites often transmitted through undercooked foods, although they are also found in other environments. Platyhelminths include tapeworms and flukes, which are often transmitted through undercooked meat.
Multiple Choice
A fluke is classified within which of the following?
1. Nematoda
2. Rotifera
3. Platyhelminthes
4. Annelida
Answer
C
A nonsegmented worm is found during a routine colonoscopy of an individual who reported having abdominal cramps, nausea, and vomiting. This worm is likely which of the following?
1. nematode
2. fluke
3. trematode
4. annelid
Answer
A
A segmented worm has male and female reproductive organs in each segment. Some use hooks to attach to the intestinal wall. Which type of worm is this?
1. fluke
2. nematode
3. cestode
4. annelid
Answer
C
Fill in the Blank
Flukes are in class _________.
Answer
Trematoda
A species of worm in which there are distinct male and female individuals is described as _________.
Answer
dioecious
Short Answer
What is the best defense against tapeworm infection?
Critical Thinking
Given the life cycle of the Schistosoma parasite, suggest a method of prevention of the disease.
5.3: Fungi
The fungi include diverse saprotrophic eukaryotic organisms with chitin cell walls. Fungi can be unicellular or multicellular; some (like yeast) and fungal spores are microscopic, whereas some are large and conspicuous. Reproductive types are important in distinguishing fungal groups. Medically important species exist in the four fungal groups Zygomycota, Ascomycota, Basidiomycota, and Microsporidia.
Multiple Choice
Mushrooms are a type of which of the following?
1. conidia
2. ascus
3. polar tubule
4. basidiocarp
Answer
D
Which of the following is the most common cause of human yeast infections?
1. Candida albicans
2. Blastomyces dermatitidis
3. Cryptococcus neoformans
4. Aspergillus fumigatus
Answer
A
Which of the following is an ascomycete fungus associated with bat droppings that can cause a respiratory infection if inhaled?
1. Candida albicans
2. Histoplasma capsulatum
3. Rhizopus stolonifera
4. Trichophyton rubrum
Answer
B
Fill in the Blank
Nonseptate hyphae are also called _________.
Answer
coenocytic
Unicellular fungi are called _________.
Answer
yeasts
Some fungi have proven medically useful because they can be used to produce _________.
Answer
antibiotics
Short Answer
Which genera of fungi are common dermatophytes (fungi that cause skin infections)?
What is a dikaryotic cell?
Critical Thinking
Which of the drawings shows septate hyphae?
Explain the benefit of research into the pathways involved in the synthesis of chitin in fungi.
5.4: Algae
Algae are a diverse group of photosynthetic eukaryotic protists. Algae may be unicellular or multicellular. Large, multicellular algae are called seaweeds but are not plants and lack plant-like tissues and organs. Although algae have little pathogenicity, they may be associated with toxic algal blooms that can harm aquatic wildlife and contaminate seafood with toxins that cause paralysis.
Multiple Choice
Which polysaccharide found in red algal cell walls is a useful solidifying agent?
1. chitin
2. cellulose
3. phycoerythrin
4. agar
Answer
D
Which is the term for the hard outer covering of some dinoflagellates?
1. theca
2. thallus
3. mycelium
4. shell
Answer
A
Which protists are associated with red tides?
1. red algae
2. brown algae
3. dinoflagellates
4. green algae
Answer
C
Fill in the Blank
Structures in chloroplasts used to synthesize and store starch are called ________.
Answer
pyrenoids
Algae with chloroplasts with three or four membranes are a result of ________ ________.
Answer
secondary endosymbiosis
Short Answer
What is a distinctive feature of diatoms?
Why are algae not considered parasitic?
Which groups contain the multicellular algae?
5.5: Lichens
Lichens are a symbiotic association between a fungus and an algae or a cyanobacterium. The symbiotic association found in lichens is currently considered to be a controlled parasitism, in which the fungus benefits and the algae or cyanobacterium is harmed. Lichens are slow growing and can live for centuries in a variety of habitats. Lichens are environmentally important, helping to create soil, providing food, and acting as indicators of air pollution.
Multiple Choice
You encounter a lichen with leafy structures. Which term describes this lichen?
1. crustose
2. foliose
3. fruticose
4. agarose
Answer
B
Which of the following is the term for the outer layer of a lichen?
1. the cortex
2. the medulla
3. the thallus
4. the theca
Answer
A
The fungus in a lichen is which of the following?
1. a basidiomycete
2. an ascomycete
3. a zygomycete
4. an apicomplexan
Answer
B
Short Answer
What are three ways that lichens are environmentally valuable? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/05%3A_The_Eukaryotes_of_Microbiology/5.E%3A_The_Eukaryotes_of_Microbiology_%28Exercises%29.txt |
Public health measures in the developed world have dramatically reduced mortality from viral epidemics. But when epidemics do occur, they can spread quickly with global air travel. In 2009, an outbreak of H1N1 influenza spread across various continents. In early 2014, cases of Ebola in Guinea led to a massive epidemic in western Africa. This included the case of an infected man who traveled to the United States, sparking fears the epidemic might spread beyond Africa.
Until the late 1930s and the advent of the electron microscope, no one had seen a virus. Yet treatments for preventing or curing viral infections were used and developed long before that. Historical records suggest that by the 17th century, and perhaps earlier, inoculation (also known as variolation) was being used to prevent the viral disease smallpox in various parts of the world. By the late 18th century, Englishman Edward Jenner was inoculating patients with cowpox to prevent smallpox, a technique he coined vaccination.1
Today, the structure and genetics of viruses are well defined, yet new discoveries continue to reveal their complexities. In this chapter, we will learn about the structure, classification, and cultivation of viruses, and how they impact their hosts. In addition, we will learn about other infective particles such as viroids and prions.
• 6.1: Viruses
Viruses are generally ultramicroscopic, typically from 20 nm to 900 nm in length. Some large viruses have been found. Virions are acellular and consist of a nucleic acid, DNA or RNA, but not both, surrounded by a protein capsid. There may also be a phospholipid membrane surrounding the capsid. Viruses are obligate intracellular parasites.
• 6.2: The Viral Life Cycle
Many viruses target specific hosts or tissues. Some may have more than one host. Many viruses follow several stages to infect host cells. These stages include attachment, penetration, uncoating, biosynthesis, maturation, and release. Bacteriophages have a lytic or lysogenic cycle. The lytic cycle leads to the death of the host, whereas the lysogenic cycle leads to integration of phage into the host genome.
• 6.3: Isolation, Culture, and Identification of Viruses
Viral cultivation requires the presence of some form of host cell (whole organism, embryo, or cell culture). Viruses can be isolated from samples by filtration. Viral filtrate is a rich source of released virions. Bacteriophages are detected by presence of clear plaques on bacterial lawn. Animal and plant viruses are detected by cytopathic effects, molecular techniques (PCR, RT-PCR), enzyme immunoassays, and serological assays (hemagglutination assay, hemagglutination inhibition assay).
• 6.4: Viroids, Virusoids, and Prions
Other acellular agents such as viroids, virusoids, and prions also cause diseases. Viroids consist of small, naked ssRNAs that cause diseases in plants. Virusoids are ssRNAs that require other helper viruses to establish an infection. Prions are proteinaceous infectious particles that cause transmissible spongiform encephalopathies. Prions are extremely resistant to chemicals, heat, and radiation.
• 6.E: Acellular Pathogens (Exercises)
Footnotes
1. 1 S. Riedel “Edward Jenner and the History of Smallpox and Vaccination.” Baylor University Medical Center Proceedings 18, no. 1 (January 2005): 21–25.
Thumbnail: This colorized transmission electron microscopic (TEM) image revealed some of the ultrastructural morphology displayed by an Ebola virus virion. (Public Domain; Frederick A. Murphy via CDC).
06: Acellular Pathogens
Learning Objectives
• Describe the general characteristics of viruses as pathogens
• Describe viral genomes
• Describe the general characteristics of viral life cycles
• Differentiate among bacteriophages, plant viruses, and animal viruses
• Describe the characteristics used to identify viruses as obligate intracellular parasites
Clinical Focus: Part 1
David, a 45-year-old journalist, has just returned to the U.S. from travels in Russia, China, and Africa. He is not feeling well, so he goes to his general practitioner complaining of weakness in his arms and legs, fever, headache, noticeable agitation, and minor discomfort. He thinks it may be related to a dog bite he suffered while interviewing a Chinese farmer. He is experiencing some prickling and itching sensations at the site of the bite wound, but he tells the doctor that the dog seemed healthy and that he had not been concerned until now. The doctor ordered a culture and sensitivity test to rule out bacterial infection of the wound, and the results came back negative for any possible pathogenic bacteria.
Exercise \(1\)
1. Based on this information, what additional tests should be performed on the patient?
2. What type of treatment should the doctor recommend?
Despite their small size, which prevented them from being seen with light microscopes, the discovery of a filterable component smaller than a bacterium that causes tobacco mosaic disease (TMD) dates back to 1892.1 At that time, Dmitri Ivanovski, a Russian botanist, discovered the source of TMD by using a porcelain filtering device first invented by Charles Chamberland and Louis Pasteur in Paris in 1884. Porcelain Chamberland filters have a pore size of 0.1 µm, which is small enough to remove all bacteria ≥0.2 µm from any liquids passed through the device. An extract obtained from TMD-infected tobacco plants was made to determine the cause of the disease. Initially, the source of the disease was thought to be bacterial. It was surprising to everyone when Ivanovski, using a Chamberland filter, found that the cause of TMD was not removed after passing the extract through the porcelain filter. So if a bacterium was not the cause of TMD, what could be causing the disease? Ivanovski concluded the cause of TMD must be an extremely small bacterium or bacterial spore. Other scientists, including Martinus Beijerinck, continued investigating the cause of TMD. It was Beijerinck, in 1899, who eventually concluded the causative agent was not a bacterium but, instead, possibly a chemical, like a biological poison we would describe today as a toxin. As a result, the word virus, Latin for poison, was used to describe the cause of TMD a few years after Ivanovski’s initial discovery. Even though he was not able to see the virus that caused TMD, and did not realize the cause was not a bacterium, Ivanovski is credited as the original discoverer of viruses and a founder of the field of virology.
Today, we can see viruses using electron microscopes (Figure \(1\)) and we know much more about them. Viruses are distinct biological entities; however, their evolutionary origin is still a matter of speculation. In terms of taxonomy, they are not included in the tree of life because they are acellular (not consisting of cells). In order to survive and reproduce, viruses must infect a cellular host, making them obligate intracellular parasites. The genome of a virus enters a host cell and directs the production of the viral components, proteins and nucleic acids, needed to form new virus particles called virions. New virions are made in the host cell by assembly of viral components. The new virions transport the viral genome to another host cell to carry out another round of infection. Table \(1\) summarizes the properties of viruses.
Table \(1\): Properties of viruses.
Characteristics of Viruses
Infectious, acellular pathogens
Obligate intracellular parasites with host and cell-type specificity
DNA or RNA genome (never both)
Genome is surrounded by a protein capsid and, in some cases, a phospholipid membrane studded with viral glycoproteins
Lack genes for many products needed for successful reproduction, requiring exploitation of host-cell genomes to reproduce
Exercise \(2\)
Why was the first virus investigated mistaken for a toxin?
Hosts and Viral Transmission
Viruses can infect every type of host cell, including those of plants, animals, fungi, protists, bacteria, and archaea. Most viruses will only be able to infect the cells of one or a few species of organism. This is called the host range. However, having a wide host range is not common and viruses will typically only infect specific hosts and only specific cell types within those hosts. The viruses that infect bacteria are called bacteriophages, or simply phages. The word phage comes from the Greek word for devour. Other viruses are just identified by their host group, such as animal or plant viruses. Once a cell is infected, the effects of the virus can vary depending on the type of virus. Viruses may cause abnormal growth of the cell or cell death, alter the cell’s genome, or cause little noticeable effect in the cell.
Viruses can be transmitted through direct contact, indirect contact with fomites, or through a vector: an animal that transmits a pathogen from one host to another. Arthropods such as mosquitoes, ticks, and flies, are typical vectors for viral diseases, and they may act as mechanical vectors or biological vectors. Mechanical transmission occurs when the arthropod carries a viral pathogen on the outside of its body and transmits it to a new host by physical contact. Biological transmission occurs when the arthropod carries the viral pathogen inside its body and transmits it to the new host through biting.
In humans, a wide variety of viruses are capable of causing various infections and diseases. Some of the deadliest emerging pathogens in humans are viruses, yet we have few treatments or drugs to deal with viral infections, making them difficult to eradicate.
Viruses that can be transmitted from an animal host to a human host can cause zoonoses. For example, the avian influenza virus originates in birds, but can cause disease in humans. Reverse zoonoses are caused by infection of an animal by a virus that originated in a human.
Fighting Bacteria With Viruses
The emergence of superbugs, or multidrug resistant bacteria, has become a major challenge for pharmaceutical companies and a serious health-care problem. According to a 2013 report by the US Centers for Disease Control and Prevention (CDC), more than 2 million people are infected with drug-resistant bacteria in the US annually, resulting in at least 23,000 deaths.2 The continued use and overuse of antibiotics will likely lead to the evolution of even more drug-resistant strains.
One potential solution is the use of phage therapy, a procedure that uses bacteria-killing viruses (bacteriophages) to treat bacterial infections. Phage therapy is not a new idea. The discovery of bacteriophages dates back to the early 20th century, and phage therapy was first used in Europe in 1915 by the English bacteriologist Frederick Twort.3 However, the subsequent discovery of penicillin and other antibiotics led to the near abandonment of this form of therapy, except in the former Soviet Union and a few countries in Eastern Europe. Interest in phage therapy outside of the countries of the former Soviet Union is only recently re-emerging because of the rise in antibiotic-resistant bacteria.4
Phage therapy has some advantages over antibiotics in that phages kill only one specific bacterium, whereas antibiotics kill not only the pathogen but also beneficial bacteria of the normal microbiota. Development of new antibiotics is also expensive for drug companies and for patients, especially for those who live in countries with high poverty rates.
Phages have also been used to prevent food spoilage. In 2006, the US Food and Drug Administration approved the use of a solution containing six bacteriophages that can be sprayed on lunch meats such as bologna, ham, and turkey to kill Listeria monocytogenes, a bacterium responsible for listeriosis, a form of food poisoning. Some consumers have concerns about the use of phages on foods, however, especially given the rising popularity of organic products. Foods that have been treated with phages must declare “bacteriophage preparation” in the list of ingredients or include a label declaring that the meat has been “treated with antimicrobial solution to reduce microorganisms.”5
Exercise \(3\)
1. Why do humans not have to be concerned about the presence of bacteriophages in their food?
2. What are three ways that viruses can be transmitted between hosts?
Viral Structures
In general, virions (viral particles) are small and cannot be observed using a regular light microscope. They are much smaller than prokaryotic and eukaryotic cells; this is an adaptation allowing viruses to infect these larger cells (see Figure \(2\)). The size of a virion can range from 20 nm for small viruses up to 900 nm for typical, large viruses (see Figure \(3\)). Recent discoveries, however, have identified new giant viral species, such as Pandoravirus salinus and Pithovirus sibericum, with sizes approaching that of a bacterial cell.6
In 1935, after the development of the electron microscope, Wendell Stanley was the first scientist to crystallize the structure of the tobacco mosaic virus and discovered that it is composed of RNA and protein. In 1943, he isolated Influenza B virus, which contributed to the development of an influenza (flu) vaccine. Stanley’s discoveries unlocked the mystery of the nature of viruses that had been puzzling scientists for over 40 years and his contributions to the field of virology led to him being awarded the Nobel Prize in 1946.
As a result of continuing research into the nature of viruses, we now know they consist of a nucleic acid (either RNA or DNA, but never both) surrounded by a protein coat called a capsid (see Figure \(4\)). The interior of the capsid is not filled with cytosol, as in a cell, but instead it contains the bare necessities in terms of genome and enzymes needed to direct the synthesis of new virions. Each capsid is composed of protein subunits called capsomeres made of one or more different types of capsomere proteins that interlock to form the closely packed capsid.
There are two categories of viruses based on general composition. Viruses formed from only a nucleic acid and capsid are called naked viruses or nonenveloped viruses. Viruses formed with a nucleic-acid packed capsid surrounded by a lipid layer are called enveloped viruses (see Figure \(4\)). The viral envelope is a small portion of phospholipid membrane obtained as the virion buds from a host cell. The viral envelope may either be intracellular or cytoplasmic in origin.
Extending outward and away from the capsid on some naked viruses and enveloped viruses are protein structures called spikes. At the tips of these spikes are structures that allow the virus to attach and enter a cell, like the influenza virus hemagglutinin spikes (H) or enzymes like the neuraminidase (N) influenza virus spikes that allow the virus to detach from the cell surface during release of new virions. Influenza viruses are often identified by their H and N spikes. For example, H1N1 influenza viruses were responsible for the pandemics in 1918 and 2009,7 H2N2 for the pandemic in 1957, and H3N2 for the pandemic in 1968.
Viruses vary in the shape of their capsids, which can be either helical, polyhedral, or complex. A helical capsid forms the shape of tobacco mosaic virus (TMV), a naked helical virus, and Ebola virus, an enveloped helical virus. The capsid is cylindrical or rod shaped, with the genome fitting just inside the length of the capsid. Polyhedral capsids form the shapes of poliovirus and rhinovirus, and consist of a nucleic acid surrounded by a polyhedral (many-sided) capsid in the form of an icosahedron. An icosahedral capsid is a three-dimensional, 20-sided structure with 12 vertices. These capsids somewhat resemble a soccer ball. Both helical and polyhedral viruses can have envelopes. Viral shapes seen in certain types of bacteriophages, such as T4 phage, and poxviruses, like vaccinia virus, may have features of both polyhedral and helical viruses so they are described as a complex viral shape (see Figure \(5\)). In the bacteriophage complex form, the genome is located within the polyhedral head and the sheath connects the head to the tail fibers and tail pins that help the virus attach to receptors on the host cell’s surface. Poxviruses that have complex shapes are often brick shaped, with intricate surface characteristics not seen in the other categories of capsid.
Exercise \(4\)
Which types of viruses have spikes?
Classification and Taxonomy of Viruses
Although viruses are not classified in the three domains of life, their numbers are great enough to require classification. Since 1971, the International Union of Microbiological Societies Virology Division has given the task of developing, refining, and maintaining a universal virus taxonomy to the International Committee on Taxonomy of Viruses (ICTV). Since viruses can mutate so quickly, it can be difficult to classify them into a genus and a species epithet using the binomial nomenclature system. Thus, the ICTV’s viral nomenclature system classifies viruses into families and genera based on viral genetics, chemistry, morphology, and mechanism of multiplication. To date, the ICTV has classified known viruses in seven orders, 96 families, and 350 genera. Viral family names end in -viridae (e.g, Parvoviridae) and genus names end in −virus (e.g., Parvovirus). The names of viral orders, families, and genera are all italicized. When referring to a viral species, we often use a genus and species epithet such as Pandoravirus dulcis or Pandoravirus salinus.
The Baltimore classification system is an alternative to ICTV nomenclature. The Baltimore system classifies viruses according to their genomes (DNA or RNA, single versus double stranded, and mode of replication). This system thus creates seven groups of viruses that have common genetics and biology.
Link to Learning
Explore the latest virus taxonomy at the ICTV website.
Aside from formal systems of nomenclature, viruses are often informally grouped into categories based on chemistry, morphology, or other characteristics they share in common. Categories may include naked or enveloped structure, single-stranded (ss) or double-stranded (ds) DNA or ss or ds RNA genomes, segmented or nonsegmented genomes, and positive-strand (+) or negative-strand (−) RNA. For example, herpes viruses can be classified as a dsDNA enveloped virus; human immunodeficiency virus (HIV) is a +ssRNA enveloped virus, and tobacco mosaic virus is a +ssRNA virus. Other characteristics such as host specificity, tissue specificity, capsid shape, and special genes or enzymes may also be used to describe groups of similar viruses. Table \(2\) lists some of the most common viruses that are human pathogens by genome type.
Table \(2\): Common Pathogenic Viruses
Genome Family Example Virus Clinical Features
dsDNA, enveloped Poxviridae Orthopoxvirus Skin papules, pustules, lesions
Poxviridae Parapoxvirus Skin lesions
Herpesviridae Simplexvirus Cold sores, genital herpes, sexually transmitted disease
dsDNA, naked Adenoviridae Atadenovirus Respiratory infection (common cold)
Papillomaviridae Papillomavirus Genital warts, cervical, vulvar, or vaginal cancer
Reoviridae Reovirus Gastroenteritis severe diarrhea (stomach flu)
ssDNA, naked Parvoviridae Adeno-associated dependoparvovirus A Respiratory tract infection
Parvoviridae Adeno-associated dependoparvovirus B Respiratory tract infection
dsRNA, naked Reoviridae Rotavirus Gastroenteritis
+ssRNA, naked Picornaviridae Enterovirus C Poliomyelitis
Picornaviridae Rhinovirus Upper respiratory tract infection (common cold)
Picornaviridae Hepatovirus Hepatitis
+ssRNA, enveloped Togaviridae Alphavirus Encephalitis, hemorrhagic fever
Togaviridae Rubivirus Rubella
Retroviridae Lentivirus Acquired immune deficiency syndrome (AIDS)
−ssRNA, enveloped Filoviridae Zaire Ebolavirus Hemorrhagic fever
Orthomyxoviridae Influenzavirus A, B, C Flu
Rhabdoviridae Lyssavirus Rabies
Exercise \(5\)
What are the types of virus genomes?
Classification of Viral Diseases
While the ICTV has been tasked with the biological classification of viruses, it has also played an important role in the classification of diseases caused by viruses. To facilitate the tracking of virus-related human diseases, the ICTV has created classifications that link to the International Classification of Diseases (ICD), the standard taxonomy of disease that is maintained and updated by the World Health Organization (WHO). The ICD assigns an alphanumeric code of up to six characters to every type of viral infection, as well as all other types of diseases, medical conditions, and causes of death. This ICD code is used in conjunction with two other coding systems (the Current Procedural Terminology, and the Healthcare Common Procedure Coding System) to categorize patient conditions for treatment and insurance reimbursement.
For example, when a patient seeks treatment for a viral infection, ICD codes are routinely used by clinicians to order laboratory tests and prescribe treatments specific to the virus suspected of causing the illness. This ICD code is then used by medical laboratories to identify tests that must be performed to confirm the diagnosis. The ICD code is used by the health-care management system to verify that all treatments and laboratory work performed are appropriate for the given virus. Medical coders use ICD codes to assign the proper code for procedures performed, and medical billers, in turn, use this information to process claims for reimbursement by insurance companies. Vital-records keepers use ICD codes to record cause of death on death certificates, and epidemiologists used ICD codes to calculate morbidity and mortality statistics.
Exercise \(6\)
Identify two locations where you would likely find an ICD code.
Clinical Focus: Part 2
David’s doctor was concerned that his symptoms included prickling and itching at the site of the dog bite; these sensations could be early symptoms of rabies. Several tests are available to diagnose rabies in live patients, but no single antemortem test is adequate. The doctor decided to take samples of David’s blood, saliva, and skin for testing. The skin sample was taken from the nape of the neck (posterior side of the neck near the hairline). It was about 6-mm long and contained at least 10 hair follicles, including the superficial cutaneous nerve. An immunofluorescent staining technique was used on the skin biopsy specimen to detect rabies antibodies in the cutaneous nerves at the base of the hair follicles. A test was also performed on a serum sample from David’s blood to determine whether any antibodies for the rabies virus had been produced.
Meanwhile, the saliva sample was used for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, a test that can detect the presence of viral nucleic acid (RNA). The blood tests came back positive for the presence of rabies virus antigen, prompting David’s doctor to prescribe prophylactic treatment. David is given a series of intramuscular injections of human rabies immunoglobulin along with a series of rabies vaccines.
Exercise \(7\)
1. Why does the immunofluorescent technique look for rabies antibodies rather than the rabies virus itself?
2. If David has contracted rabies, what is his prognosis?
Summary
• Viruses are generally ultramicroscopic, typically from 20 nm to 900 nm in length. Some large viruses have been found.
• Virions are acellular and consist of a nucleic acid, DNA or RNA, but not both, surrounded by a protein capsid. There may also be a phospholipid membrane surrounding the capsid.
• Viruses are obligate intracellular parasites.
• Viruses are known to infect various types of cells found in plants, animals, fungi, protists, bacteria, and archaea. Viruses typically have limited host ranges and infect specific cell types.
• Viruses may have helical, polyhedral, or complex shapes.
• Classification of viruses is based on morphology, type of nucleic acid, host range, cell specificity, and enzymes carried within the virion.
• Like other diseases, viral diseases are classified using ICD codes.
Footnotes
1. 1 H. Lecoq. “[Discovery of the First Virus, the Tobacco Mosaic Virus: 1892 or 1898?].” Comptes Rendus de l’Academie des Sciences – Serie III – Sciences de la Vie 324, no. 10 (2001): 929–933.
2. 2 US Department of Health and Human Services, Centers for Disease Control and Prevention. “Antibiotic Resistance Threats in the United States, 2013.” www.cdc.gov/drugresistance/pd...s-2013-508.pdf (accessed September 22, 2015).
3. 3 M. Clokie et al. “Phages in Nature.” Bacteriophage 1, no. 1 (2011): 31–45.
4. 4 A. Sulakvelidze et al. “Bacteriophage Therapy.” Antimicrobial Agents and Chemotherapy 45, no. 3 (2001): 649–659.
5. 5 US Food and Drug Administration. “FDA Approval of Listeria-specific Bacteriophage Preparation on Ready-to-Eat (RTE) Meat and Poultry Products.” www.fda.gov/food/ingredientsp.../ucm083572.htm (accessed September 22, 2015).
6. 6 N. Philippe et al. “Pandoraviruses: Amoeba Viruses with Genomes up to 2.5 Mb Reaching that of Parasitic Eukaryotes.” Science 341, no. 6143 (2013): 281–286.
7. 7 J. Cohen. “What’s Old Is New: 1918 Virus Matches 2009 H1N1 Strain. Science 327, no. 5973 (2010): 1563–1564. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/06%3A_Acellular_Pathogens/6.01%3A_Viruses.txt |
Learning Objectives
• Describe the lytic and lysogenic life cycles
• Describe the replication process of animal viruses
• Describe unique characteristics of retroviruses and latent viruses
• Discuss human viruses and their virus-host cell interactions
• Explain the process of transduction
• Describe the replication process of plant viruses
All viruses depend on cells for reproduction and metabolic processes. By themselves, viruses do not encode for all of the enzymes necessary for viral replication. But within a host cell, a virus can commandeer cellular machinery to produce more viral particles. Bacteriophages replicate only in the cytoplasm, since prokaryotic cells do not have a nucleus or organelles. In eukaryotic cells, most DNA viruses can replicate inside the nucleus, with an exception observed in the large DNA viruses, such as the poxviruses, that can replicate in the cytoplasm. RNA viruses that infect animal cells often replicate in the cytoplasm.
The Life Cycle of Viruses with Prokaryote Hosts
The life cycle of bacteriophages has been a good model for understanding how viruses affect the cells they infect, since similar processes have been observed for eukaryotic viruses, which can cause immediate death of the cell or establish a latent or chronic infection. Virulent phages typically lead to the death of the cell through cell lysis. Temperate phages, on the other hand, can become part of a host chromosome and are replicated with the cell genome until such time as they are induced to make newly assembled viruses, or progeny viruses.
The Lytic Cycle
During the lytic cycle of virulent phage, the bacteriophage takes over the cell, reproduces new phages, and destroys the cell. T-even phage is a good example of a well-characterized class of virulent phages. There are five stages in the bacteriophage lytic cycle (see Figure \(1\)). Attachment is the first stage in the infection process in which the phage interacts with specific bacterial surface receptors (e.g., lipopolysaccharides and OmpC protein on host surfaces). Most phages have a narrow host range and may infect one species of bacteria or one strain within a species. This unique recognition can be exploited for targeted treatment of bacterial infection by phage therapy or for phage typing to identify unique bacterial subspecies or strains. The second stage of infection is entry or penetration. This occurs through contraction of the tail sheath, which acts like a hypodermic needle to inject the viral genome through the cell wall and membrane. The phage head and remaining components remain outside the bacteria.
The third stage of infection is biosynthesis of new viral components. After entering the host cell, the virus synthesizes virus-encoded endonucleases to degrade the bacterial chromosome. It then hijacks the host cell to replicate, transcribe, and translate the necessary viral components (capsomeres, sheath, base plates, tail fibers, and viral enzymes) for the assembly of new viruses. Polymerase genes are usually expressed early in the cycle, while capsid and tail proteins are expressed later. During the maturation phase, new virions are created. To liberate free phages, the bacterial cell wall is disrupted by phage proteins such as holin or lysozyme. The final stage is release. Mature viruses burst out of the host cell in a process called lysis and the progeny viruses are liberated into the environment to infect new cells.
The Lysogenic Cycle
In a lysogenic cycle, the phage genome also enters the cell through attachment and penetration. A prime example of a phage with this type of life cycle is the lambda phage. During the lysogenic cycle, instead of killing the host, the phage genome integrates into the bacterial chromosome and becomes part of the host. The integrated phage genome is called a prophage. A bacterial host with a prophage is called a lysogen. The process in which a bacterium is infected by a temperate phage is called lysogeny. It is typical of temperate phages to be latent or inactive within the cell. As the bacterium replicates its chromosome, it also replicates the phage’s DNA and passes it on to new daughter cells during reproduction. The presence of the phage may alter the phenotype of the bacterium, since it can bring in extra genes (e.g., toxin genes that can increase bacterial virulence). This change in the host phenotype is called lysogenic conversion or phage conversion. Some bacteria, such as Vibrio cholerae and Clostridium botulinum, are less virulent in the absence of the prophage. The phages infecting these bacteria carry the toxin genes in their genome and enhance the virulence of the host when the toxin genes are expressed. In the case of V. cholera, phage encoded toxin can cause severe diarrhea; in C. botulinum, the toxin can cause paralysis. During lysogeny, the prophage will persist in the host chromosome until induction, which results in the excision of the viral genome from the host chromosome. After induction has occurred the temperate phage can proceed through a lytic cycle and then undergo lysogeny in a newly infected cell (see Figure \(2\)).
Link to Learning
This video illustrates the stages of the lysogenic life cycle of a bacteriophage and the transition to a lytic phase.
Exercise \(1\)
Is a latent phage undetectable in a bacterium?
Transduction
Transduction occurs when a bacteriophage transfers bacterial DNA from one bacterium to another during sequential infections. There are two types of transduction: generalized and specialized transduction. During the lytic cycle of viral replication, the virus hijacks the host cell, degrades the host chromosome, and makes more viral genomes. As it assembles and packages DNA into the phage head, packaging occasionally makes a mistake. Instead of packaging viral DNA, it takes a random piece of host DNA and inserts it into the capsid. Once released, this virion will then inject the former host’s DNA into a newly infected host. The asexual transfer of genetic information can allow for DNA recombination to occur, thus providing the new host with new genes (e.g., an antibiotic-resistance gene, or a sugar-metabolizing gene). Generalized transduction occurs when a random piece of bacterial chromosomal DNA is transferred by the phage during the lytic cycle. Specialized transduction occurs at the end of the lysogenic cycle, when the prophage is excised and the bacteriophage enters the lytic cycle. Since the phage is integrated into the host genome, the prophage can replicate as part of the host. However, some conditions (e.g., ultraviolet light exposure or chemical exposure) stimulate the prophage to undergo induction, causing the phage to excise from the genome, enter the lytic cycle, and produce new phages to leave host cells. During the process of excision from the host chromosome, a phage may occasionally remove some bacterial DNA near the site of viral integration. The phage and host DNA from one end or both ends of the integration site are packaged within the capsid and are transferred to the new, infected host. Since the DNA transferred by the phage is not randomly packaged but is instead a specific piece of DNA near the site of integration, this mechanism of gene transfer is referred to as specialized transduction (see Figure \(3\)). The DNA can then recombine with host chromosome, giving the latter new characteristics. Transduction seems to play an important role in the evolutionary process of bacteria, giving them a mechanism for asexual exchange of genetic information.
Exercise \(2\)
Which phage life cycle is associated with which forms of transduction?
Life Cycle of Viruses with Animal Hosts
Lytic animal viruses follow similar infection stages to bacteriophages: attachment, penetration, biosynthesis, maturation, and release (see Figure \(4\)). However, the mechanisms of penetration, nucleic-acid biosynthesis, and release differ between bacterial and animal viruses. After binding to host receptors, animal viruses enter through endocytosis(engulfment by the host cell) or through membrane fusion (viral envelope with the host cell membrane). Many viruses are host specific, meaning they only infect a certain type of host; and most viruses only infect certain types of cells within tissues. This specificity is called a tissue tropism. Examples of this are demonstrated by the poliovirus, which exhibits tropism for the tissues of the brain and spinal cord, or the influenza virus, which has a primary tropism for the respiratory tract.
Animal viruses do not always express their genes using the normal flow of genetic information—from DNA to RNA to protein. Some viruses have a dsDNA genome like cellular organisms and can follow the normal flow. However, others may have ssDNA, dsRNA, or ssRNA genomes. The nature of the genome determines how the genome is replicated and expressed as viral proteins. If a genome is ssDNA, host enzymes will be used to synthesize a second strand that is complementary to the genome strand, thus producing dsDNA. The dsDNA can now be replicated, transcribed, and translated similar to host DNA.
If the viral genome is RNA, a different mechanism must be used. There are three types of RNA genome: dsRNA, positive (+) single-strand (+ssRNA) or negative (−) single-strand RNA (−ssRNA). If a virus has a +ssRNA genome, it can be translated directly to make viral proteins. Viral genomic +ssRNA acts like cellular mRNA. However, if a virus contains a −ssRNA genome, the host ribosomes cannot translate it until the −ssRNA is replicated into +ssRNA by viral RNA-dependent RNA polymerase (RdRP) (see Figure \(5\)). The RdRP is brought in by the virus and can be used to make +ssRNA from the original −ssRNA genome. The RdRP is also an important enzyme for the replication of dsRNA viruses, because it uses the negative strand of the double-stranded genome as a template to create +ssRNA. The newly synthesized +ssRNA copies can then be translated by cellular ribosomes.
An alternative mechanism for viral nucleic acid synthesis is observed in the retroviruses, which are +ssRNA viruses (see Figure \(6\)). Single-stranded RNA viruses such as HIV carry a special enzyme called reverse transcriptase within the capsid that synthesizes a complementary ssDNA (cDNA) copy using the +ssRNA genome as a template. The ssDNA is then made into dsDNA, which can integrate into the host chromosome and become a permanent part of the host. The integrated viral genome is called a provirus. The virus now can remain in the host for a long time to establish a chronic infection. The provirus stage is similar to the prophage stage in a bacterial infection during the lysogenic cycle. However, unlike prophage, the provirus does not undergo excision after splicing into the genome.
Exercise \(3\)
Is RNA-dependent RNA polymerase made from a viral gene or a host gene?
Persistent Infections
Persistent infection occurs when a virus is not completely cleared from the system of the host but stays in certain tissues or organs of the infected person. The virus may remain silent or undergo productive infection without seriously harming or killing the host. Mechanisms of persistent infection may involve the regulation of the viral or host gene expressions or the alteration of the host immune response. The two primary categories of persistent infections are latent infection and chronic infection. Examples of viruses that cause latent infections include herpes simplex virus (oral and genital herpes), varicella-zoster virus (chickenpox and shingles), and Epstein-Barr virus (mononucleosis). Hepatitis C virus and HIV are two examples of viruses that cause long-term chronic infections.
Latent Infection
Not all animal viruses undergo replication by the lytic cycle. There are viruses that are capable of remaining hidden or dormant inside the cell in a process called latency. These types of viruses are known as latent viruses and may cause latent infections. Viruses capable of latency may initially cause an acute infection before becoming dormant.
For example, the varicella-zoster virus infects many cells throughout the body and causes chickenpox, characterized by a rash of blisters covering the skin. About 10 to 12 days postinfection, the disease resolves and the virus goes dormant, living within nerve-cell ganglia for years. During this time, the virus does not kill the nerve cells or continue replicating. It is not clear why the virus stops replicating within the nerve cells and expresses few viral proteins but, in some cases, typically after many years of dormancy, the virus is reactivated and causes a new disease called shingles (Figure \(7\)). Whereas chickenpox affects many areas throughout the body, shingles is a nerve cell-specific disease emerging from the ganglia in which the virus was dormant.
Latent viruses may remain dormant by existing as circular viral genome molecules outside of the host chromosome. Others become proviruses by integrating into the host genome. During dormancy, viruses do not cause any symptoms of disease and may be difficult to detect. A patient may be unaware that he or she is carrying the virus unless a viral diagnostic test has been performed.
Chronic Infection
A chronic infection is a disease with symptoms that are recurrent or persistent over a long time. Some viral infections can be chronic if the body is unable to eliminate the virus. HIV is an example of a virus that produces a chronic infection, often after a long period of latency. Once a person becomes infected with HIV, the virus can be detected in tissues continuously thereafter, but untreated patients often experience no symptoms for years. However, the virus maintains chronic persistence through several mechanisms that interfere with immune function, including preventing expression of viral antigens on the surface of infected cells, altering immune cells themselves, restricting expression of viral genes, and rapidly changing viral antigens through mutation. Eventually, the damage to the immune system results in progression of the disease leading to acquired immunodeficiency syndrome (AIDS). The various mechanisms that HIV uses to avoid being cleared by the immune system are also used by other chronically infecting viruses, including the hepatitis C virus.
Exercise \(4\)
In what two ways can a virus manage to maintain a persistent infection?
Life Cycle of Viruses with Plant Hosts
Plant viruses are more similar to animal viruses than they are to bacteriophages. Plant viruses may be enveloped or non-enveloped. Like many animal viruses, plant viruses can have either a DNA or RNA genome and be single stranded or double stranded. However, most plant viruses do not have a DNA genome; the majority have a +ssRNA genome, which acts like messenger RNA (mRNA). Only a minority of plant viruses have other types of genomes.
Plant viruses may have a narrow or broad host range. For example, the citrus tristeza virus infects only a few plants of the Citrus genus, whereas the cucumber mosaic virus infects thousands of plants of various plant families. Most plant viruses are transmitted by contact between plants, or by fungi, nematodes, insects, or other arthropods that act as mechanical vectors. However, some viruses can only be transferred by a specific type of insect vector; for example, a particular virus might be transmitted by aphids but not whiteflies. In some cases, viruses may also enter healthy plants through wounds, as might occur due to pruning or weather damage.
Viruses that infect plants are considered biotrophic parasites, which means that they can establish an infection without killing the host, similar to what is observed in the lysogenic life cycles of bacteriophages. Viral infection can be asymptomatic (latent) or can lead to cell death (lytic infection). The life cycle begins with the penetration of the virus into the host cell. Next, the virus is uncoated within the cytoplasm of the cell when the capsid is removed. Depending on the type of nucleic acid, cellular components are used to replicate the viral genome and synthesize viral proteins for assembly of new virions. To establish a systemic infection, the virus must enter a part of the vascular system of the plant, such as the phloem. The time required for systemic infection may vary from a few days to a few weeks depending on the virus, the plant species, and the environmental conditions. The virus life cycle is complete when it is transmitted from an infected plant to a healthy plant.
Exercise \(5\)
What is the structure and genome of a typical plant virus?
Viral Growth Curve
Unlike the growth curve for a bacterial population, the growth curve for a virus population over its life cycle does not follow a sigmoidal curve. During the initial stage, an inoculum of virus causes infection. In the eclipse phase, viruses bind and penetrate the cells with no virions detected in the medium. The chief difference that next appears in the viral growth curve compared to a bacterial growth curve occurs when virions are released from the lysed host cell at the same time. Such an occurrence is called a burst, and the number of virions per bacterium released is described as the burst size. In a one-step multiplication curve for bacteriophage, the host cells lyse, releasing many viral particles to the medium, which leads to a very steep rise in viral titer (the number of virions per unit volume). If no viable host cells remain, the viral particles begin to degrade during the decline of the culture (see Figure \(8\)).
Exercise \(6\)
What aspect of the life cycle of a virus leads to the sudden increase in the growth curve?
Unregistered Treatments
Ebola is incurable and deadly. The outbreak in West Africa in 2014 was unprecedented, dwarfing other human Ebola epidemics in the level of mortality. Of 24,666 suspected or confirmed cases reported, 10,179 people died.1
No approved treatments or vaccines for Ebola are available. While some drugs have shown potential in laboratory studies and animal models, they have not been tested in humans for safety and effectiveness. Not only are these drugs untested or unregistered but they are also in short supply.
Given the great suffering and high mortality rates, it is fair to ask whether unregistered and untested medications are better than none at all. Should such drugs be dispensed and, if so, who should receive them, in light of their extremely limited supplies? Is it ethical to treat untested drugs on patients with Ebola? On the other hand, is it ethical to withhold potentially life-saving drugs from dying patients? Or should the drugs perhaps be reserved for health-care providers working to contain the disease?
In August 2014, two infected US aid workers and a Spanish priest were treated with ZMapp, an unregistered drug that had been tested in monkeys but not in humans. The two American aid workers recovered, but the priest died. Later that month, the WHO released a report on the ethics of treating patients with the drug. Since Ebola is often fatal, the panel reasoned that it is ethical to give the unregistered drugs and unethical to withhold them for safety concerns. This situation is an example of “compassionate use” outside the well-established system of regulation and governance of therapies.
Ebola in the US
On September 24, 2014, Thomas Eric Duncan arrived at the Texas Health Presbyterian Hospital in Dallas complaining of a fever, headache, vomiting, and diarrhea—symptoms commonly observed in patients with the cold or the flu. After examination, an emergency department doctor diagnosed him with sinusitis, prescribed some antibiotics, and sent him home. Two days later, Duncan returned to the hospital by ambulance. His condition had deteriorated and additional blood tests confirmed that he has been infected with the Ebola virus.
Further investigations revealed that Duncan had just returned from Liberia, one of the countries in the midst of a severe Ebola epidemic. On September 15, nine days before he showed up at the hospital in Dallas, Duncan had helped transport an Ebola-stricken neighbor to a hospital in Liberia. The hospital continued to treat Duncan, but he died several days after being admitted.
The timeline of the Duncan case is indicative of the life cycle of the Ebola virus. The incubation time for Ebola ranges from 2 days to 21 days. Nine days passed between Duncan’s exposure to the virus infection and the appearance of his symptoms. This corresponds, in part, to the eclipse period in the growth of the virus population. During the eclipse phase, Duncan would have been unable to transmit the disease to others. However, once an infected individual begins exhibiting symptoms, the disease becomes very contagious. Ebola virus is transmitted through direct contact with droplets of bodily fluids such as saliva, blood, and vomit. Duncan could conceivably have transmitted the disease to others at any time after he began having symptoms, presumably some time before his arrival at the hospital in Dallas. Once a hospital realizes a patient like Duncan is infected with Ebola virus, the patient is immediately quarantined, and public health officials initiate a back trace to identify everyone with whom a patient like Duncan might have interacted during the period in which he was showing symptoms.
Public health officials were able to track down 10 high-risk individuals (family members of Duncan) and 50 low-risk individuals to monitor them for signs of infection. None contracted the disease. However, one of the nurses charged with Duncan’s care did become infected. This, along with Duncan’s initial misdiagnosis, made it clear that US hospitals needed to provide additional training to medical personnel to prevent a possible Ebola outbreak in the US.
Exercise \(7\)
1. What types of training can prepare health professionals to contain emerging epidemics like the Ebola outbreak of 2014?
2. What is the difference between a contagious pathogen and an infectious pathogen?
Link to Learning
For additional information about Ebola, please visit the CDC website.
Summary
• Many viruses target specific hosts or tissues. Some may have more than one host.
• Many viruses follow several stages to infect host cells. These stages include attachment, penetration, uncoating, biosynthesis, maturation, and release.
• Bacteriophages have a lytic or lysogenic cycle. The lytic cycle leads to the death of the host, whereas the lysogenic cycle leads to integration of phage into the host genome.
• Bacteriophages inject DNA into the host cell, whereas animal viruses enter by endocytosis or membrane fusion.
• Animal viruses can undergo latency, similar to lysogeny for a bacteriophage.
• The majority of plant viruses are positive-strand ssRNA and can undergo latency, chronic, or lytic infection, as observed for animal viruses.
• The growth curve of bacteriophage populations is a one-step multiplication curve and not a sigmoidal curve, as compared to the bacterial growth curve.
• Bacteriophages transfer genetic information between hosts using either generalized or specialized transduction.
Footnotes
1. 1 World Health Organization. “WHO Ebola Data and Statistics.” March 18, 2005. http://apps.who.int/gho/data/view.eb...150318?lang=en | textbooks/bio/Microbiology/Microbiology_(OpenStax)/06%3A_Acellular_Pathogens/6.02%3A_The_Viral_Life_Cycle.txt |
Learning Objectives
• Discuss why viruses were originally described as filterable agents
• Describe the cultivation of viruses and specimen collection and handling
• Compare in vivo and in vitro techniques used to cultivate viruses
At the beginning of this chapter, we described how porcelain Chamberland filters with pores small enough to allow viruses to pass through were used to discover TMV. Today, porcelain filters have been replaced with membrane filters and other devices used to isolate and identify viruses.
Isolation of Viruses
Unlike bacteria, many of which can be grown on an artificial nutrient medium, viruses require a living host cell for replication. Infected host cells (eukaryotic or prokaryotic) can be cultured and grown, and then the growth medium can be harvested as a source of virus. Virions in the liquid medium can be separated from the host cells by either centrifugation or filtration. Filters can physically remove anything present in the solution that is larger than the virions; the viruses can then be collected in the filtrate (Figure \(1\)).
Exercise \(1\)
What size filter pore is needed to collect a virus?
Cultivation of Viruses
Viruses can be grown in vivo (within a whole living organism, plant, or animal) or in vitro (outside a living organism in cells in an artificial environment, such as a test tube, cell culture flask, or agar plate). Bacteriophages can be grown in the presence of a dense layer of bacteria (also called a bacterial lawn) grown in a 0.7 % soft agar in a Petri dish or flat (horizontal) flask (Figure \(\PageIndex{2a}\)). The agar concentration is decreased from the 1.5% usually used in culturing bacteria. The soft 0.7% agar allows the bacteriophages to easily diffuse through the medium. For lytic bacteriophages, lysing of the bacterial hosts can then be readily observed when a clear zone called a plaque is detected (Figure \(\PageIndex{1b}\)). As the phage kills the bacteria, many plaques are observed among the cloudy bacterial lawn.
Animal viruses require cells within a host animal or tissue-culture cells derived from an animal. Animal virus cultivation is important for 1) identification and diagnosis of pathogenic viruses in clinical specimens, 2) production of vaccines, and 3) basic research studies. In vivo host sources can be a developing embryo in an embryonated bird’s egg (e.g., chicken, turkey) or a whole animal. For example, most of the influenza vaccine manufactured for annual flu vaccination programs is cultured in hens’ eggs.
The embryo or host animal serves as an incubator for viral replication (Figure \(3\)). Location within the embryo or host animal is important. Many viruses have a tissue tropism, and must therefore be introduced into a specific site for growth. Within an embryo, target sites include the amniotic cavity, the chorioallantoic membrane, or the yolk sac. Viral infection may damage tissue membranes, producing lesions called pox; disrupt embryonic development; or cause the death of the embryo.
For in vitro studies, various types of cells can be used to support the growth of viruses. A primary cell culture is freshly prepared from animal organs or tissues. Cells are extracted from tissues by mechanical scraping or mincing to release cells or by an enzymatic method using trypsin or collagenase to break up tissue and release single cells into suspension. Because of anchorage-dependence requirements, primary cell cultures require a liquid culture medium in a Petri dish or tissue-culture flask so cells have a solid surface such as glass or plastic for attachment and growth. Primary cultures usually have a limited life span. When cells in a primary culture undergo mitosis and a sufficient density of cells is produced, cells come in contact with other cells. When this cell-to-cell-contact occurs, mitosis is triggered to stop. This is called contact inhibition and it prevents the density of the cells from becoming too high. To prevent contact inhibition, cells from the primary cell culture must be transferred to another vessel with fresh growth medium. This is called a secondary cell culture. Periodically, cell density must be reduced by pouring off some cells and adding fresh medium to provide space and nutrients to maintain cell growth. In contrast to primary cell cultures, continuous cell lines, usually derived from transformed cells or tumors, are often able to be subcultured many times or even grown indefinitely (in which case they are called immortal). Continuous cell lines may not exhibit anchorage dependency (they will grow in suspension) and may have lost their contact inhibition. As a result, continuous cell lines can grow in piles or lumps resembling small tumor growths (Figure \(4\)).
An example of an immortal cell line is the HeLa cell line, which was originally cultivated from tumor cells obtained from Henrietta Lacks, a patient who died of cervical cancer in 1951. HeLa cells were the first continuous tissue-culture cell line and were used to establish tissue culture as an important technology for research in cell biology, virology, and medicine. Prior to the discovery of HeLa cells, scientists were not able to establish tissue cultures with any reliability or stability. More than six decades later, this cell line is still alive and being used for medical research. See The Immortal Cell Line of Henrietta Lacks to read more about this important cell line and the controversial means by which it was obtained.
Exercise \(2\)
What property of cells makes periodic dilutions of primary cell cultures necessary?
The Immortal Cell Line of Henrietta Lacks
In January 1951, Henrietta Lacks, a 30-year-old African American woman from Baltimore, was diagnosed with cervical cancer at John Hopkins Hospital. We now know her cancer was caused by the human papillomavirus (HPV). Cytopathic effects of the virus altered the characteristics of her cells in a process called transformation, which gives the cells the ability to divide continuously. This ability, of course, resulted in a cancerous tumor that eventually killed Mrs. Lacks in October at age 31. Before her death, samples of her cancerous cells were taken without her knowledge or permission. The samples eventually ended up in the possession of Dr. George Gey, a biomedical researcher at Johns Hopkins University. Gey was able to grow some of the cells from Lacks’s sample, creating what is known today as the immortal HeLa cell line. These cells have the ability to live and grow indefinitely and, even today, are still widely used in many areas of research.
According to Lacks’s husband, neither Henrietta nor the family gave the hospital permission to collect her tissue specimen. Indeed, the family was not aware until 20 years after Lacks’s death that her cells were still alive and actively being used for commercial and research purposes. Yet HeLa cells have been pivotal in numerous research discoveries related to polio, cancer, and AIDS, among other diseases. The cells have also been commercialized, although they have never themselves been patented. Despite this, Henrietta Lacks’s estate has never benefited from the use of the cells, although, in 2013, the Lacks family was given control over the publication of the genetic sequence of her cells.
This case raises several bioethical issues surrounding patients’ informed consent and the right to know. At the time Lacks’s tissues were taken, there were no laws or guidelines about informed consent. Does that mean she was treated fairly at the time? Certainly by today’s standards, the answer would be no. Harvesting tissue or organs from a dying patient without consent is not only considered unethical but illegal, regardless of whether such an act could save other patients’ lives. Is it ethical, then, for scientists to continue to use Lacks’s tissues for research, even though they were obtained illegally by today’s standards?
Ethical or not, Lacks’s cells are widely used today for so many applications that it is impossible to list them all. Is this a case in which the ends justify the means? Would Lacks be pleased to know about her contribution to science and the millions of people who have benefited? Would she want her family to be compensated for the commercial products that have been developed using her cells? Or would she feel violated and exploited by the researchers who took part of her body without her consent? Because she was never asked, we will never know.
Detection of a Virus
Regardless of the method of cultivation, once a virus has been introduced into a whole host organism, embryo, or tissue-culture cell, a sample can be prepared from the infected host, embryo, or cell line for further analysis under a brightfield, electron, or fluorescent microscope. Cytopathic effects (CPEs) are distinct observable cell abnormalities due to viral infection. CPEs can include loss of adherence to the surface of the container, changes in cell shape from flat to round, shrinkage of the nucleus, vacuoles in the cytoplasm, fusion of cytoplasmic membranes and the formation of multinucleated syncytia, inclusion bodies in the nucleus or cytoplasm, and complete cell lysis (see Figure \(6\)).
Further pathological changes include viral disruption of the host genome and altering normal cells into transformed cells, which are the types of cells associated with carcinomas and sarcomas. The type or severity of the CPE depends on the type of virus involved. Figure \(6\) lists CPEs for specific viruses.
Link to Learning
Watch this video to learn about the effects of viruses on cells.
Hemagglutination Assay
A serological assay is used to detect the presence of certain types of viruses in patient serum. Serum is the straw-colored liquid fraction of blood plasma from which clotting factors have been removed. Serum can be used in a direct assay called a hemagglutination assay to detect specific types of viruses in the patient’s sample. Hemagglutination is the agglutination (clumping) together of erythrocytes (red blood cells). Many viruses produce surface proteins or spikes called hemagglutinins that can bind to receptors on the membranes of erythrocytes and cause the cells to agglutinate. Hemagglutination is observable without using the microscope, but this method does not always differentiate between infectious and noninfectious viral particles, since both can agglutinate erythrocytes.
To identify a specific pathogenic virus using hemagglutination, we must use an indirect approach. Proteins called antibodies, generated by the patient’s immune system to fight a specific virus, can be used to bind to components such as hemagglutinins that are uniquely associated with specific types of viruses. The binding of the antibodies with the hemagglutinins found on the virus subsequently prevent erythrocytes from directly interacting with the virus. So when erythrocytes are added to the antibody-coated viruses, there is no appearance of agglutination; agglutination has been inhibited. We call these types of indirect assays for virus-specific antibodies hemagglutination inhibition (HAI) assays. HAI can be used to detect the presence of antibodies specific to many types of viruses that may be causing or have caused an infection in a patient even months or years after infection (see Figure \(7\)). This assay is described in greater detail in Agglutination Assays.
Exercise \(3\)
What is the outcome of a positive HIA test?
Nucleic Acid Amplification Test
Nucleic acid amplification tests (NAAT) are used in molecular biology to detect unique nucleic acid sequences of viruses in patient samples. Polymerase chain reaction (PCR) is an NAAT used to detect the presence of viral DNA in a patient’s tissue or body fluid sample. PCR is a technique that amplifies (i.e., synthesizes many copies) of a viral DNA segment of interest. Using PCR, short nucleotide sequences called primers bind to specific sequences of viral DNA, enabling identification of the virus.
Reverse transcriptase-PCR (RT-PCR) is an NAAT used to detect the presence of RNA viruses. RT-PCR differs from PCR in that the enzyme reverse transcriptase (RT) is used to make a cDNA from the small amount of viral RNA in the specimen. The cDNA can then be amplified by PCR. Both PCR and RT-PCR are used to detect and confirm the presence of the viral nucleic acid in patient specimens.
HPV Scare
Michelle, a 21-year-old nursing student, came to the university clinic worried that she might have been exposed to a sexually transmitted disease (STD). Her sexual partner had recently developed several bumps on the base of his penis. He had put off going to the doctor, but Michelle suspects they are genital warts caused by HPV. She is especially concerned because she knows that HPV not only causes warts but is a prominent cause of cervical cancer. She and her partner always use condoms for contraception, but she is not confident that this precaution will protect her from HPV.
Michelle’s physician finds no physical signs of genital warts or any other STDs, but recommends that Michelle get a Pap smear along with an HPV test. The Pap smear will screen for abnormal cervical cells and the CPEs associated with HPV; the HPV test will test for the presence of the virus. If both tests are negative, Michelle can be more assured that she most likely has not become infected with HPV. However, her doctor suggests it might be wise for Michelle to get vaccinated against HPV to protect herself from possible future exposure.
Exercise \(4\)
Why does Michelle’s physician order two different tests instead of relying on one or the other?
Enzyme Immunoassay
Enzyme immunoassays (EIAs) rely on the ability of antibodies to detect and attach to specific biomolecules called antigens. The detecting antibody attaches to the target antigen with a high degree of specificity in what might be a complex mixture of biomolecules. Also included in this type of assay is a colorless enzyme attached to the detecting antibody. The enzyme acts as a tag on the detecting antibody and can interact with a colorless substrate, leading to the production of a colored end product. EIAs often rely on layers of antibodies to capture and react with antigens, all of which are attached to a membrane filter (see Figure \(8\)). EIAs for viral antigens are often used as preliminary screening tests. If the results are positive, further confirmation will require tests with even greater sensitivity, such as a western blot or an NAAT. EIAs are discussed in more detail in EIAs and ELISAs.
Exercise \(5\)
What typically indicates a positive EIA test?
Clinical Focus: Part 3
Along with the RT/PCR analysis, David’s saliva was also collected for viral cultivation. In general, no single diagnostic test is sufficient for antemortem diagnosis, since the results will depend on the sensitivity of the assay, the quantity of virions present at the time of testing, and the timing of the assay, since release of virions in the saliva can vary. As it turns out, the result was negative for viral cultivation from the saliva. This is not surprising to David’s doctor, because one negative result is not an absolute indication of the absence of infection. It may be that the number of virions in the saliva is low at the time of sampling. It is not unusual to repeat the test at intervals to enhance the chance of detecting higher virus loads.
Exercise \(6\)
Should David’s doctor modify his course of treatment based on these test results?
Summary
• Viral cultivation requires the presence of some form of host cell (whole organism, embryo, or cell culture).
• Viruses can be isolated from samples by filtration.
• Viral filtrate is a rich source of released virions.
• Bacteriophages are detected by presence of clear plaques on bacterial lawn.
• Animal and plant viruses are detected by cytopathic effects, molecular techniques (PCR, RT-PCR), enzyme immunoassays, and serological assays (hemagglutination assay, hemagglutination inhibition assay). | textbooks/bio/Microbiology/Microbiology_(OpenStax)/06%3A_Acellular_Pathogens/6.03%3A_Isolation_Culture_and_Identification_of_Viruses.txt |
Learning Objectives
• Describe viroids and their unique characteristics
• Describe virusoids and their unique characteristics
• Describe prions and their unique characteristics
Research attempts to discover the causative agents of previously uninvestigated diseases have led to the discovery of nonliving disease agents quite different from viruses. These include particles consisting only of RNA or only of protein that, nonetheless, are able to self-propagate at the expense of a host—a key similarity to viruses that allows them to cause disease conditions. To date, these discoveries include viroids, virusoids, and the proteinaceous prions.
Viroids
In 1971, Theodor Diener, a pathologist working at the Agriculture Research Service, discovered an acellular particle that he named a viroid, meaning “virus-like.” Viroids consist only of a short strand of circular RNA capable of self-replication. The first viroid discovered was found to cause potato tuber spindle disease, which causes slower sprouting and various deformities in potato plants (see Figure \(1\)). Like viruses, potato spindle tuber viroids (PSTVs) take control of the host machinery to replicate their RNA genome. Unlike viruses, viroids do not have a protein coat to protect their genetic information.
Viroids can result in devastating losses of commercially important agricultural food crops grown in fields and orchards. Since the discovery of PSTV, other viroids have been discovered that cause diseases in plants. Tomato planta macho viroid (TPMVd) infects tomato plants, which causes loss of chlorophyll, disfigured and brittle leaves, and very small tomatoes, resulting in loss of productivity in this field crop. Avocado sunblotch viroid (ASBVd) results in lower yields and poorer-quality fruit. ASBVd is the smallest viroid discovered thus far that infects plants. Peach latent mosaic viroid(PLMVd) can cause necrosis of flower buds and branches, and wounding of ripened fruit, which leads to fungal and bacterial growth in the fruit. PLMVd can also cause similar pathological changes in plums, nectarines, apricots, and cherries, resulting in decreased productivity in these orchards, as well. Viroids, in general, can be dispersed mechanically during crop maintenance or harvesting, vegetative reproduction, and possibly via seeds and insects, resulting in a severe drop in food availability and devastating economic consequences.
Exercise \(1\)
What is the genome of a viroid made of?
Virusoids
A second type of pathogenic RNA that can infect commercially important agricultural crops are the virusoids, which are subviral particles best described as non–self-replicating ssRNAs. RNA replication of virusoids is similar to that of viroids but, unlike viroids, virusoids require that the cell also be infected with a specific “helper” virus. There are currently only five described types of virusoids and their associated helper viruses. The helper viruses are all from the family of Sobemoviruses. An example of a helper virus is the subterranean clover mottle virus, which has an associated virusoid packaged inside the viral capsid. Once the helper virus enters the host cell, the virusoids are released and can be found free in plant cell cytoplasm, where they possess ribozyme activity. The helper virus undergoes typical viral replication independent of the activity of the virusoid. The virusoid genomes are small, only 220 to 388 nucleotides long. A virusoid genome does not code for any proteins, but instead serves only to replicate virusoid RNA.
Virusoids belong to a larger group of infectious agents called satellite RNAs, which are similar pathogenic RNAs found in animals. Unlike the plant virusoids, satellite RNAs may encode for proteins; however, like plant virusoids, satellite RNAs must coinfect with a helper virus to replicate. One satellite RNA that infects humans and that has been described by some scientists as a virusoid is the hepatitis delta virus (HDV), which, by some reports, is also called hepatitis delta virusoid. Much larger than a plant virusoid, HDV has a circular, ssRNA genome of 1,700 nucleotides and can direct the biosynthesis of HDV-associated proteins. The HDV helper virus is the hepatitis B virus (HBV). Coinfection with HBV and HDV results in more severe pathological changes in the liver during infection, which is how HDV was first discovered.
Exercise \(2\)
What is the main difference between a viroid and a virusoid?
Prions
At one time, scientists believed that any infectious particle must contain DNA or RNA. Then, in 1982, Stanley Prusiner, a medical doctor studying scrapie (a fatal, degenerative disease in sheep) discovered that the disease was caused by proteinaceous infectious particles, or prions. Because proteins are acellular and do not contain DNA or RNA, Prusiner’s findings were originally met with resistance and skepticism; however, his research was eventually validated, and he received the Nobel Prize in Physiology or Medicine in 1997.
A prion is a misfolded rogue form of a normal protein (PrPc) found in the cell. This rogue prion protein (PrPsc), which may be caused by a genetic mutation or occur spontaneously, can be infectious, stimulating other endogenous normal proteins to become misfolded, forming plaques (see Figure \(2\)). Today, prions are known to cause various forms of transmissible spongiform encephalopathy (TSE) in human and animals. TSE is a rare degenerative disorder that affects the brain and nervous system. The accumulation of rogue proteins causes the brain tissue to become sponge-like, killing brain cells and forming holes in the tissue, leading to brain damage, loss of motor coordination, and dementia (see Figure \(3\)). Infected individuals are mentally impaired and become unable to move or speak. There is no cure, and the disease progresses rapidly, eventually leading to death within a few months or years.
TSEs in humans include kuru, fatal familial insomnia, Gerstmann-Straussler-Scheinker disease, and Creutzfeldt-Jakob disease (see Figure \(3\)). TSEs in animals include mad cow disease, scrapie (in sheep and goats), and chronic wasting disease (in elk and deer). TSEs can be transmitted between animals and from animals to humans by eating contaminated meat or animal feed. Transmission between humans can occur through heredity (as is often the case with GSS and CJD) or by contact with contaminated tissue, as might occur during a blood transfusion or organ transplant. There is no evidence for transmission via casual contact with an infected person. Table \(1\) lists TSEs that affect humans and their modes of transmission.
Table \(1\): Transmissible Spongiform Encephalopathies (TSEs) in Humans
Disease Mechanism(s) of Transmission1
Sporadic CJD (sCJD) Not known; possibly by alteration of normal prior protein (PrP) to rogue form due to somatic mutation
Variant CJD (vCJD) Eating contaminated cattle products and by secondary bloodborne transmission
Familial CJD (fCJD) Mutation in germline PrP gene
Iatrogenic CJD (iCJD) Contaminated neurosurgical instruments, corneal graft, gonadotrophic hormone, and, secondarily, by blood transfusion
Kuru Eating infected meat through ritualistic cannibalism
Gerstmann-Straussler-Scheinker disease (GSS) Mutation in germline PrP gene
Fatal familial insomnia (FFI) Mutation in germline PrP gene
Prions are extremely difficult to destroy because they are resistant to heat, chemicals, and radiation. Even standard sterilization procedures do not ensure the destruction of these particles. Currently, there is no treatment or cure for TSE disease, and contaminated meats or infected animals must be handled according to federal guidelines to prevent transmission.
Exercise \(3\)
Does a prion have a genome?
Clinical Focus: Resolution
A few days later, David’s doctor receives the results of the immunofluorescence test on his skin sample. The test is negative for rabies antigen. A second viral antigen test on his saliva sample also comes back negative. Despite these results, the doctor decides to continue David’s current course of treatment. Given the positive RT-PCR test, it is best not to rule out a possible rabies infection.
Near the site of the bite, David receives an injection of rabies immunoglobulin, which attaches to and inactivates any rabies virus that may be present in his tissues. Over the next 14 days, he receives a series of four rabies-specific vaccinations in the arm. These vaccines activate David’s immune response and help his body recognize and fight the virus. Thankfully, with treatment, David symptoms improve and he makes a full recovery.
Not all rabies cases have such a fortunate outcome. In fact, rabies is usually fatal once the patient starts to exhibit symptoms, and postbite treatments are mainly palliative (i.e., sedation and pain management).
Summary
• Other acellular agents such as viroids, virusoids, and prions also cause diseases. Viroids consist of small, naked ssRNAs that cause diseases in plants. Virusoids are ssRNAs that require other helper viruses to establish an infection. Prions are proteinaceous infectious particles that cause transmissible spongiform encephalopathies.
• Prions are extremely resistant to chemicals, heat, and radiation.
• There are no treatments for prion infection.
Footnotes
1. 1 National Institute of Neurological Disorders and Stroke. “Creutzfeldt-Jakob Disease Fact Sheet.” http://www.ninds.nih.gov/disorders/cjd/detail_cjd.htm (accessed December 31, 2015). | textbooks/bio/Microbiology/Microbiology_(OpenStax)/06%3A_Acellular_Pathogens/6.04%3A_Viroids_Virusoids_and_Prions.txt |
6.1: Viruses
Viruses are generally ultramicroscopic, typically from 20 nm to 900 nm in length. Some large viruses have been found. Virions are acellular and consist of a nucleic acid, DNA or RNA, but not both, surrounded by a protein capsid. There may also be a phospholipid membrane surrounding the capsid. Viruses are obligate intracellular parasites.
Multiple Choice
The component(s) of a virus that is/are extended from the envelope for attachment is/are the:
1. capsomeres
2. spikes
3. nucleic acid
4. viral whiskers
Answer
B
Which of the following does a virus lack? Select all that apply.
1. ribosomes
2. metabolic processes
3. nucleic acid
4. glycoprotein
Answer
A and B
The envelope of a virus is derived from the host’s
1. nucleic acids
2. membrane structures
3. cytoplasm
4. genome
Answer
B
In naming viruses, the family name ends with ________ and genus name ends with _________.
1. −virus; −viridae
2. −viridae; −virus
3. −virion; virus
4. −virus; virion
Answer
B
What is another name for a nonenveloped virus?
1. enveloped virus
2. provirus
3. naked virus
4. latent virus
Answer
C
True/False
True or False: Scientists have identified viruses that are able to infect fungal cells.
Answer
True
Fill in the Blank
A virus that infects a bacterium is called a/an ___________________.
Answer
bacteriophage
A/an __________ virus possesses characteristics of both a polyhedral and helical virus.
Answer
complex
A virus containing only nucleic acid and a capsid is called a/an ___________________ virus or __________________ virus.
Answer
naked or nonenveloped
The ____________ _____________ on the bacteriophage allow for binding to the bacterial cell.
Answer
tail fibers
Short Answer
Discuss the geometric differences among helical, polyhedral, and complex viruses.
What was the meaning of the word “virus” in the 1880s and why was it used to describe the cause of tobacco mosaic disease?
Critical Thinking
Name each labeled part of the illustrated bacteriophage.
In terms of evolution, which do you think arises first? The virus or the host? Explain your answer.
Do you think it is possible to create a virus in the lab? Imagine that you are a mad scientist. Describe how you would go about creating a new virus.
6.2: The Viral Life Cycle
Many viruses target specific hosts or tissues. Some may have more than one host. Many viruses follow several stages to infect host cells. These stages include attachment, penetration, uncoating, biosynthesis, maturation, and release. Bacteriophages have a lytic or lysogenic cycle. The lytic cycle leads to the death of the host, whereas the lysogenic cycle leads to integration of phage into the host genome.
Multiple Choice
Which of the following leads to the destruction of the host cells?
1. lysogenic cycle
2. lytic cycle
3. prophage
4. temperate phage
Answer
B
A virus obtains its envelope during which of the following phases?
1. attachment
2. penetration
3. assembly
4. release
Answer
D
Which of the following components is brought into a cell by HIV?
1. a DNA-dependent DNA polymerase
2. RNA polymerase
3. ribosome
4. reverse transcriptase
Answer
D
A positive-strand RNA virus:
1. must first be converted to a mRNA before it can be translated.
2. can be used directly to translate viral proteins.
3. will be degraded by host enzymes.
4. is not recognized by host ribosomes.
Answer
B
What is the name for the transfer of genetic information from one bacterium to another bacterium by a phage?
1. transduction
2. penetration
3. excision
4. translation
Answer
A
Fill in the Blank
An enzyme from HIV that can make a copy of DNA from RNA is called _______________________.
Answer
reverse transcriptase
For lytic viruses, _________________ is a phase during a viral growth curve when the virus is not detected.
Answer
eclipse
Short Answer
Briefly explain the difference between the mechanism of entry of a T-even bacteriophage and an animal virus.
Discuss the difference between generalized and specialized transduction.
Differentiate between lytic and lysogenic cycles.
Critical Thinking
Label the five stages of a bacteriophage infection in the figure:
Bacteriophages have lytic and lysogenic cycles. Discuss the advantages and disadvantages for the phage.
How does reverse transcriptase aid a retrovirus in establishing a chronic infection?
Discuss some methods by which plant viruses are transmitted from a diseased plant to a healthy one.
6.3: Isolation, Culture, and Identification of Viruses
Viral cultivation requires the presence of some form of host cell (whole organism, embryo, or cell culture). Viruses can be isolated from samples by filtration. Viral filtrate is a rich source of released virions. Bacteriophages are detected by presence of clear plaques on bacterial lawn. Animal and plant viruses are detected by cytopathic effects, molecular techniques (PCR, RT-PCR), enzyme immunoassays, and serological assays (hemagglutination assay, hemagglutination inhibition assay).
Multiple Choice
Which of the followings cannot be used to culture viruses?
1. tissue culture
2. liquid medium only
3. embryo
4. animal host
Answer
B
Which of the following tests can be used to detect the presence of a specific virus?
1. EIA
2. RT-PCR
3. PCR
4. all of the above
Answer
D
Which of the following is NOT a cytopathic effect?
1. transformation
2. cell fusion
3. mononucleated cell
4. inclusion bodies
Answer
C
Fill in the Blank
Viruses can be diagnosed and observed using a(n) _____________ microscope.
Answer
Electron
Cell abnormalities resulting from a viral infection are called ____________ _____________.
Answer
cytopathic effects
Short Answer
Briefly explain the various methods of culturing viruses.
Critical Thinking
Label the components indicated by arrows.
(credit: modification of work by American Society for Microbiology)
What are some characteristics of the viruses that are similar to a computer virus?
6.4: Viroids, Virusoids, and Prions
Other acellular agents such as viroids, virusoids, and prions also cause diseases. Viroids consist of small, naked ssRNAs that cause diseases in plants. Virusoids are ssRNAs that require other helper viruses to establish an infection. Prions are proteinaceous infectious particles that cause transmissible spongiform encephalopathies. Prions are extremely resistant to chemicals, heat, and radiation.
Multiple Choice
Which of these infectious agents do not have nucleic acid?
1. viroids
2. viruses
3. bacteria
4. prions
Answer
D
Which of the following is true of prions?
1. They can be inactivated by boiling at 100 °C.
2. They contain a capsid.
3. They are a rogue form of protein, PrP.
4. They can be reliably inactivated by an autoclave.
Answer
C
Fill in the Blank
Both viroids and virusoids have a(n) _________ genome, but virusoids require a(n) _________ to reproduce.
Answer
RNA, helper virus
Short Answer
Describe the disease symptoms observed in animals infected with prions.
Critical Thinking
Does a prion replicate? Explain. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/06%3A_Acellular_Pathogens/6.E%3A_Acellular_Pathogens_%28Exercises%29.txt |
The earth is estimated to be 4.6 billion years old, but for the first 2 billion years, the atmosphere lacked oxygen, without which the earth could not support life as we know it. One hypothesis about how life emerged on earth involves the concept of a “primordial soup.” This idea proposes that life began in a body of water when metals and gases from the atmosphere combined with a source of energy, such as lightning or ultraviolet light, to form the carbon compounds that are the chemical building blocks of life. In 1952, Stanley Miller (1930–2007), a graduate student at the University of Chicago, and his professor Harold Urey (1893–1981), set out to confirm this hypothesis in a now-famous experiment. Miller and Urey combined what they believed to be the major components of the earth’s early atmosphere—water (H2O), methane (CH4), hydrogen (H2), and ammonia (NH3)—and sealed them in a sterile flask. Next, they heated the flask to produce water vapor and passed electric sparks through the mixture to mimic lightning in the atmosphere (Figure \(1\)). When they analyzed the contents of the flask a week later, they found amino acids, the structural units of proteins—molecules essential to the function of all organisms.
• 7.1: Organic Molecules
Biochemistry is the discipline that studies the chemistry of life, and its objective is to explain form and function based on chemical principles. Organic chemistry is the discipline devoted to the study of carbon-based chemistry, which is the foundation for the study of biomolecules and the discipline of biochemistry. Both biochemistry and organic chemistry are based on the concepts of general chemistry.
• 7.2: Carbohydrates
The most abundant biomolecules on earth are carbohydrates. From a chemical viewpoint, carbohydrates are primarily a combination of carbon and water, and many of them have the empirical formula (CH₂O)ₙ, where n is the number of repeated units. This view represents these molecules simply as “hydrated” carbon atom chains in which water molecules attach to each carbon atom, leading to the term “carbohydrates.”
• 7.3: Lipids
Although they are composed primarily of carbon and hydrogen, lipid molecules may also contain oxygen, nitrogen, sulfur, and phosphorous. Lipids serve numerous and diverse purposes in the structure and functions of organisms. They can be a source of nutrients, a storage form for carbon, energy-storage molecules, or structural components of membranes and hormones. Lipids comprise a broad class of many chemically distinct compounds, the most common of which are discussed in this section.
• 7.4: Proteins
Amino acids are capable of bonding together in essentially any number, yielding molecules of essentially any size that possess a wide array of physical and chemical properties and perform numerous functions vital to all organisms. The molecules derived from amino acids can function as structural components of cells and subcellular entities, as sources of nutrients, as atom- and energy-storage reservoirs, and as functional species such as hormones, enzymes, receptors, and transport molecules.
• 7.5: Using Biochemistry to Identify Microorganisms
Accurate identification of bacteria is essential in a clinical laboratory for diagnostic and management of epidemics, pandemics, and food poisoning caused by bacterial outbreaks. In this section, we will discuss a few methods that use biochemical characteristics to identify microorganisms.
• 7.E: Microbial Biochemistry (Exercises)
Thumbnail: An enzyme binding site that would normally bind substrate can alternatively bind a competitive inhibitor, preventing substrate access. Dihydrofolate reductase is inhibited by methotrexate which prevents binding of its substrate, folic acid. Binding site in blue, inhibitor in green, and substrate in black (PDB: 4QI9). (CC BY 4.0; Thomas Shafee).
07: Microbial Biochemistry
Learning Objectives
• Identify common elements and structures found in organic molecules
• Explain the concept of isomerism
• Identify examples of functional groups
• Describe the role of functional groups in synthesizing polymers
Clinical Focus: Part 1
Penny is a 16-year-old student who visited her doctor, complaining about an itchy skin rash. She had a history of allergic episodes. The doctor looked at her sun-tanned skin and asked her if she switched to a different sunscreen. She said she had, so the doctor diagnosed an allergic eczema. The symptoms were mild so the doctor told Penny to avoid using the sunscreen that caused the reaction and prescribed an over-the-counter moisturizing cream to keep her skin hydrated and to help with itching.
Exercise $1$
1. What kinds of substances would you expect to find in a moisturizing cream?
2. What physical or chemical properties of these substances would help alleviate itching and inflammation of the skin?
Biochemistry is the discipline that studies the chemistry of life, and its objective is to explain form and function based on chemical principles. Organic chemistry is the discipline devoted to the study of carbon-based chemistry, which is the foundation for the study of biomolecules and the discipline of biochemistry. Both biochemistry and organic chemistry are based on the concepts of general chemistry, some of which are presented in Appendix A.
Elements in Living Cells
The most abundant element in cells is hydrogen (H), followed by carbon (C), oxygen (O), nitrogen (N), phosphorous (P), and sulfur (S). We call these elements macronutrients, and they account for about 99% of the dry weight of cells. Some elements, such as sodium (Na), potassium (K), magnesium (Mg), zinc (Zn), iron (Fe), calcium (Ca), molybdenum (Mo), copper (Cu), cobalt (Co), manganese (Mn), or vanadium (Va), are required by some cells in very small amounts and are called micronutrients or trace elements. All of these elements are essential to the function of many biochemical reactions, and, therefore, are essential to life.
The four most abundant elements in living matter (C, N, O, and H) have low atomic numbers and are thus light elements capable of forming strong bonds with other atoms to produce molecules (Figure $1$). Carbon forms four chemical bonds, whereas nitrogen forms three, oxygen forms two, and hydrogen forms one. When bonded together within molecules, oxygen, sulfur, and nitrogen often have one or more “lone pairs” of electrons that play important roles in determining many of the molecules’ physical and chemical properties (see Appendix A). These traits in combination permit the formation of a vast number of diverse molecular species necessary to form the structures and enable the functions of living organisms.
Living organisms contain inorganic compounds (mainly water and salts; see Appendix A) and organic molecules. Organic molecules contain carbon; inorganic compounds do not. Carbon oxides and carbonates are exceptions; they contain carbon but are considered inorganic because they do not contain hydrogen. The atoms of an organic molecule are typically organized around chains of carbon atoms.
Inorganic compounds make up 1%–1.5% of a living cell’s mass. They are small, simple compounds that play important roles in the cell, although they do not form cell structures. Most of the carbon found in organic molecules originates from inorganic carbon sources such as carbon dioxide captured via carbon fixation by microorganisms.
Exercise $2$
1. Describe the most abundant elements in nature.
2. What are the differences between organic and inorganic molecules?
Organic Molecules and Isomerism
Organic molecules in organisms are generally larger and more complex than inorganic molecules. Their carbon skeletons are held together by covalent bonds. They form the cells of an organism and perform the chemical reactions that facilitate life. All of these molecules, called biomolecules because they are part of living matter, contain carbon, which is the building block of life. Carbon is a very unique element in that it has four valence electrons in its outer orbitals and can form four single covalent bonds with up to four other atoms at the same time (see Appendix A). These atoms are usually oxygen, hydrogen, nitrogen, sulfur, phosphorous, and carbon itself; the simplest organic compound is methane, in which carbon binds only to hydrogen (Figure $2$).
As a result of carbon’s unique combination of size and bonding properties, carbon atoms can bind together in large numbers, thus producing a chain or carbon skeleton. The carbon skeleton of organic molecules can be straight, branched, or ring shaped (cyclic). Organic molecules are built on chains of carbon atoms of varying lengths; most are typically very long, which allows for a huge number and variety of compounds. No other element has the ability to form so many different molecules of so many different sizes and shapes.
Molecules with the same atomic makeup but different structural arrangement of atoms are called isomers. The concept of isomerism is very important in chemistry because the structure of a molecule is always directly related to its function. Slight changes in the structural arrangements of atoms in a molecule may lead to very different properties. Chemists represent molecules by their structural formula, which is a graphic representation of the molecular structure, showing how the atoms are arranged. Compounds that have identical molecular formulas but differ in the bonding sequence of the atoms are called structural isomers. The monosaccharides glucose, galactose, and fructose all have the same molecular formula, C6H12O6, but we can see from Figure $3$ that the atoms are bonded together differently.
Isomers that differ in the spatial arrangements of atoms are called stereoisomers; one unique type is enantiomers. The properties of enantiomers were originally discovered by Louis Pasteur in 1848 while using a microscope to analyze crystallized fermentation products of wine. Enantiomers are molecules that have the characteristic of chirality, in which their structures are nonsuperimposable mirror images of each other. Chirality is an important characteristic in many biologically important molecules, as illustrated by the examples of structural differences in the enantiomeric forms of the monosaccharide glucose or the amino acid alanine (Figure $4$).
Many organisms are only able to use one enantiomeric form of certain types of molecules as nutrients and as building blocks to make structures within a cell. Some enantiomeric forms of amino acids have distinctly different tastes and smells when consumed as food. For example, L-aspartame, commonly called aspartame, tastes sweet, whereas D-aspartame is tasteless. Drug enantiomers can have very different pharmacologic affects. For example, the compound methorphan exists as two enantiomers, one of which acts as an antitussive (dextromethorphan, a cough suppressant), whereas the other acts as an analgesic (levomethorphan, a drug similar in effect to codeine).
Enantiomers are also called optical isomers because they can rotate the plane of polarized light. Some of the crystals Pasteur observed from wine fermentation rotated light clockwise whereas others rotated the light counterclockwise. Today, we denote enantiomers that rotate polarized light clockwise (+) as d forms, and the mirror image of the same molecule that rotates polarized light counterclockwise (−) as the l form. The d and l labels are derived from the Latin words dexter (on the right) and laevus (on the left), respectively. These two different optical isomers often have very different biological properties and activities. Certain species of molds, yeast, and bacteria, such as Rhizopus, Yarrowia, and Lactobacillus spp., respectively, can only metabolize one type of optical isomer; the opposite isomer is not suitable as a source of nutrients. Another important reason to be aware of optical isomers is the therapeutic use of these types of chemicals for drug treatment, because some microorganisms can only be affected by one specific optical isomer.
Exercise $3$
We say that life is carbon based. What makes carbon so suitable to be part of all the macromolecules of living organisms?
Biologically Significant Functional Groups
In addition to containing carbon atoms, biomolecules also contain functional groups—groups of atoms within molecules that are categorized by their specific chemical composition and the chemical reactions they perform, regardless of the molecule in which the group is found. Some of the most common functional groups are listed in Figure $5$. In the formulas, the symbol R stands for “residue” and represents the remainder of the molecule. R might symbolize just a single hydrogen atom or it may represent a group of many atoms. Notice that some functional groups are relatively simple, consisting of just one or two atoms, while some comprise two of these simpler functional groups. For example, a carbonyl group is a functional group composed of a carbon atom double bonded to an oxygen atom: C=O. It is present in several classes of organic compounds as part of larger functional groups such as ketones, aldehydes, carboxylic acids, and amides. In ketones, the carbonyl is present as an internal group, whereas in aldehydes it is a terminal group.
Macromolecules
Carbon chains form the skeletons of most organic molecules. Functional groups combine with the chain to form biomolecules. Because these biomolecules are typically large, we call them macromolecules. Many biologically relevant macromolecules are formed by linking together a great number of identical, or very similar, smaller organic molecules. The smaller molecules act as building blocks and are called monomers, and the macromolecules that result from their linkage are called polymers. Cells and cell structures include four main groups of carbon-containing macromolecules: polysaccharides, proteins, lipids, and nucleic acids. The first three groups of molecules will be studied throughout this chapter. The biochemistry of nucleic acids will be discussed in Biochemistry of the Genome.
Of the many possible ways that monomers may be combined to yield polymers, one common approach encountered in the formation of biological macromolecules is dehydration synthesis. In this chemical reaction, monomer molecules bind end to end in a process that results in the formation of water molecules as a byproduct:
$\text{H—monomer—OH} + \text{H—monomer—OH} ⟶ \text{H—monomer—monomer—OH} + \ce{H2O}$
Figure $6$ shows dehydration synthesis of glucose binding together to form maltose and a water molecule. Table $1$ summarizes macromolecules and some of their functions.
Table $1$: Functions of Macromolecules
Macromolecule Functions
Carbohydrates Energy storage, receptors, food, structural role in plants, fungal cell walls, exoskeletons of insects
Lipids Energy storage, membrane structure, insulation, hormones, pigments
Nucleic acids Storage and transfer of genetic information
Proteins Enzymes, structure, receptors, transport, structural role in the cytoskeleton of a cell and the extracellular matrix
Exercise $4$
What is the byproduct of a dehydration synthesis reaction?
Key Concepts and Summary
• The most abundant elements in cells are hydrogen, carbon, oxygen, nitrogen, phosphorus, and sulfur.
• Life is carbon based. Each carbon atom can bind to another one producing a carbon skeleton that can be straight, branched, or ring shaped.
• The same numbers and types of atoms may bond together in different ways to yield different molecules called isomers. Isomers may differ in the bonding sequence of their atoms (structural isomers) or in the spatial arrangement of atoms whose bonding sequences are the same (stereoisomers), and their physical and chemical properties may vary slightly or drastically.
• Functional groups confer specific chemical properties to molecules bearing them. Common functional groups in biomolecules are hydroxyl, methyl, carbonyl, carboxyl, amino, phosphate, and sulfhydryl.
• Macromolecules are polymers assembled from individual units, the monomers, which bind together like building blocks. Many biologically significant macromolecules are formed by dehydration synthesis, a process in which monomers bind together by combining their functional groups and generating water molecules as byproducts. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/07%3A_Microbial_Biochemistry/7.01%3A_Organic_Molecules.txt |
Learning Objectives
• Give examples of monosaccharides and polysaccharides
• Describe the function of monosaccharides and polysaccharides within a cell
The most abundant biomolecules on earth are carbohydrates. From a chemical viewpoint, carbohydrates are primarily a combination of carbon and water, and many of them have the empirical formula (CH2O)n, where n is the number of repeated units. This view represents these molecules simply as “hydrated” carbon atom chains in which water molecules attach to each carbon atom, leading to the term “carbohydrates.” Although all carbohydrates contain carbon, hydrogen, and oxygen, there are some that also contain nitrogen, phosphorus, and/or sulfur. Carbohydrates have myriad different functions. They are abundant in terrestrial ecosystems, many forms of which we use as food sources. These molecules are also vital parts of macromolecular structures that store and transmit genetic information (i.e., DNA and RNA). They are the basis of biological polymers that impart strength to various structural components of organisms (e.g., cellulose and chitin), and they are the primary source of energy storage in the form of starch and glycogen.
Monosaccharides: The Sweet Ones
In biochemistry, carbohydrates are often called saccharides, from the Greek sakcharon, meaning sugar, although not all the saccharides are sweet. The simplest carbohydrates are called monosaccharides, or simple sugars. They are the building blocks (monomers) for the synthesis of polymers or complex carbohydrates, as will be discussed further in this section. Monosaccharides are classified based on the number of carbons in the molecule. General categories are identified using a prefix that indicates the number of carbons and the suffix –ose, which indicates a saccharide; for example, triose (three carbons), tetrose (four carbons), pentose (five carbons), and hexose (six carbons) (Figure $1$). The hexose D-glucose is the most abundant monosaccharide in nature. Other very common and abundant hexose monosaccharides are galactose, used to make the disaccharide milk sugar lactose, and the fruit sugar fructose.
Monosaccharides of four or more carbon atoms are typically more stable when they adopt cyclic, or ring, structures. These ring structures result from a chemical reaction between functional groups on opposite ends of the sugar’s flexible carbon chain, namely the carbonyl group and a relatively distant hydroxyl group. Glucose, for example, forms a six-membered ring (Figure $2$).
Exercise $1$
Why do monosaccharides form ring structures?
Disaccharides
Two monosaccharide molecules may chemically bond to form a disaccharide. The name given to the covalent bond between the two monosaccharides is a glycosidic bond. Glycosidic bonds form between hydroxyl groups of the two saccharide molecules, an example of the dehydration synthesis described in the previous section of this chapter:
$\text{monosaccharide—OH} + \text{HO—monosaccharide} ⟶ \underbrace{\text{monosaccharide—O—monosaccharide}}_{\text{disaccharide}}$
Common disaccharides are the grain sugar maltose, made of two glucose molecules; the milk sugar lactose, made of a galactose and a glucose molecule; and the table sugar sucrose, made of a glucose and a fructose molecule (Figure $3$).
Polysaccharides
Polysaccharides, also called glycans, are large polymers composed of hundreds of monosaccharide monomers. Unlike mono- and disaccharides, polysaccharides are not sweet and, in general, they are not soluble in water. Like disaccharides, the monomeric units of polysaccharides are linked together by glycosidic bonds.
Polysaccharides are very diverse in their structure. Three of the most biologically important polysaccharides—starch, glycogen, and cellulose—are all composed of repetitive glucose units, although they differ in their structure (Figure $4$). Cellulose consists of a linear chain of glucose molecules and is a common structural component of cell walls in plants and other organisms. Glycogen and starch are branched polymers; glycogen is the primary energy-storage molecule in animals and bacteria, whereas plants primarily store energy in starch. The orientation of the glycosidic linkages in these three polymers is different as well and, as a consequence, linear and branched macromolecules have different properties.
Modified glucose molecules can be fundamental components of other structural polysaccharides. Examples of these types of structural polysaccharides are N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) found in bacterial cell wall peptidoglycan. Polymers of NAG form chitin, which is found in fungal cell walls and in the exoskeleton of insects.
Exercise $2$
What are the most biologically important polysaccharides and why are they important?
Key Concepts and Summary
• Carbohydrates, the most abundant biomolecules on earth, are widely used by organisms for structural and energy-storage purposes.
• Carbohydrates include individual sugar molecules (monosaccharides) as well as two or more molecules chemically linked by glycosidic bonds. Monosaccharides are classified based on the number of carbons in the molecule as trioses (3 C), tetroses (4 C), pentoses (5 C), and hexoses (6 C). They are the building blocks for the synthesis of polymers or complex carbohydrates.
• Disaccharides such as sucrose, lactose, and maltose are molecules composed of two monosaccharides linked together by a glycosidic bond.
• Polysaccharides, or glycans, are polymers composed of hundreds of monosaccharide monomers linked together by glycosidic bonds. The energy-storage polymers starch and glycogen are examples of polysaccharides and are all composed of branched chains of glucose molecules.
• The polysaccharide cellulose is a common structural component of the cell walls of organisms. Other structural polysaccharides, such as N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM), incorporate modified glucose molecules and are used in the construction of peptidoglycan or chitin. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/07%3A_Microbial_Biochemistry/7.02%3A_Carbohydrates.txt |
Learning Objectives
• Describe the chemical composition of lipids
• Describe the unique characteristics and diverse structures of lipids
• Compare and contrast triacylglycerides (triglycerides) and phospholipids
• Describe how phospholipids are used to construct biological membranes
Although they are composed primarily of carbon and hydrogen, lipid molecules may also contain oxygen, nitrogen, sulfur, and phosphorous. Lipids serve numerous and diverse purposes in the structure and functions of organisms. They can be a source of nutrients, a storage form for carbon, energy-storage molecules, or structural components of membranes and hormones. Lipids comprise a broad class of many chemically distinct compounds, the most common of which are discussed in this section.
Fatty Acids and Triacylglycerides
The fatty acids are lipids that contain long-chain hydrocarbons terminated with a carboxylic acid functional group. Because of the long hydrocarbon chain, fatty acids are hydrophobic (“water fearing”) or nonpolar. Fatty acids with hydrocarbon chains that contain only single bonds are called saturated fatty acids because they have the greatest number of hydrogen atoms possible and are, therefore, “saturated” with hydrogen. Fatty acids with hydrocarbon chains containing at least one double bond are called unsaturated fatty acids because they have fewer hydrogen atoms. Saturated fatty acids have a straight, flexible carbon backbone, whereas unsaturated fatty acids have “kinks” in their carbon skeleton because each double bond causes a rigid bend of the carbon skeleton. These differences in saturated versus unsaturated fatty acid structure result in different properties for the corresponding lipids in which the fatty acids are incorporated. For example, lipids containing saturated fatty acids are solids at room temperature, whereas lipids containing unsaturated fatty acids are liquids.
A triacylglycerol, or triglyceride, is formed when three fatty acids are chemically linked to a glycerol molecule (Figure \(1\)). Triglycerides are the primary components of adipose tissue (body fat), and are major constituents of sebum (skin oils). They play an important metabolic role, serving as efficient energy-storage molecules that can provide more than double the caloric content of both carbohydrates and proteins.
Exercise \(1\)
Explain why fatty acids with hydrocarbon chains that contain only single bonds are called saturated fatty acids.
Phospholipids and Biological Membranes
Triglycerides are classified as simple lipids because they are formed from just two types of compounds: glycerol and fatty acids. In contrast, complex lipids contain at least one additional component, for example, a phosphate group (phospholipids) or a carbohydrate moiety (glycolipids). Figure \(2\) depicts a typical phospholipid composed of two fatty acids linked to glycerol (a diglyceride). The two fatty acid carbon chains may be both saturated, both unsaturated, or one of each. Instead of another fatty acid molecule (as for triglycerides), the third binding position on the glycerol molecule is occupied by a modified phosphate group.
The molecular structure of lipids results in unique behavior in aqueous environments. Figure \(1\) depicts the structure of a triglyceride. Because all three substituents on the glycerol backbone are long hydrocarbon chains, these compounds are nonpolar and not significantly attracted to polar water molecules—they are hydrophobic. Conversely, phospholipids such as the one shown in Figure \(2\) have a negatively charged phosphate group. Because the phosphate is charged, it is capable of strong attraction to water molecules and thus is hydrophilic, or “water loving.” The hydrophilic portion of the phospholipid is often referred to as a polar “head,” and the long hydrocarbon chains as nonpolar “tails.” A molecule presenting a hydrophobic portion and a hydrophilic moiety is said to be amphipathic. Notice the “R” designation within the hydrophilic head depicted in Figure \(2\), indicating that a polar head group can be more complex than a simple phosphate moiety. Glycolipids are examples in which carbohydrates are bonded to the lipids’ head groups.
The amphipathic nature of phospholipids enables them to form uniquely functional structures in aqueous environments. As mentioned, the polar heads of these molecules are strongly attracted to water molecules, and the nonpolar tails are not. Because of their considerable lengths, these tails are, in fact, strongly attracted to one another. As a result, energetically stable, large-scale assemblies of phospholipid molecules are formed in which the hydrophobic tails congregate within enclosed regions, shielded from contact with water by the polar heads (Figure \(3\)). The simplest of these structures are micelles, spherical assemblies containing a hydrophobic interior of phospholipid tails and an outer surface of polar head groups. Larger and more complex structures are created from lipid-bilayer sheets, or unit membranes, which are large, two-dimensional assemblies of phospholipids congregated tail to tail. The cell membranes of nearly all organisms are made from lipid-bilayer sheets, as are the membranes of many intracellular components. These sheets may also form lipid-bilayer spheres that are the structural basis of vesicles and liposomes, subcellular components that play a role in numerous physiological functions.
Exercise \(2\)
How is the amphipathic nature of phospholipids significant?
Isoprenoids and Sterols
The isoprenoids are branched lipids, also referred to as terpenoids, that are formed by chemical modifications of the isoprene molecule (Figure \(4\)). These lipids play a wide variety of physiological roles in plants and animals, with many technological uses as pharmaceuticals (capsaicin), pigments (e.g., orange beta carotene, xanthophylls), and fragrances (e.g., menthol, camphor, limonene [lemon fragrance], and pinene [pine fragrance]). Long-chain isoprenoids are also found in hydrophobic oils and waxes. Waxes are typically water resistant and hard at room temperature, but they soften when heated and liquefy if warmed adequately. In humans, the main wax production occurs within the sebaceous glands of hair follicles in the skin, resulting in a secreted material called sebum, which consists mainly of triacylglycerol, wax esters, and the hydrocarbon squalene. There are many bacteria in the microbiota on the skin that feed on these lipids. One of the most prominent bacteria that feed on lipids is Propionibacterium acnes, which uses the skin’s lipids to generate short-chain fatty acids and is involved in the production of acne.
Another type of lipids are steroids, complex, ringed structures that are found in cell membranes; some function as hormones. The most common types of steroids are sterols, which are steroids containing an OH group. These are mainly hydrophobic molecules, but also have hydrophilic hydroxyl groups. The most common sterol found in animal tissues is cholesterol. Its structure consists of four rings with a double bond in one of the rings, and a hydroxyl group at the sterol-defining position. The function of cholesterol is to strengthen cell membranes in eukaryotes and in bacteria without cell walls, such as Mycoplasma. Prokaryotes generally do not produce cholesterol, although bacteria produce similar compounds called hopanoids, which are also multiringed structures that strengthen bacterial membranes (Figure \(5\)). Fungi and some protozoa produce a similar compound called ergosterol, which strengthens the cell membranes of these organisms.
Link to Learning: Liposomes
This video provides additional information about phospholipids and liposomes.
Exercise \(3\)
How are isoprenoids used in technology?
Clinical Focus: Part 2
The moisturizing cream prescribed by Penny’s doctor was a topical corticosteroid cream containing hydrocortisone. Hydrocortisone is a synthetic form of cortisol, a corticosteroid hormone produced in the adrenal glands, from cholesterol. When applied directly to the skin, it can reduce inflammation and temporarily relieve minor skin irritations, itching, and rashes by reducing the secretion of histamine, a compound produced by cells of the immune system in response to the presence of pathogens or other foreign substances. Because histamine triggers the body’s inflammatory response, the ability of hydrocortisone to reduce the local production of histamine in the skin effectively suppresses the immune system and helps limit inflammation and accompanying symptoms such as pruritus (itching) and rashes.
Exercise \(4\)
Does the corticosteroid cream treat the cause of Penny’s rash, or just the symptoms?
Key Concepts and Summary
• Lipids are composed mainly of carbon and hydrogen, but they can also contain oxygen, nitrogen, sulfur, and phosphorous. They provide nutrients for organisms, store carbon and energy, play structural roles in membranes, and function as hormones, pharmaceuticals, fragrances, and pigments.
• Fatty acids are long-chain hydrocarbons with a carboxylic acid functional group. Their relatively long nonpolar hydrocarbon chains make them hydrophobic. Fatty acids with no double bonds are saturated; those with double bonds are unsaturated.
• Fatty acids chemically bond to glycerol to form structurally essential lipids such as triglycerides and phospholipids. Triglycerides comprise three fatty acids bonded to glycerol, yielding a hydrophobic molecule. Phospholipids contain both hydrophobic hydrocarbon chains and polar head groups, making them amphipathic and capable of forming uniquely functional large scale structures.
• Biological membranes are large-scale structures based on phospholipid bilayers that provide hydrophilic exterior and interior surfaces suitable for aqueous environments, separated by an intervening hydrophobic layer. These bilayers are the structural basis for cell membranes in most organisms, as well as subcellular components such as vesicles.
• Isoprenoids are lipids derived from isoprene molecules that have many physiological roles and a variety of commercial applications.
• A wax is a long-chain isoprenoid that is typically water resistant; an example of a wax-containing substance is sebum, produced by sebaceous glands in the skin. Steroids are lipids with complex, ringed structures that function as structural components of cell membranes and as hormones. Sterols are a subclass of steroids containing a hydroxyl group at a specific location on one of the molecule’s rings; one example is cholesterol.
• Bacteria produce hopanoids, structurally similar to cholesterol, to strengthen bacterial membranes. Fungi and protozoa produce a strengthening agent called ergosterol. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/07%3A_Microbial_Biochemistry/7.03%3A_Lipids.txt |
Learning Objectives
• Describe the fundamental structure of an amino acid
• Describe the chemical structures of proteins
• Summarize the unique characteristics of proteins
At the beginning of this chapter, a famous experiment was described in which scientists synthesized amino acids under conditions simulating those present on earth long before the evolution of life as we know it. These compounds are capable of bonding together in essentially any number, yielding molecules of essentially any size that possess a wide array of physical and chemical properties and perform numerous functions vital to all organisms. The molecules derived from amino acids can function as structural components of cells and subcellular entities, as sources of nutrients, as atom- and energy-storage reservoirs, and as functional species such as hormones, enzymes, receptors, and transport molecules.
Amino Acids and Peptide Bonds
An amino acid is an organic molecule in which a hydrogen atom, a carboxyl group (–COOH), and an amino group (–NH2) are all bonded to the same carbon atom, the so-called α carbon. The fourth group bonded to the α carbon varies among the different amino acids and is called a residue or a side chain, represented in structural formulas by the letter R. A residue is a monomer that results when two or more amino acids combine and remove water molecules. The primary structure of a protein, a peptide chain, is made of amino acid residues. The unique characteristics of the functional groups and R groups allow these components of the amino acids to form hydrogen, ionic, and disulfide bonds, along with polar/nonpolar interactions needed to form secondary, tertiary, and quaternary protein structures. These groups are composed primarily of carbon, hydrogen, oxygen, nitrogen, and sulfur, in the form of hydrocarbons, acids, amides, alcohols, and amines. A few examples illustrating these possibilities are provided in Figure \(1\).
Amino acids may chemically bond together by reaction of the carboxylic acid group of one molecule with the amine group of another. This reaction forms a peptide bond and a water molecule and is another example of dehydration synthesis (Figure \(2\)). Molecules formed by chemically linking relatively modest numbers of amino acids (approximately 50 or fewer) are called peptides, and prefixes are often used to specify these numbers: dipeptides (two amino acids), tripeptides (three amino acids), and so forth. More generally, the approximate number of amino acids is designated: oligopeptides are formed by joining up to approximately 20 amino acids, whereas polypeptides are synthesized from up to approximately 50 amino acids. When the number of amino acids linked together becomes very large, or when multiple polypeptides are used as building subunits, the macromolecules that result are called proteins. The continuously variable length (the number of monomers) of these biopolymers, along with the variety of possible R groups on each amino acid, allows for a nearly unlimited diversity in the types of proteins that may be formed.
Exercise \(1\)
How many amino acids are in polypeptides?
Protein Structure
The size (length) and specific amino acid sequence of a protein are major determinants of its shape, and the shape of a protein is critical to its function. For example, in the process of biological nitrogen fixation (see Biogeochemical Cycles), soil microorganisms collectively known as rhizobia symbiotically interact with roots of legume plants such as soybeans, peanuts, or beans to form a novel structure called a nodule on the plant roots. The plant then produces a carrier protein called leghemoglobin, a protein that carries nitrogen or oxygen. Leghemoglobin binds with a very high affinity to its substrate oxygen at a specific region of the protein where the shape and amino acid sequence are appropriate (the active site). If the shape or chemical environment of the active site is altered, even slightly, the substrate may not be able to bind as strongly, or it may not bind at all. Thus, for the protein to be fully active, it must have the appropriate shape for its function.
Protein structure is categorized in terms of four levels: primary, secondary, tertiary, and quaternary. The primary structure is simply the sequence of amino acids that make up the polypeptide chain. Figure \(3\) depicts the primary structure of a protein.
The chain of amino acids that defines a protein’s primary structure is not rigid, but instead is flexible because of the nature of the bonds that hold the amino acids together. When the chain is sufficiently long, hydrogen bonding may occur between amine and carbonyl functional groups within the peptide backbone (excluding the R side group), resulting in localized folding of the polypeptide chain into helices and sheets. These shapes constitute a protein’s secondary structure. The most common secondary structures are the α-helix and β-pleated sheet. In the α-helix structure, the helix is held by hydrogen bonds between the oxygen atom in a carbonyl group of one amino acid and the hydrogen atom of the amino group that is just four amino acid units farther along the chain. In the β-pleated sheet, the pleats are formed by similar hydrogen bonds between continuous sequences of carbonyl and amino groups that are further separated on the backbone of the polypeptide chain (Figure \(4\)).
The next level of protein organization is the tertiary structure, which is the large-scale three-dimensional shape of a single polypeptide chain. Tertiary structure is determined by interactions between amino acid residues that are far apart in the chain. A variety of interactions give rise to protein tertiary structure, such as disulfide bridges, which are bonds between the sulfhydryl (–SH) functional groups on amino acid side groups; hydrogen bonds; ionic bonds; and hydrophobic interactions between nonpolar side chains. All these interactions, weak and strong, combine to determine the final three-dimensional shape of the protein and its function (Figure \(5\)).
The process by which a polypeptide chain assumes a large-scale, three-dimensional shape is called protein folding. Folded proteins that are fully functional in their normal biological role are said to possess a native structure. When a protein loses its three-dimensional shape, it may no longer be functional. These unfolded proteins are denatured. Denaturation implies the loss of the secondary structure and tertiary structure (and, if present, the quaternary structure) without the loss of the primary structure.
Some proteins are assemblies of several separate polypeptides, also known as protein subunits. These proteins function adequately only when all subunits are present and appropriately configured. The interactions that hold these subunits together constitute the quaternary structure of the protein. The overall quaternary structure is stabilized by relatively weak interactions. Hemoglobin, for example, has a quaternary structure of four globular protein subunits: two α and two β polypeptides, each one containing an iron-based heme (Figure \(6\)).
Another important class of proteins is the conjugated proteins that have a nonprotein portion. If the conjugated protein has a carbohydrate attached, it is called a glycoprotein. If it has a lipid attached, it is called a lipoprotein. These proteins are important components of membranes. Figure \(7\) summarizes the four levels of protein structure.
Exercise \(2\)
What can happen if a protein’s primary, secondary, tertiary, or quaternary structure is changed?
Primary Structure, Dysfunctional Proteins, and Cystic Fibrosis
Proteins associated with biological membranes are classified as extrinsic or intrinsic. Extrinsic proteins, also called peripheral proteins, are loosely associated with one side of the membrane. Intrinsic proteins, or integral proteins, are embedded in the membrane and often function as part of transport systems as transmembrane proteins. Cystic fibrosis (CF) is a human genetic disorder caused by a change in the transmembrane protein. It affects mostly the lungs but may also affect the pancreas, liver, kidneys, and intestine. CF is caused by a loss of the amino acid phenylalanine in a cystic fibrosis transmembrane protein (CFTR). The loss of one amino acid changes the primary structure of a protein that normally helps transport salt and water in and out of cells (Figure \(8\)).
The change in the primary structure prevents the protein from functioning properly, which causes the body to produce unusually thick mucus that clogs the lungs and leads to the accumulation of sticky mucus. The mucus obstructs the pancreas and stops natural enzymes from helping the body break down food and absorb vital nutrients.
In the lungs of individuals with cystic fibrosis, the altered mucus provides an environment where bacteria can thrive. This colonization leads to the formation of biofilms in the small airways of the lungs. The most common pathogens found in the lungs of patients with cystic fibrosis are Pseudomonas aeruginosa (Figure \(9\)) and Burkholderia cepacia. Pseudomonas differentiates within the biofilm in the lung and forms large colonies, called “mucoid” Pseudomonas. The colonies have a unique pigmentation that shows up in laboratory tests (Figure \(9\)) and provides physicians with the first clue that the patient has CF (such colonies are rare in healthy individuals).
Link to Learning
For more information about cystic fibrosis, visit the Cystic Fibrosis Foundation website.
Key Concepts and Summary
• Amino acids are small molecules essential to all life. Each has an α carbon to which a hydrogen atom, carboxyl group, and amine group are bonded. The fourth bonded group, represented by R, varies in chemical composition, size, polarity, and charge among different amino acids, providing variation in properties.
• Peptides are polymers formed by the linkage of amino acids via dehydration synthesis. The bonds between the linked amino acids are called peptide bonds. The number of amino acids linked together may vary from a few to many.
• Proteins are polymers formed by the linkage of a very large number of amino acids. They perform many important functions in a cell, serving as nutrients and enzymes; storage molecules for carbon, nitrogen, and energy; and structural components.
• The structure of a protein is a critical determinant of its function and is described by a graduated classification: primary, secondary, tertiary, and quaternary. The native structure of a protein may be disrupted by denaturation, resulting in loss of its higher-order structure and its biological function.
• Some proteins are formed by several separate protein subunits, the interaction of these subunits composing the quaternary structure of the protein complex.
• Conjugated proteins have a nonpolypeptide portion that can be a carbohydrate (forming a glycoprotein) or a lipid fraction (forming a lipoprotein). These proteins are important components of membranes. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/07%3A_Microbial_Biochemistry/7.04%3A_Proteins.txt |
Learning Objectives
• Describe examples of biosynthesis products within a cell that can be detected to identify bacteria
Accurate identification of bacterial isolates is essential in a clinical microbiology laboratory because the results often inform decisions about treatment that directly affect patient outcomes. For example, cases of food poisoning require accurate identification of the causative agent so that physicians can prescribe appropriate treatment. Likewise, it is important to accurately identify the causative pathogen during an outbreak of disease so that appropriate strategies can be employed to contain the epidemic.
There are many ways to detect, characterize, and identify microorganisms. Some methods rely on phenotypic biochemical characteristics, while others use genotypic identification. The biochemical characteristics of a bacterium provide many traits that are useful for classification and identification. Analyzing the nutritional and metabolic capabilities of the bacterial isolate is a common approach for determining the genus and the species of the bacterium. Some of the most important metabolic pathways that bacteria use to survive will be discussed in Microbial Metabolism. In this section, we will discuss a few methods that use biochemical characteristics to identify microorganisms.
Some microorganisms store certain compounds as granules within their cytoplasm, and the contents of these granules can be used for identification purposes. For example, poly-β-hydroxybutyrate (PHB) is a carbon- and energy-storage compound found in some nonfluorescent bacteria of the genus Pseudomonas. Different species within this genus can be classified by the presence or the absence of PHB and fluorescent pigments. The human pathogen P. aeruginosa and the plant pathogen P. syringae are two examples of fluorescent Pseudomonas species that do not accumulate PHB granules.
Other systems rely on biochemical characteristics to identify microorganisms by their biochemical reactions, such as carbon utilization and other metabolic tests. In small laboratory settings or in teaching laboratories, those assays are carried out using a limited number of test tubes. However, more modern systems, such as the one developed by Biolog, Inc., are based on panels of biochemical reactions performed simultaneously and analyzed by software. Biolog’s system identifies cells based on their ability to metabolize certain biochemicals and on their physiological properties, including pH and chemical sensitivity. It uses all major classes of biochemicals in its analysis. Identifications can be performed manually or with the semi- or fully automated instruments.
Another automated system identifies microorganisms by determining the specimen’s mass spectrum and then comparing it to a database that contains known mass spectra for thousands of microorganisms. This method is based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and uses disposable MALDI plates on which the microorganism is mixed with a specialized matrix reagent (Figure \(1\)). The sample/reagent mixture is irradiated with a high-intensity pulsed ultraviolet laser, resulting in the ejection of gaseous ions generated from the various chemical constituents of the microorganism. These gaseous ions are collected and accelerated through the mass spectrometer, with ions traveling at a velocity determined by their mass-to-charge ratio (m/z), thus, reaching the detector at different times. A plot of detector signal versus m/z yields a mass spectrum for the organism that is uniquely related to its biochemical composition. Comparison of the mass spectrum to a library of reference spectra obtained from identical analyses of known microorganisms permits identification of the unknown microbe.
Microbes can also be identified by measuring their unique lipid profiles. As we have learned, fatty acids of lipids can vary in chain length, presence or absence of double bonds, and number of double bonds, hydroxyl groups, branches, and rings. To identify a microbe by its lipid composition, the fatty acids present in their membranes are analyzed. A common biochemical analysis used for this purpose is a technique used in clinical, public health, and food laboratories. It relies on detecting unique differences in fatty acids and is called fatty acid methyl ester (FAME) analysis. In a FAME analysis, fatty acids are extracted from the membranes of microorganisms, chemically altered to form volatile methyl esters, and analyzed by gas chromatography (GC). The resulting GC chromatogram is compared with reference chromatograms in a database containing data for thousands of bacterial isolates to identify the unknown microorganism (Figure \(2\)).
A related method for microorganism identification is called phospholipid-derived fatty acids (PLFA) analysis. Membranes are mostly composed of phospholipids, which can be saponified (hydrolyzed with alkali) to release the fatty acids. The resulting fatty acid mixture is then subjected to FAME analysis, and the measured lipid profiles can be compared with those of known microorganisms to identify the unknown microorganism.
Bacterial identification can also be based on the proteins produced under specific growth conditions within the human body. These types of identification procedures are called proteomic analysis. To perform proteomic analysis, proteins from the pathogen are first separated by high-pressure liquid chromatography (HPLC), and the collected fractions are then digested to yield smaller peptide fragments. These peptides are identified by mass spectrometry and compared with those of known microorganisms to identify the unknown microorganism in the original specimen.
Microorganisms can also be identified by the carbohydrates attached to proteins (glycoproteins) in the plasma membrane or cell wall. Antibodies and other carbohydrate-binding proteins can attach to specific carbohydrates on cell surfaces, causing the cells to clump together. Serological tests (e.g., the Lancefield groups tests, which are used for identification of Streptococcus species) are performed to detect the unique carbohydrates located on the surface of the cell.
Clinical Focus: Resolution
Penny stopped using her new sunscreen and applied the corticosteroid cream to her rash as directed. However, after several days, her rash had not improved and actually seemed to be getting worse. She made a follow-up appointment with her doctor, who observed a bumpy red rash and pus-filled blisters around hair follicles (Figure \(3\)). The rash was especially concentrated in areas that would have been covered by a swimsuit. After some questioning, Penny told the physician that she had recently attended a pool party and spent some time in a hot tub. In light of this new information, the doctor suspected a case of hot tub rash, an infection frequently caused by the bacterium Pseudomonas aeruginosa, an opportunistic pathogen that can thrive in hot tubs and swimming pools, especially when the water is not sufficiently chlorinated. P. aeruginosa is the same bacterium that is associated with infections in the lungs of patients with cystic fibrosis.
The doctor collected a specimen from Penny’s rash to be sent to the clinical microbiology lab. Confirmatory tests were carried out to distinguish P. aeruginosa from enteric pathogens that can also be present in pool and hot-tub water. The test included the production of the blue-green pigment pyocyanin on cetrimide agar and growth at 42 °C. Cetrimide is a selective agent that inhibits the growth of other species of microbial flora and also enhances the production of P. aeruginosa pigments pyocyanin and fluorescein, which are a characteristic blue-green and yellow-green, respectively.
Tests confirmed the presence of P. aeruginosa in Penny’s skin sample, but the doctor decided not to prescribe an antibiotic. Even though P. aeruginosa is a bacterium, Pseudomonas species are generally resistant to many antibiotics. Luckily, skin infections like Penny’s are usually self-limiting; the rash typically lasts about 2 weeks and resolves on its own, with or without medical treatment. The doctor advised Penny to wait it out and keep using the corticosteroid cream. The cream will not kill the P. aeruginosa on Penny’s skin, but it should calm her rash and minimize the itching by suppressing her body’s inflammatory response to the bacteria.
Key Concepts and Summary
• Accurate identification of bacteria is essential in a clinical laboratory for diagnostic and management of epidemics, pandemics, and food poisoning caused by bacterial outbreaks.
• The phenotypic identification of microorganisms involves using observable traits, including profiles of structural components such as lipids, biosynthetic products such as sugars or amino acids, or storage compounds such as poly-β-hydroxybutyrate.
• An unknown microbe may be identified from the unique mass spectrum produced when it is analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF).
• Microbes can be identified by determining their lipid compositions, using fatty acid methyl esters (FAME) or phospholipid-derived fatty acids (PLFA) analysis.
• Proteomic analysis, the study of all accumulated proteins of an organism, can also be used for bacterial identification.
• Glycoproteins in the plasma membrane or cell wall structures can bind to lectins or antibodies and can be used for identification. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/07%3A_Microbial_Biochemistry/7.05%3A_Using_Biochemistry_to_Identify_Microorganisms.txt |
7.1: Organic Molecules
Biochemistry is the discipline that studies the chemistry of life, and its objective is to explain form and function based on chemical principles. Organic chemistry is the discipline devoted to the study of carbon-based chemistry, which is the foundation for the study of biomolecules and the discipline of biochemistry. Both biochemistry and organic chemistry are based on the concepts of general chemistry.
Multiple Choice
Which of these elements is not a micronutrient?
1. C
2. Ca
3. Co
4. Cu
Answer
A
Which of the following is the name for molecules whose structures are nonsuperimposable mirror images?
1. structural isomers
2. monomers
3. polymers
4. enantiomers
Answer
D
True/False
Aldehydes, amides, carboxylic acids, esters, and ketones all contain carbonyl groups.
Answer
True
Two molecules containing the same types and numbers of atoms but different bonding sequences are called enantiomers.
Answer
False
Short Answer
Why are carbon, nitrogen, oxygen, and hydrogen the most abundant elements in living matter and, therefore, considered macronutrients?
Identify the functional group in each of the depicted structural formulas.
Critical Thinking
The structural formula shown corresponds to penicillin G, a narrow-spectrum antibiotic that is given intravenously or intramuscularly as a treatment for several bacterial diseases. The antibiotic is produced by fungi of the genus Penicillium. (a) Identify three major functional groups in this molecule that each comprise two simpler functional groups. (b) Name the two simpler functional groups composing each of the major functional groups identified in (a).
7.2: Carbohydrates
The most abundant biomolecules on earth are carbohydrates. From a chemical viewpoint, carbohydrates are primarily a combination of carbon and water, and many of them have the empirical formula (CH2O)n, where n is the number of repeated units. This view represents these molecules simply as “hydrated” carbon atom chains in which water molecules attach to each carbon atom, leading to the term “carbohydrates.”
Multiple Choice
By definition, carbohydrates contain which elements?
1. carbon and hydrogen
2. carbon, hydrogen, and nitrogen
3. carbon, hydrogen, and oxygen
4. carbon and oxygen
Answer
C
Monosaccharides may link together to form polysaccharides by forming which type of bond?
1. hydrogen
2. peptide
3. ionic
4. glycosidic
Answer
D
Matching
Match each polysaccharide with its description.
___chitin A. energy storage polymer in plants
___glycogen B. structural polymer found in plants
___starch C. structural polymer found in cell walls of fungi and exoskeletons of some animals
___cellulose D. energy storage polymer found in animal cells and bacteria
Answer
C, D, A, B
Short Answer
What are monosaccharides, disaccharides, and polysaccharides?
Critical Thinking
The figure depicts the structural formulas of glucose, galactose, and fructose. (a) Circle the functional groups that classify the sugars either an aldose or a ketose, and identify each sugar as one or the other. (b) The chemical formula of these compounds is the same, although the structural formula is different. What are such compounds called?
Structural diagrams for the linear and cyclic forms of a monosaccharide are shown. (a) What is the molecular formula for this monosaccharide? (Count the C, H and O atoms in each to confirm that these two molecules have the same formula, and report this formula.) (b) Identify which hydroxyl group in the linear structure undergoes the ring-forming reaction with the carbonyl group.
The term “dextrose” is commonly used in medical settings when referring to the biologically relevant isomer of the monosaccharide glucose. Explain the logic of this alternative name.
7.3: Lipids
Although they are composed primarily of carbon and hydrogen, lipid molecules may also contain oxygen, nitrogen, sulfur, and phosphorous. Lipids serve numerous and diverse purposes in the structure and functions of organisms. They can be a source of nutrients, a storage form for carbon, energy-storage molecules, or structural components of membranes and hormones. Lipids comprise a broad class of many chemically distinct compounds, the most common of which are discussed in this section.
Multiple Choice
Which of the following describes lipids?
1. a source of nutrients for organisms
2. energy-storage molecules
3. molecules having structural role in membranes
4. molecules that are part of hormones and pigments
5. all of the above
Answer
E
Molecules bearing both polar and nonpolar groups are said to be which of the following?
1. hydrophilic
2. amphipathic
3. hydrophobic
4. polyfunctional
Answer
B
True/False
Lipids are a naturally occurring group of substances that are not soluble in water but are freely soluble in organic solvents.
Answer
False
Fatty acids having no double bonds are called “unsaturated.”
Answer
False
A triglyceride is formed by joining three glycerol molecules to a fatty acid backbone in a dehydration reaction.
Answer
False
Fill in the Blank
Waxes contain esters formed from long-chain __________ and saturated __________, and they may also contain substituted hydrocarbons.
Answer
alcohols; fatty acids
Cholesterol is the most common member of the __________ group, found in animal tissues; it has a tetracyclic carbon ring system with a __________ bond in one of the rings and one free __________group.
Answer
steroid; double; hydroxyl
Critical Thinking
Microorganisms can thrive under many different conditions, including high-temperature environments such as hot springs. To function properly, cell membranes have to be in a fluid state. How do you expect the fatty acid content (saturated versus unsaturated) of bacteria living in high-temperature environments might compare with that of bacteria living in more moderate temperatures?
Short Answer
Describe the structure of a typical phospholipid. Are these molecules polar or nonpolar?
7.4: Proteins
Amino acids are capable of bonding together in essentially any number, yielding molecules of essentially any size that possess a wide array of physical and chemical properties and perform numerous functions vital to all organisms. The molecules derived from amino acids can function as structural components of cells and subcellular entities, as sources of nutrients, as atom- and energy-storage reservoirs, and as functional species such as hormones, enzymes, receptors, and transport molecules.
Multiple Choice
Which of the following groups varies among different amino acids?
1. hydrogen atom
2. carboxyl group
3. R group
4. amino group
Answer
C
The amino acids present in proteins differ in which of the following?
1. size
2. shape
3. side groups
4. all of the above
Answer
D
Which of the following bonds are not involved in tertiary structure?
1. peptide bonds
2. ionic bonds
3. hydrophobic interactions
4. hydrogen bonds
Answer
A
Fill in the Blank
The sequence of amino acids in a protein is called its __________.
Answer
Primary structure
Denaturation implies the loss of the __________ and __________ structures without the loss of the __________ structure.
Answer
secondary, tertiary, primary
True/False
A change in one amino acid in a protein sequence always results in a loss of function.
Answer
False
Critical Thinking
Heating a protein sufficiently may cause it to denature. Considering the definition of denaturation, what does this statement say about the strengths of peptide bonds in comparison to hydrogen bonds?
The image shown represents a tetrapeptide. (a) How many peptide bonds are in this molecule? (b) Identify the side groups of the four amino acids composing this peptide.
7.5: Using Biochemistry to Identify Microorganisms
Accurate identification of bacteria is essential in a clinical laboratory for diagnostic and management of epidemics, pandemics, and food poisoning caused by bacterial outbreaks. In this section, we discuss a few methods that use biochemical characteristics to identify microorganisms.
Multiple Choice
Which of the following characteristics/compounds is not considered to be a phenotypic biochemical characteristic used of microbial identification?
1. poly-β-hydroxybutyrate
2. small-subunit (16S) rRNA gene
3. carbon utilization
4. lipid composition
Answer
B
Proteomic analysis is a methodology that deals with which of the following?
1. the analysis of proteins functioning as enzymes within the cell
2. analysis of transport proteins in the cell
3. the analysis of integral proteins of the cell membrane
4. the study of all accumulated proteins of an organism
Answer
D
Which method involves the generation of gas phase ions from intact microorganisms?
1. FAME
2. PLFA
3. MALDI-TOF
4. Lancefield group testing
Answer
C
Which method involves the analysis of membrane-bound carbohydrates?
1. FAME
2. PLFA
3. MALDI-TOF
4. Lancefield group testing
Answer
D
Which method involves conversion of a microbe’s lipids to volatile compounds for analysis by gas chromatography?
1. FAME
2. proteomic analysis
3. MALDI-TOF
4. Lancefield group testing
Answer
A
Fill in the Blank
A FAME analysis involves the conversion of _______ to more volatile _____ for analysis using ____________.
Answer
fatty acids, methyl esters, gas chromatography
True/False
MALDI-TOF relies on obtaining a unique mass spectrum for the bacteria tested and then checking the acquired mass spectrum against the spectrum databases registered in the analysis software to identify the microorganism.
Answer
True
Lancefield group tests can identify microbes using antibodies that specifically bind cell-surface proteins.
Answer
False
Short Answer
Compare MALDI-TOF, FAME, and PLFA, and explain how each technique would be used to identify pathogens. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/07%3A_Microbial_Biochemistry/7.E%3A_Microbial_Biochemistry_%28Exercises%29.txt |
Throughout earth’s history, microbial metabolism has been a driving force behind the development and maintenance of the planet’s biosphere. Eukaryotic organisms such as plants and animals typically depend on organic molecules for energy, growth, and reproduction. Prokaryotes, on the other hand, can metabolize a wide range of organic as well as inorganic matter, from complex organic molecules like cellulose to inorganic molecules and ions such as atmospheric nitrogen (N2), molecular hydrogen (H2), sulfide (S2−), manganese (II) ions (Mn2+), ferrous iron (Fe2+), and ferric iron (Fe3+), to name a few. By metabolizing such substances, microbes chemically convert them to other forms. In some cases, microbial metabolism produces chemicals that can be harmful to other organisms; in others, it produces substances that are essential to the metabolism and survival of other life forms (Figure \(1\)).
• 8.1: Energy, Matter, and Enzymes
Cellular processes such as the building or breaking down of complex molecules occur through series of stepwise, interconnected chemical reactions called metabolic pathways. The term anabolism refers to those endergonic metabolic pathways involved in biosynthesis, converting simple molecular building blocks into more complex molecules, and fueled by the use of cellular energy.
• 8.2: Catabolism of Carbohydrates
Glycolysis is the first step in the breakdown of glucose, resulting in the formation of ATP, which is produced by substrate-level phosphorylation; NADH; and two pyruvate molecules. Glycolysis does not use oxygen and is not oxygen dependent. After glycolysis, a three-carbon pyruvate is decarboxylated to form a two-carbon acetyl group, coupled with the formation of NADH. The acetyl group is attached to a large carrier compound called coenzyme A.
• 8.3: Cellular Respiration
Cellular respiration begins when electrons are transferred from NADH and FADH₂—through a series of chemical reactions to a final inorganic electron acceptor (either oxygen in aerobic respiration or non-oxygen inorganic molecules in anaerobic respiration). These electron transfers take place on the inner part of the cell membrane of prokaryotic cells or in specialized protein complexes in the inner membrane of the mitochondria of eukaryotic cells.
• 8.4: Fermentation
Fermentation uses an organic molecule as a final electron acceptor to regenerate NAD⁺ from NADH so that glycolysis can continue. Fermentation does not involve an electron transport system, and no ATP is made by the fermentation process directly. Fermenters make very little ATP—only two ATP molecules per glucose molecule during glycolysis. Microbial fermentation processes have been used for the production of foods and pharmaceuticals, and for the identification of microbes.
• 8.5: Catabolism of Lipids and Proteins
Collectively, microbes have the ability to degrade a wide variety of carbon sources besides carbohydrates, including lipids and proteins. The catabolic pathways for all of these molecules eventually connect into glycolysis and the Krebs cycle. Several types of lipids can be microbially degraded. Triglycerides are degraded by extracellular lipases, releasing fatty acids from the glycerol backbone. Phospholipids are degraded by phospholipases, releasing fatty acids and phosphorylated head groups.
• 8.6: Photosynthesis and the Importance of Light
Heterotrophic organisms ranging from E. coli to humans rely on the chemical energy found mainly in carbohydrate molecules. Many of these carbohydrates are produced by photosynthesis, the biochemical process by which phototrophic organisms convert solar energy (sunlight) into chemical energy. Although photosynthesis is most commonly associated with plants, microbial photosynthesis is also a significant supplier of chemical energy, fueling many diverse ecosystems.
• 8.7: Biogeochemical Cycles
Energy flows directionally through ecosystems, entering as sunlight for phototrophs or as inorganic molecules for chemoautotrophs. The six most common elements associated with organic molecules—carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur—take a variety of chemical forms and may exist for long periods in the atmosphere, on land, in water, or beneath earth’s surface.
• 8.E: Microbial Metabolism (Exercises)
Thumbnail: The Krebs cycle, also known as the citric acid cycle, is summarized here. Note incoming two-carbon acetyl results in the main outputs per turn of two CO2, three NADH, one FADH2, and one ATP (or GTP) molecules made by substrate-level phosphorylation. Two turns of the Krebs cycle are required to process all of the carbon from one glucose molecule. (CC BY 4.0; OpenStax)
08: Microbial Metabolism
Learning Objectives
• Define and describe metabolism
• Compare and contrast autotrophs and heterotrophs
• Describe the importance of oxidation-reduction reactions in metabolism
• Describe why ATP, FAD, NAD+, and NADP+ are important in a cell
• Identify the structure and structural components of an enzyme
• Describe the differences between competitive and noncompetitive enzyme inhibitors
Clinical Focus: Part 1
Hannah is a 15-month-old girl from Washington state. She is spending the summer in Gambia, where her parents are working for a nongovernmental organization. About 3 weeks after her arrival in Gambia, Hannah’s appetite began to diminish and her parents noticed that she seemed unusually sluggish, fatigued, and confused. She also seemed very irritable when she was outdoors, especially during the day. When she began vomiting, her parents figured she had caught a 24-hour virus, but when her symptoms persisted, they took her to a clinic. The local physician noticed that Hannah’s reflexes seemed abnormally slow, and when he examined her eyes with a light, she seemed unusually light sensitive. She also seemed to be experiencing a stiff neck.
Exercise \(1\)
What are some possible causes of Hannah’s symptoms?
The term used to describe all of the chemical reactions inside a cell is metabolism (Figure \(1\)). Cellular processes such as the building or breaking down of complex molecules occur through series of stepwise, interconnected chemical reactions called metabolic pathways. Reactions that are spontaneous and release energy are exergonic reactions, whereas endergonic reactions require energy to proceed. The term anabolism refers to those endergonic metabolic pathways involved in biosynthesis, converting simple molecular building blocks into more complex molecules, and fueled by the use of cellular energy. Conversely, the term catabolism refers to exergonic pathways that break down complex molecules into simpler ones. Molecular energy stored in the bonds of complex molecules is released in catabolic pathways and harvested in such a way that it can be used to produce high-energy molecules, which are used to drive anabolic pathways. Thus, in terms of energy and molecules, cells are continually balancing catabolism with anabolism.
Classification by Carbon and Energy Source
Organisms can be identified according to the source of carbon they use for metabolism as well as their energy source. The prefixes auto- (“self”) and hetero- (“other”) refer to the origins of the carbon sources various organisms can use. Organisms that convert inorganic carbon dioxide (CO2) into organic carbon compounds are autotrophs. Plants and cyanobacteria are well-known examples of autotrophs. Conversely, heterotrophs rely on more complex organic carbon compounds as nutrients; these are provided to them initially by autotrophs. Many organisms, ranging from humans to many prokaryotes, including the well-studied Escherichia coli, are heterotrophic.
Organisms can also be identified by the energy source they use. All energy is derived from the transfer of electrons, but the source of electrons differs between various types of organisms. The prefixes photo- (“light”) and chemo- (“chemical”) refer to the energy sources that various organisms use. Those that get their energy for electron transfer from light are phototrophs, whereas chemotrophs obtain energy for electron transfer by breaking chemical bonds. There are two types of chemotrophs: organotrophs and lithotrophs. Organotrophs, including humans, fungi, and many prokaryotes, are chemotrophs that obtain energy from organic compounds. Lithotrophs (“litho” means “rock”) are chemotrophs that get energy from inorganic compounds, including hydrogen sulfide (H2S) and reduced iron. Lithotrophy is unique to the microbial world.
The strategies used to obtain both carbon and energy can be combined for the classification of organisms according to nutritional type. Most organisms are chemoheterotrophs because they use organic molecules as both their electron and carbon sources. Table \(1\) summarizes this and the other classifications.
Table \(1\): Classifications of Organisms by Energy and Carbon Source
Classifications Energy Source Carbon Source Examples
Chemotrophs Chemoautotrophs Chemical Inorganic Hydrogen-, sulfur-, iron-, nitrogen-, and carbon monoxide-oxidizing bacteria
Chemoheterotrophs Chemical Organic compounds All animals, most fungi, protozoa, and bacteria
Phototrophs Photoautotrophs Light Inorganic All plants, algae, cyanobacteria, and green and purple sulfur bacteria
Photoheterotrophs Light Organic compounds Green and purple nonsulfur bacteria, heliobacteria
Exercise \(2\)
1. Explain the difference between catabolism and anabolism.
2. Explain the difference between autotrophs and heterotrophs.
Oxidation and Reduction in Metabolism
The transfer of electrons between molecules is important because most of the energy stored in atoms and used to fuel cell functions is in the form of high-energy electrons. The transfer of energy in the form of electrons allows the cell to transfer and use energy incrementally; that is, in small packages rather than a single, destructive burst. Reactions that remove electrons from donor molecules, leaving them oxidized, are oxidation reactions; those that add electrons to acceptor molecules, leaving them reduced, are reduction reactions. Because electrons can move from one molecule to another, oxidation and reduction occur in tandem. These pairs of reactions are called oxidation-reduction reactions, or redox reactions.
Energy Carriers: NAD+, NADP+, FAD, and ATP
The energy released from the breakdown of the chemical bonds within nutrients can be stored either through the reduction of electron carriers or in the bonds of adenosine triphosphate (ATP). In living systems, a small class of compounds functions as mobile electron carriers, molecules that bind to and shuttle high-energy electrons between compounds in pathways. The principal electron carriers we will consider originate from the B vitamin group and are derivatives of nucleotides; they are nicotinamide adenine dinucleotide, nicotine adenine dinucleotide phosphate, and flavin adenine dinucleotide. These compounds can be easily reduced or oxidized. Nicotinamide adenine dinucleotide (NAD+/NADH) is the most common mobile electron carrier used in catabolism. NAD+ is the oxidized form of the molecule; NADH is the reduced form of the molecule. Nicotine adenine dinucleotide phosphate (NADP+), the oxidized form of an NAD+ variant that contains an extra phosphate group, is another important electron carrier; it forms NADPH when reduced. The oxidized form of flavin adenine dinucleotide is FAD, and its reduced form is FADH2. Both NAD+/NADH and FAD/FADH2 are extensively used in energy extraction from sugars during catabolism in chemoheterotrophs, whereas NADP+/NADPH plays an important role in anabolic reactions and photosynthesis. Collectively, FADH2, NADH, and NADPH are often referred to as having reducing power due to their ability to donate electrons to various chemical reactions.
A living cell must be able to handle the energy released during catabolism in a way that enables the cell to store energy safely and release it for use only as needed. Living cells accomplish this by using the compound adenosine triphosphate (ATP). ATP is often called the “energy currency” of the cell, and, like currency, this versatile compound can be used to fill any energy need of the cell. At the heart of ATP is a molecule of adenosine monophosphate (AMP), which is composed of an adenine molecule bonded to a ribose molecule and a single phosphate group. Ribose is a five-carbon sugar found in RNA, and AMP is one of the nucleotides in RNA. The addition of a second phosphate group to this core molecule results in the formation of adenosine diphosphate (ADP); the addition of a third phosphate group forms ATP (Figure \(2\)). Adding a phosphate group to a molecule, a process called phosphorylation, requires energy. Phosphate groups are negatively charged and thus repel one another when they are arranged in series, as they are in ADP and ATP. This repulsion makes the ADP and ATP molecules inherently unstable. Thus, the bonds between phosphate groups (one in ADP and two in ATP) are called high-energy phosphate bonds. When these high-energy bonds are broken to release one phosphate (called inorganic phosphate [Pi]) or two connected phosphate groups (called pyrophosphate [PPi]) from ATP through a process called dephosphorylation, energy is released to drive endergonic reactions (Figure \(3\)).
Exercise \(3\)
What is the function of an electron carrier?
Enzyme Structure and Function
A substance that helps speed up a chemical reaction is a catalyst. Catalysts are not used or changed during chemical reactions and, therefore, are reusable. Whereas inorganic molecules may serve as catalysts for a wide range of chemical reactions, proteins called enzymes serve as catalysts for biochemical reactions inside cells. Enzymes thus play an important role in controlling cellular metabolism.
An enzyme functions by lowering the activation energy of a chemical reaction inside the cell. Activation energy is the energy needed to form or break chemical bonds and convert reactants to products (Figure \(4\)). Enzymes lower the activation energy by binding to the reactant molecules and holding them in such a way as to speed up the reaction.
The chemical reactants to which an enzyme binds are called substrates, and the location within the enzyme where the substrate binds is called the enzyme’s active site. The characteristics of the amino acids near the active site create a very specific chemical environment within the active site that induces suitability to binding, albeit briefly, to a specific substrate (or substrates). Due to this jigsaw puzzle-like match between an enzyme and its substrates, enzymes are known for their specificity. In fact, as an enzyme binds to its substrate(s), the enzyme structure changes slightly to find the best fit between the transition state (a structural intermediate between the substrate and product) and the active site, just as a rubber glove molds to a hand inserted into it. This active-site modification in the presence of substrate, along with the simultaneous formation of the transition state, is called induced fit (Figure \(5\)). Overall, there is a specifically matched enzyme for each substrate and, thus, for each chemical reaction; however, there is some flexibility as well. Some enzymes have the ability to act on several different structurally related substrates.
Enzymes are subject to influences by local environmental conditions such as pH, substrate concentration, and temperature. Although increasing the environmental temperature generally increases reaction rates, enzyme catalyzed or otherwise, increasing or decreasing the temperature outside of an optimal range can affect chemical bonds within the active site, making them less well suited to bind substrates. High temperatures will eventually cause enzymes, like other biological molecules, to denature, losing their three-dimensional structure and function. Enzymes are also suited to function best within a certain pH range, and, as with temperature, extreme environmental pH values (acidic or basic) can cause enzymes to denature. Active-site amino-acid side chains have their own acidic or basic properties that are optimal for catalysis and, therefore, are sensitive to changes in pH.
Another factor that influences enzyme activity is substrate concentration: Enzyme activity is increased at higher concentrations of substrate until it reaches a saturation point at which the enzyme can bind no additional substrate. Overall, enzymes are optimized to work best under the environmental conditions in which the organisms that produce them live. For example, while microbes that inhabit hot springs have enzymes that work best at high temperatures, human pathogens have enzymes that work best at 37°C. Similarly, while enzymes produced by most organisms work best at a neutral pH, microbes growing in acidic environments make enzymes optimized to low pH conditions, allowing for their growth at those conditions.
Many enzymes do not work optimally, or even at all, unless bound to other specific nonprotein helper molecules, either temporarily through ionic or hydrogen bonds or permanently through stronger covalent bonds. Binding to these molecules promotes optimal conformation and function for their respective enzymes. Two types of helper molecules are cofactors and coenzymes. Cofactors are inorganic ions such as iron (Fe2+) and magnesium (Mg2+) that help stabilize enzyme conformation and function. One example of an enzyme that requires a metal ion as a cofactor is the enzyme that builds DNA molecules, DNA polymerase, which requires a bound zinc ion (Zn2+) to function.
Coenzymes are organic helper molecules that are required for enzyme action. Like enzymes, they are not consumed and, hence, are reusable. The most common sources of coenzymes are dietary vitamins. Some vitamins are precursors to coenzymes and others act directly as coenzymes.
Some cofactors and coenzymes, like coenzyme A (CoA), often bind to the enzyme’s active site, aiding in the chemistry of the transition of a substrate to a product (Figure \(6\)). In such cases, an enzyme lacking a necessary cofactor or coenzyme is called an apoenzyme and is inactive. Conversely, an enzyme with the necessary associated cofactor or coenzyme is called a holoenzyme and is active. NADH and ATP are also both examples of commonly used coenzymes that provide high-energy electrons or phosphate groups, respectively, which bind to enzymes, thereby activating them.
Exercise \(4\)
What role do enzymes play in a chemical reaction?
Enzyme Inhibitors
Enzymes can be regulated in ways that either promote or reduce their activity. There are many different kinds of molecules that inhibit or promote enzyme function, and various mechanisms exist for doing so (Figure \(7\)). A competitive inhibitor is a molecule similar enough to a substrate that it can compete with the substrate for binding to the active site by simply blocking the substrate from binding. For a competitive inhibitor to be effective, the inhibitor concentration needs to be approximately equal to the substrate concentration. Sulfa drugs provide a good example of competitive competition. They are used to treat bacterial infections because they bind to the active site of an enzyme within the bacterial folic acid synthesis pathway. When present in a sufficient dose, a sulfa drug prevents folic acid synthesis, and bacteria are unable to grow because they cannot synthesize DNA, RNA, and proteins. Humans are unaffected because we obtain folic acid from our diets.
On the other hand, a noncompetitive (allosteric) inhibitor binds to the enzyme at an allosteric site, a location other than the active site, and still manages to block substrate binding to the active site by inducing a conformational change that reduces the affinity of the enzyme for its substrate (Figure \(8\)). Because only one inhibitor molecule is needed per enzyme for effective inhibition, the concentration of inhibitors needed for noncompetitive inhibition is typically much lower than the substrate concentration.
In addition to allosteric inhibitors, there are allosteric activators that bind to locations on an enzyme away from the active site, inducing a conformational change that increases the affinity of the enzyme’s active site(s) for its substrate(s).
Allosteric control is an important mechanism of regulation of metabolic pathways involved in both catabolism and anabolism. In a most efficient and elegant way, cells have evolved also to use the products of their own metabolic reactions for feedback inhibition of enzyme activity. Feedback inhibition involves the use of a pathway product to regulate its own further production. The cell responds to the abundance of specific products by slowing production during anabolic or catabolic reactions (Figure \(8\)).
Exercise \(5\)
Explain the difference between a competitive inhibitor and a noncompetitive inhibitor.
Key Concepts and Summary
• Metabolism includes chemical reactions that break down complex molecules (catabolism) and those that build complex molecules (anabolism).
• Organisms may be classified according to their source of carbon. Autotrophs convert inorganic carbon dioxide into organic carbon; heterotrophs use fixed organic carbon compounds.
• Organisms may also be classified according to their energy source. Phototrophs obtain their energy from light. Chemotrophs get their energy from chemical compounds. Organotrophs use organic molecules, and lithotrophs use inorganic chemicals.
• Cellular electron carriers accept high-energy electrons from foods and later serve as electron donors in subsequent redox reactions. FAD/FADH2, NAD+/NADH, and NADP+/NADPH are important electron carriers.
• Adenosine triphosphate (ATP) serves as the energy currency of the cell, safely storing chemical energy in its two high-energy phosphate bonds for later use to drive processes requiring energy.
• Enzymes are biological catalysts that increase the rate of chemical reactions inside cells by lowering the activation energy required for the reaction to proceed.
• In nature, exergonic reactions do not require energy beyond activation energy to proceed, and they release energy. They may proceed without enzymes, but at a slow rate. Conversely, endergonic reactions require energy beyond activation energy to occur. In cells, endergonic reactions are coupled to exergonic reactions, making the combination energetically favorable.
• Substrates bind to the enzyme’s active site. This process typically alters the structures of both the active site and the substrate, favoring transition-state formation; this is known as induced fit.
• Cofactors are inorganic ions that stabilize enzyme conformation and function. Coenzymes are organic molecules required for proper enzyme function and are often derived from vitamins. An enzyme lacking a cofactor or coenzyme is an apoenzyme; an enzyme with a bound cofactor or coenzyme is a holoenzyme.
• Competitive inhibitors regulate enzymes by binding to an enzyme’s active site, preventing substrate binding. Noncompetitive (allosteric) inhibitors bind to allosteric sites, inducing a conformational change in the enzyme that prevents it from functioning. Feedback inhibition occurs when the product of a metabolic pathway noncompetitively binds to an enzyme early on in the pathway, ultimately preventing the synthesis of the product. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/08%3A_Microbial_Metabolism/8.01%3A_Energy_Matter_and_Enzymes.txt |
Learning Objectives
• Describe why glycolysis is not oxygen dependent
• Define and describe the net yield of three-carbon molecules, ATP, and NADH from glycolysis
• Explain how three-carbon pyruvate molecules are converted into two-carbon acetyl groups that can be funneled into the Krebs cycle.
• Define and describe the net yield of CO2, GTP/ATP, FADH2, and NADH from the Krebs cycle
• Explain how intermediate carbon molecules of the Krebs cycle can be used in a cell
Extensive enzyme pathways exist for breaking down carbohydrates to capture energy in ATP bonds. In addition, many catabolic pathways produce intermediate molecules that are also used as building blocks for anabolism. Understanding these processes is important for several reasons. First, because the main metabolic processes involved are common to a wide range of chemoheterotrophic organisms, we can learn a great deal about human metabolism by studying metabolism in more easily manipulated bacteria like E. coli. Second, because animal and human pathogens are also chemoheterotrophs, learning about the details of metabolism in these bacteria, including possible differences between bacterial and human pathways, is useful for the diagnosis of pathogens as well as for the discovery of antimicrobial therapies targeting specific pathogens. Last, learning specifically about the pathways involved in chemoheterotrophic metabolism also serves as a basis for comparing other more unusual metabolic strategies used by microbes. Although the chemical source of electrons initiating electron transfer is different between chemoheterorophs and chemoautotrophs, many similar processes are used in both types of organisms.
The typical example used to introduce concepts of metabolism to students is carbohydrate catabolism. For chemoheterotrophs, our examples of metabolism start with the catabolism of polysaccharides such as glycogen, starch, or cellulose. Enzymes such as amylase, which breaks down glycogen or starch, and cellulases, which break down cellulose, can cause the hydrolysis of glycosidic bonds between the glucose monomers in these polymers, releasing glucose for further catabolism.
Glycolysis
For bacteria, eukaryotes, and most archaea, glycolysis is the most common pathway for the catabolism of glucose; it produces energy, reduced electron carriers, and precursor molecules for cellular metabolism. Every living organism carries out some form of glycolysis, suggesting this mechanism is an ancient universal metabolic process. The process itself does not use oxygen; however, glycolysis can be coupled with additional metabolic processes that are either aerobic or anaerobic. Glycolysis takes place in the cytoplasm of prokaryotic and eukaryotic cells. It begins with a single six-carbon glucose molecule and ends with two molecules of a three-carbon sugar called pyruvate. Pyruvate may be broken down further after glycolysis to harness more energy through aerobic or anaerobic respiration, but many organisms, including many microbes, may be unable to respire; for these organisms, glycolysis may be their only source of generating ATP.
The type of glycolysis found in animals and that is most common in microbes is the Embden-Meyerhof-Parnas (EMP) pathway, named after Gustav Embden (1874–1933), Otto Meyerhof (1884–1951), and Jakub Parnas (1884–1949). Glycolysis using the EMP pathway consists of two distinct phases (Figure \(1\)). The first part of the pathway, called the energy investment phase, uses energy from two ATP molecules to modify a glucose molecule so that the six-carbon sugar molecule can be split evenly into two phosphorylated three-carbon molecules called glyceraldehyde 3-phosphate (G3P). The second part of the pathway, called the energy payoff phase, extracts energy by oxidizing G3P to pyruvate, producing four ATP molecules and reducing two molecules of NAD+ to two molecules of NADH, using electrons that originated from glucose. (A discussion and illustration of the full EMP pathway with chemical structures and enzyme names appear in Appendix C.)
The ATP molecules produced during the energy payoff phase of glycolysis are formed by substrate-level phosphorylation (Figure \(1\)), one of two mechanisms for producing ATP. In substrate-level phosphorylation, a phosphate group is removed from an organic molecule and is directly transferred to an available ADP molecule, producing ATP. During glycolysis, high-energy phosphate groups from the intermediate molecules are added to ADP to make ATP.
Overall, in this process of glycolysis, the net gain from the breakdown of a single glucose molecule is:
• two ATP molecules
• two NADH molecule, and
• two pyruvate molecules.
Other Glycolytic Pathways
When we refer to glycolysis, unless otherwise indicated, we are referring to the EMP pathway used by animals and many bacteria. However, some prokaryotes use alternative glycolytic pathways. One important alternative is the Entner-Doudoroff (ED) pathway, named after its discoverers Nathan Entner and Michael Doudoroff (1911–1975). Although some bacteria, including the opportunistic gram-negative pathogen Pseudomonas aeruginosa, contain only the ED pathway for glycolysis, other bacteria, like E. coli, have the ability to use either the ED pathway or the EMP pathway.
A third type of glycolytic pathway that occurs in all cells, which is quite different from the previous two pathways, is the pentose phosphate pathway (PPP) also called the phosphogluconate pathway or the hexose monophosphate shunt. Evidence suggests that the PPP may be the most ancient universal glycolytic pathway. The intermediates from the PPP are used for the biosynthesis of nucleotides and amino acids. Therefore, this glycolytic pathway may be favored when the cell has need for nucleic acid and/or protein synthesis, respectively. A discussion and illustration of the complete ED pathway and PPP with chemical structures and enzyme names appear in Appendix C.
Exercise \(1\)
When might an organism use the ED pathway or the PPP for glycolysis?
Transition Reaction, Coenzyme A, and the Krebs Cycle
Glycolysis produces pyruvate, which can be further oxidized to capture more energy. For pyruvate to enter the next oxidative pathway, it must first be decarboxylated by the enzyme complex pyruvate dehydrogenase to a two-carbon acetyl group in the transition reaction, also called the bridge reaction (see Appendix C and Figure \(3\)). In the transition reaction, electrons are also transferred to NAD+ to form NADH. To proceed to the next phase of this metabolic process, the comparatively tiny two-carbon acetyl must be attached to a very large carrier compound called coenzyme A (CoA). The transition reaction occurs in the mitochondrial matrix of eukaryotes; in prokaryotes, it occurs in the cytoplasm because prokaryotes lack membrane-enclosed organelles.
The Krebs cycle transfers remaining electrons from the acetyl group produced during the transition reaction to electron carrier molecules, thus reducing them. The Krebs cycle also occurs in the cytoplasm of prokaryotes along with glycolysis and the transition reaction, but it takes place in the mitochondrial matrix of eukaryotic cells where the transition reaction also occurs. The Krebs cycle is named after its discoverer, British scientist Hans Adolf Krebs (1900–1981) and is also called the citric acid cycle, or the tricarboxylic acid cycle (TCA) because citric acid has three carboxyl groups in its structure. Unlike glycolysis, the Krebs cycle is a closed loop: The last part of the pathway regenerates the compound used in the first step (Figure \(4\)). The eight steps of the cycle are a series of chemical reactions that capture the two-carbon acetyl group (the CoA carrier does not enter the Krebs cycle) from the transition reaction, which is added to a four-carbon intermediate in the Krebs cycle, producing the six-carbon intermediate citric acid (giving the alternate name for this cycle). As one turn of the cycle returns to the starting point of the four-carbon intermediate, the cycle produces two CO2 molecules, one ATP molecule (or an equivalent, such as guanosine triphosphate [GTP]) produced by substrate-level phosphorylation, and three molecules of NADH and one of FADH2. (A discussion and detailed illustration of the full Krebs cycle appear in Appendix C.)
Although many organisms use the Krebs cycle as described as part of glucose metabolism, several of the intermediate compounds in the Krebs cycle can be used in synthesizing a wide variety of important cellular molecules, including amino acids, chlorophylls, fatty acids, and nucleotides; therefore, the cycle is both anabolic and catabolic (Figure \(5\)).
Key Concepts and Summary
• Glycolysis is the first step in the breakdown of glucose, resulting in the formation of ATP, which is produced by substrate-level phosphorylation; NADH; and two pyruvate molecules. Glycolysis does not use oxygen and is not oxygen dependent.
• After glycolysis, a three-carbon pyruvate is decarboxylated to form a two-carbon acetyl group, coupled with the formation of NADH. The acetyl group is attached to a large carrier compound called coenzyme A.
• After the transition step, coenzyme A transports the two-carbon acetyl to the Krebs cycle, where the two carbons enter the cycle. Per turn of the cycle, one acetyl group derived from glycolysis is further oxidized, producing three NADH molecules, one FADH2, and one ATP by substrate-level phosphorylation, and releasing two CO2 molecules.
• The Krebs cycle may be used for other purposes. Many of the intermediates are used to synthesize important cellular molecules, including amino acids, chlorophylls, fatty acids, and nucleotides. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/08%3A_Microbial_Metabolism/8.02%3A_Catabolism_of_Carbohydrates.txt |
Learning Objectives
• Compare and contrast the electron transport system location and function in a prokaryotic cell and a eukaryotic cell
• Compare and contrast the differences between substrate-level and oxidative phosphorylation
• Explain the relationship between chemiosmosis and proton motive force
• Describe the function and location of ATP synthase in a prokaryotic versus eukaryotic cell
• Compare and contrast aerobic and anaerobic respiration
We have just discussed two pathways in glucose catabolism—glycolysis and the Krebs cycle—that generate ATP by substrate-level phosphorylation. Most ATP, however, is generated during a separate process called oxidative phosphorylation, which occurs during cellular respiration. Cellular respiration begins when electrons are transferred from NADH and FADH2—made in glycolysis, the transition reaction, and the Krebs cycle—through a series of chemical reactions to a final inorganic electron acceptor (either oxygen in aerobic respiration or non-oxygen inorganic molecules in anaerobic respiration). These electron transfers take place on the inner part of the cell membrane of prokaryotic cells or in specialized protein complexes in the inner membrane of the mitochondria of eukaryotic cells. The energy of the electrons is harvested to generate an electrochemical gradient across the membrane, which is used to make ATP by oxidative phosphorylation.
Electron Transport System
The electron transport system (ETS) is the last component involved in the process of cellular respiration; it comprises a series of membrane-associated protein complexes and associated mobile accessory electron carriers. Electron transport is a series of chemical reactions that resembles a bucket brigade in that electrons from NADH and FADH2 are passed rapidly from one ETS electron carrier to the next. These carriers can pass electrons along in the ETS because of their redox potential. For a protein or chemical to accept electrons, it must have a more positive redox potential than the electron donor. Therefore, electrons move from electron carriers with more negative redox potential to those with more positive redox potential. The four major classes of electron carriers involved in both eukaryotic and prokaryotic electron transport systems are the cytochromes, flavoproteins, iron-sulfur proteins, and the quinones.
In aerobic respiration, the final electron acceptor (i.e., the one having the most positive redox potential) at the end of the ETS is an oxygen molecule (O2) that becomes reduced to water (H2O) by the final ETS carrier. This electron carrier, cytochrome oxidase, differs between bacterial types and can be used to differentiate closely related bacteria for diagnoses. For example, the gram-negative opportunist Pseudomonas aeruginosa and the gram-negative cholera-causing Vibrio cholerae use cytochrome c oxidase, which can be detected by the oxidase test, whereas other gram-negative Enterobacteriaceae, like E. coli, are negative for this test because they produce different cytochrome oxidase types.
There are many circumstances under which aerobic respiration is not possible, including any one or more of the following:
• The cell lacks genes encoding an appropriate cytochrome oxidase for transferring electrons to oxygen at the end of the electron transport system.
• The cell lacks genes encoding enzymes to minimize the severely damaging effects of dangerous oxygen radicals produced during aerobic respiration, such as hydrogen peroxide (H2O2) or superoxide \((\ce{O2-})\).
• The cell lacks a sufficient amount of oxygen to carry out aerobic respiration.
One possible alternative to aerobic respiration is anaerobic respiration, using an inorganic molecule other than oxygen as a final electron acceptor. There are many types of anaerobic respiration found in bacteria and archaea. Denitrifiers are important soil bacteria that use nitrate \((\ce{NO3-})\) and nitrite \((\ce{NO2-})\) as final electron acceptors, producing nitrogen gas (N2). Many aerobically respiring bacteria, including E. coli, switch to using nitrate as a final electron acceptor and producing nitrite when oxygen levels have been depleted.
Microbes using anaerobic respiration commonly have an intact Krebs cycle, so these organisms can access the energy of the NADH and FADH2 molecules formed. However, anaerobic respirers use altered ETS carriers encoded by their genomes, including distinct complexes for electron transfer to their final electron acceptors. Smaller electrochemical gradients are generated from these electron transfer systems, so less ATP is formed through anaerobic respiration.
Exercise \(1\)
Do both aerobic respiration and anaerobic respiration use an electron transport chain?
Chemiosmosis, Proton Motive Force, and Oxidative Phosphorylation
In each transfer of an electron through the ETS, the electron loses energy, but with some transfers, the energy is stored as potential energy by using it to pump hydrogen ions (H+) across a membrane. In prokaryotic cells, H+ is pumped to the outside of the cytoplasmic membrane (called the periplasmic space in gram-negative and gram-positive bacteria), and in eukaryotic cells, they are pumped from the mitochondrial matrix across the inner mitochondrial membrane into the intermembrane space. There is an uneven distribution of H+ across the membrane that establishes an electrochemical gradient because H+ ions are positively charged (electrical) and there is a higher concentration (chemical) on one side of the membrane. This electrochemical gradient formed by the accumulation of H+ (also known as a proton) on one side of the membrane compared with the other is referred to as the proton motive force (PMF). Because the ions involved are H+, a pH gradient is also established, with the side of the membrane having the higher concentration of H+ being more acidic. Beyond the use of the PMF to make ATP, as discussed in this chapter, the PMF can also be used to drive other energetically unfavorable processes, including nutrient transport and flagella rotation for motility.
The potential energy of this electrochemical gradient generated by the ETS causes the H+ to diffuse across a membrane (the plasma membrane in prokaryotic cells and the inner membrane in mitochondria in eukaryotic cells). This flow of hydrogen ions across the membrane, called chemiosmosis, must occur through a channel in the membrane via a membrane-bound enzyme complex called ATP synthase (Figure \(1\)). The tendency for movement in this way is much like water accumulated on one side of a dam, moving through the dam when opened. ATP synthase (like a combination of the intake and generator of a hydroelectric dam) is a complex protein that acts as a tiny generator, turning by the force of the H+ diffusing through the enzyme, down their electrochemical gradient from where there are many mutually repelling H+ to where there are fewer H+. In prokaryotic cells, H+ flows from the outside of the cytoplasmic membrane into the cytoplasm, whereas in eukaryotic mitochondria, H+ flows from the intermembrane space to the mitochondrial matrix. The turning of the parts of this molecular machine regenerates ATP from ADP and inorganic phosphate (Pi) by oxidative phosphorylation, a second mechanism for making ATP that harvests the potential energy stored within an electrochemical gradient.
The number of ATP molecules generated from the catabolism of glucose varies. For example, the number of hydrogen ions that the electron transport system complexes can pump through the membrane varies between different species of organisms. In aerobic respiration in mitochondria, the passage of electrons from one molecule of NADH generates enough proton motive force to make three ATP molecules by oxidative phosphorylation, whereas the passage of electrons from one molecule of FADH2 generates enough proton motive force to make only two ATP molecules. Thus, the 10 NADH molecules made per glucose during glycolysis, the transition reaction, and the Krebs cycle carry enough energy to make 30 ATP molecules, whereas the two FADH2 molecules made per glucose during these processes provide enough energy to make four ATP molecules. Overall, the theoretical maximum yield of ATP made during the complete aerobic respiration of glucose is 38 molecules, with four being made by substrate-level phosphorylation and 34 being made by oxidative phosphorylation (Figure \(2\)). In reality, the total ATP yield is usually less, ranging from one to 34 ATP molecules, depending on whether the cell is using aerobic respiration or anaerobic respiration; in eukaryotic cells, some energy is expended to transport intermediates from the cytoplasm into the mitochondria, affecting ATP yield.
Figure \(2\) summarizes the theoretical maximum yields of ATP from various processes during the complete aerobic respiration of one glucose molecule.
Exercise \(1\)
What are the functions of the proton motive force?
Summary
• Most ATP generated during the cellular respiration of glucose is made by oxidative phosphorylation.
• An electron transport system (ETS) is composed of a series of membrane-associated protein complexes and associated mobile accessory electron carriers. The ETS is embedded in the cytoplasmic membrane of prokaryotes and the inner mitochondrial membrane of eukaryotes.
• Each ETS complex has a different redox potential, and electrons move from electron carriers with more negative redox potential to those with more positive redox potential.
• To carry out aerobic respiration, a cell requires oxygen as the final electron acceptor. A cell also needs a complete Krebs cycle, an appropriate cytochrome oxidase, and oxygen detoxification enzymes to prevent the harmful effects of oxygen radicals produced during aerobic respiration.
• Organisms performing anaerobic respiration use alternative electron transport system carriers for the ultimate transfer of electrons to the final non-oxygen electron acceptors.
• Microbes show great variation in the composition of their electron transport systems, which can be used for diagnostic purposes to help identify certain pathogens.
• As electrons are passed from NADH and FADH2 through an ETS, the electron loses energy. This energy is stored through the pumping of H+ across the membrane, generating a proton motive force.
• The energy of this proton motive force can be harnessed by allowing hydrogen ions to diffuse back through the membrane by chemiosmosis using ATP synthase. As hydrogen ions diffuse through down their electrochemical gradient, components of ATP synthase spin, making ATP from ADP and Pi by oxidative phosphorylation.
• Aerobic respiration forms more ATP (a maximum of 34 ATP molecules) during oxidative phosphorylation than does anaerobic respiration (between one and 32 ATP molecules). | textbooks/bio/Microbiology/Microbiology_(OpenStax)/08%3A_Microbial_Metabolism/8.03%3A_Cellular_Respiration.txt |
Learning Objectives
• Define fermentation and explain why it does not require oxygen
• Describe the fermentation pathways and their end products and give examples of microorganisms that use these pathways
• Compare and contrast fermentation and anaerobic respiration
Many cells are unable to carry out respiration because of one or more of the following circumstances:
1. The cell lacks a sufficient amount of any appropriate, inorganic, final electron acceptor to carry out cellular respiration.
2. The cell lacks genes to make appropriate complexes and electron carriers in the electron transport system.
3. The cell lacks genes to make one or more enzymes in the Krebs cycle.
Whereas lack of an appropriate inorganic final electron acceptor is environmentally dependent, the other two conditions are genetically determined. Thus, many prokaryotes, including members of the clinically important genus Streptococcus, are permanently incapable of respiration, even in the presence of oxygen. Conversely, many prokaryotes are facultative, meaning that, should the environmental conditions change to provide an appropriate inorganic final electron acceptor for respiration, organisms containing all the genes required to do so will switch to cellular respiration for glucose metabolism because respiration allows for much greater ATP production per glucose molecule.
If respiration does not occur, NADH must be reoxidized to NAD+ for reuse as an electron carrier for glycolysis, the cell’s only mechanism for producing any ATP, to continue. Some living systems use an organic molecule (commonly pyruvate) as a final electron acceptor through a process called fermentation. Fermentation does not involve an electron transport system and does not directly produce any additional ATP beyond that produced during glycolysis by substrate-level phosphorylation. Organisms carrying out fermentation, called fermenters, produce a maximum of two ATP molecules per glucose during glycolysis. Table \(1\) compares the final electron acceptors and methods of ATP synthesis in aerobic respiration, anaerobic respiration, and fermentation. Note that the number of ATP molecules shown for glycolysis assumes the Embden-Meyerhof-Parnas pathway. The number of ATP molecules made by substrate-level phosphorylation (SLP) versus oxidative phosphorylation (OP) are indicated.
Table \(1\): Comparison of Respiration Versus Fermentation
Type of Metabolism Example Final Electron Acceptor Pathways Involved in ATP Synthesis (Type of Phosphorylation) Maximum Yield of ATP Molecules
Aerobic respiration Pseudomonas aeruginosa \(\ce{O2}\)
EMP glycolysis (SLP)
Krebs cycle (SLP)
Electron transport and chemiosmosis (OP):
2
2
34
Total 38
Anaerobic respiration Paracoccus denitrificans
\(\ce{NO3-}\), \(\ce{SO4^{-2}}\), \(\ce{Fe^{+3}}\), \(\ce{CO2}\),
other inorganics
EMP glycolysis (SLP)
Krebs cycle (SLP)
Electron transport and chemiosmosis (OP):
2
2
1–32
Total 536
Fermentation Candida albicans
Organics
(usually pyruvate)
EMP glycolysis (SLP)
Fermentation
2
0
Total 2
Microbial fermentation processes have been manipulated by humans and are used extensively in the production of various foods and other commercial products, including pharmaceuticals. Microbial fermentation can also be useful for identifying microbes for diagnostic purposes.
Fermentation by some bacteria, like those in yogurt and other soured food products, and by animals in muscles during oxygen depletion, is lactic acid fermentation. The chemical reaction of lactic acid fermentation is as follows:
\[\ce{Pyruvate + NADH \leftrightarrow lactic\: acid + NAD+}\]
Bacteria of several gram-positive genera, including Lactobacillus, Leuconostoc, and Streptococcus, are collectively known as the lactic acid bacteria (LAB), and various strains are important in food production. During yogurt and cheese production, the highly acidic environment generated by lactic acid fermentation denatures proteins contained in milk, causing it to solidify. When lactic acid is the only fermentation product, the process is said to be homolactic fermentation; such is the case for Lactobacillus delbrueckii and S. thermophiles used in yogurt production. However, many bacteria perform heterolactic fermentation, producing a mixture of lactic acid, ethanol and/or acetic acid, and CO2 as a result, because of their use of the branched pentose phosphate pathway instead of the EMP pathway for glycolysis. One important heterolactic fermenter is Leuconostoc mesenteroides, which is used for souring vegetables like cucumbers and cabbage, producing pickles and sauerkraut, respectively.
Lactic acid bacteria are also important medically. The production of low pH environments within the body inhibits the establishment and growth of pathogens in these areas. For example, the vaginal microbiota is composed largely of lactic acid bacteria, but when these bacteria are reduced, yeast can proliferate, causing a yeast infection. Additionally, lactic acid bacteria are important in maintaining the health of the gastrointestinal tract and, as such, are the primary component of probiotics.
Another familiar fermentation process is alcohol fermentation, which produces ethanol. The ethanol fermentation reaction is shown in Figure \(1\). In the first reaction, the enzyme pyruvate decarboxylase removes a carboxyl group from pyruvate, releasing CO2 gas while producing the two-carbon molecule acetaldehyde. The second reaction, catalyzed by the enzyme alcohol dehydrogenase, transfers an electron from NADH to acetaldehyde, producing ethanol and NAD+. The ethanol fermentation of pyruvate by the yeast Saccharomyces cerevisiae is used in the production of alcoholic beverages and also makes bread products rise due to CO2 production. Outside of the food industry, ethanol fermentation of plant products is important in biofuel production.
Beyond lactic acid fermentation and alcohol fermentation, many other fermentation methods occur in prokaryotes, all for the purpose of ensuring an adequate supply of NAD+ for glycolysis (Table \(2\)). Without these pathways, glycolysis would not occur and no ATP would be harvested from the breakdown of glucose. It should be noted that most forms of fermentation besides homolactic fermentation produce gas, commonly CO2 and/or hydrogen gas. Many of these different types of fermentation pathways are also used in food production and each results in the production of different organic acids, contributing to the unique flavor of a particular fermented food product. The propionic acid produced during propionic acid fermentation contributes to the distinctive flavor of Swiss cheese, for example.
Several fermentation products are important commercially outside of the food industry. For example, chemical solvents such as acetone and butanol are produced during acetone-butanol-ethanol fermentation. Complex organic pharmaceutical compounds used in antibiotics (e.g., penicillin), vaccines, and vitamins are produced through mixed acid fermentation. Fermentation products are used in the laboratory to differentiate various bacteria for diagnostic purposes. For example, enteric bacteria are known for their ability to perform mixed acid fermentation, reducing the pH, which can be detected using a pH indicator. Similarly, the bacterial production of acetoin during butanediol fermentation can also be detected. Gas production from fermentation can also be seen in an inverted Durham tube that traps produced gas in a broth culture.
Microbes can also be differentiated according to the substrates they can ferment. For example, E. coli can ferment lactose, forming gas, whereas some of its close gram-negative relatives cannot. The ability to ferment the sugar alcohol sorbitol is used to identify the pathogenic enterohemorrhagic O157:H7 strain of E. coli because, unlike other E. coli strains, it is unable to ferment sorbitol. Last, mannitol fermentation differentiates the mannitol-fermenting Staphylococcus aureus from other non–mannitol-fermenting staphylococci.
Table \(2\): Common Fermentation Pathways
Pathway End Products Example Microbes Commercial Products
Acetone-butanol-ethanol Acetone, butanol, ethanol, CO2 Clostridium acetobutylicum Commercial solvents, gasoline alternative
Alcohol Ethanol, CO2 Candida, Saccharomyces Beer, bread
Butanediol Formic and lactic acid; ethanol; acetoin; 2,3 butanediol; CO2; hydrogen gas Klebsiella, Enterobacter Chardonnay wine
Butyric acid Butyric acid, CO2, hydrogen gas Clostridium butyricum Butter
Lactic acid Lactic acid Streptococcus, Lactobacillus Sauerkraut, yogurt, cheese
Mixed acid Acetic, formic, lactic, and succinic acids; ethanol, CO2, hydrogen gas Escherichia, Shigella Vinegar, cosmetics, pharmaceuticals
Propionic acid Acetic acid, propionic acid, CO2 Propionibacterium, Bifidobacterium Swiss cheese
Exercise \(1\)
When would a metabolically versatile microbe perform fermentation rather than cellular respiration?
Identifying Bacteria by Using API Test Panels
Identification of a microbial isolate is essential for the proper diagnosis and appropriate treatment of patients. Scientists have developed techniques that identify bacteria according to their biochemical characteristics. Typically, they either examine the use of specific carbon sources as substrates for fermentation or other metabolic reactions, or they identify fermentation products or specific enzymes present in reactions. In the past, microbiologists have used individual test tubes and plates to conduct biochemical testing. However, scientists, especially those in clinical laboratories, now more frequently use plastic, disposable, multitest panels that contain a number of miniature reaction tubes, each typically including a specific substrate and pH indicator. After inoculation of the test panel with a small sample of the microbe in question and incubation, scientists can compare the results to a database that includes the expected results for specific biochemical reactions for known microbes, thus enabling rapid identification of a sample microbe. These test panels have allowed scientists to reduce costs while improving efficiency and reproducibility by performing a larger number of tests simultaneously.
Many commercial, miniaturized biochemical test panels cover a number of clinically important groups of bacteria and yeasts. One of the earliest and most popular test panels is the Analytical Profile Index (API) panel invented in the 1970s. Once some basic laboratory characterization of a given strain has been performed, such as determining the strain’s Gram morphology, an appropriate test strip that contains 10 to 20 different biochemical tests for differentiating strains within that microbial group can be used. Currently, the various API strips can be used to quickly and easily identify more than 600 species of bacteria, both aerobic and anaerobic, and approximately 100 different types of yeasts. Based on the colors of the reactions when metabolic end products are present, due to the presence of pH indicators, a metabolic profile is created from the results (Figure \(2\)). Microbiologists can then compare the sample’s profile to the database to identify the specific microbe.
Clinical Focus: Part 2
Many of Hannah’s symptoms are consistent with several different infections, including influenza and pneumonia. However, her sluggish reflexes along with her light sensitivity and stiff neck suggest some possible involvement of the central nervous system, perhaps indicating meningitis. Meningitis is an infection of the cerebrospinal fluid (CSF) around the brain and spinal cord that causes inflammation of the meninges, the protective layers covering the brain. Meningitis can be caused by viruses, bacteria, or fungi. Although all forms of meningitis are serious, bacterial meningitis is particularly serious. Bacterial meningitis may be caused by several different bacteria, but the bacterium Neisseria meningitidis, a gram-negative, bean-shaped diplococcus, is a common cause and leads to death within 1 to 2 days in 5% to 10% of patients.
Given the potential seriousness of Hannah’s conditions, her physician advised her parents to take her to the hospital in the Gambian capital of Banjul and there have her tested and treated for possible meningitis. After a 3-hour drive to the hospital, Hannah was immediately admitted. Physicians took a blood sample and performed a lumbar puncture to test her CSF. They also immediately started her on a course of the antibiotic ceftriaxone, the drug of choice for treatment of meningitis caused by N. meningitidis, without waiting for laboratory test results.
Exercise \(2\)
1. How might biochemical testing be used to confirm the identity of N. meningitidis?
2. Why did Hannah’s doctors decide to administer antibiotics without waiting for the test results?
Key Concepts and Summary
• Fermentation uses an organic molecule as a final electron acceptor to regenerate NAD+ from NADH so that glycolysis can continue.
• Fermentation does not involve an electron transport system, and no ATP is made by the fermentation process directly. Fermenters make very little ATP—only two ATP molecules per glucose molecule during glycolysis.
• Microbial fermentation processes have been used for the production of foods and pharmaceuticals, and for the identification of microbes.
• During lactic acid fermentation, pyruvate accepts electrons from NADH and is reduced to lactic acid. Microbes performing homolactic fermentation produce only lactic acid as the fermentation product; microbes performing heterolactic fermentation produce a mixture of lactic acid, ethanol and/or acetic acid, and CO2.
• Lactic acid production by the normal microbiota prevents growth of pathogens in certain body regions and is important for the health of the gastrointestinal tract.
• During ethanol fermentation, pyruvate is first decarboxylated (releasing CO2) to acetaldehyde, which then accepts electrons from NADH, reducing acetaldehyde to ethanol. Ethanol fermentation is used for the production of alcoholic beverages, for making bread products rise, and for biofuel production.
• Fermentation products of pathways (e.g., propionic acid fermentation) provide distinctive flavors to food products. Fermentation is used to produce chemical solvents (acetone-butanol-ethanol fermentation) and pharmaceuticals (mixed acid fermentation).
• Specific types of microbes may be distinguished by their fermentation pathways and products. Microbes may also be differentiated according to the substrates they are able to ferment. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/08%3A_Microbial_Metabolism/8.04%3A_Fermentation.txt |
Learning Objectives
• Describe how lipids are catabolized
• Describe how lipid catabolism can be used to identify microbes
• Describe how proteins are catabolized
• Describe how protein catabolism can be used to identify bacteria
Previous sections have discussed the catabolism of glucose, which provides energy to living cells, as well as how polysaccharides like glycogen, starch, and cellulose are degraded to glucose monomers. But microbes consume more than just carbohydrates for food. In fact, the microbial world is known for its ability to degrade a wide range of molecules, both naturally occurring and those made by human processes, for use as carbon sources. In this section, we will see that the pathways for both lipid and protein catabolism connect to those used for carbohydrate catabolism, eventually leading into glycolysis, the transition reaction, and the Krebs cycle pathways. Metabolic pathways should be considered to be porous—that is, substances enter from other pathways, and intermediates leave for other pathways. These pathways are not closed systems. Many of the substrates, intermediates, and products in a particular pathway are reactants in other pathways.
Lipid Catabolism
Triglycerides are a form of long-term energy storage in animals. They are made of glycerol and three fatty acids (see Figure 7.3.1). Phospholipids compose the cell and organelle membranes of all organisms except the archaea. Phospholipid structure is similar to triglycerides except that one of the fatty acids is replaced by a phosphorylated head group (see Figure 7.3.2). Triglycerides and phospholipids are broken down first by releasing fatty acid chains (and/or the phosphorylated head group, in the case of phospholipids) from the three-carbon glycerol backbone. The reactions breaking down triglycerides are catalyzed by lipases and those involving phospholipids are catalyzed by phospholipases. These enzymes contribute to the virulence of certain microbes, such as the bacterium Staphylococcus aureus and the fungus Cryptococcus neoformans. These microbes use phospholipases to destroy lipids and phospholipids in host cells and then use the catabolic products for energy (see Virulence Factors of Bacterial and Viral Pathogens).
The resulting products of lipid catabolism, glycerol and fatty acids, can be further degraded. Glycerol can be phosphorylated to glycerol-3-phosphate and easily converted to glyceraldehyde 3-phosphate, which continues through glycolysis. The released fatty acids are catabolized in a process called β-oxidation, which sequentially removes two-carbon acetyl groups from the ends of fatty acid chains, reducing NAD+ and FAD to produce NADH and FADH2, respectively, whose electrons can be used to make ATP by oxidative phosphorylation. The acetyl groups produced during β-oxidation are carried by coenzyme A to the Krebs cycle, and their movement through this cycle results in their degradation to CO2, producing ATP by substrate-level phosphorylation and additional NADH and FADH2 molecules (see Appendix C for a detailed illustration of β-oxidation).
Other types of lipids can also be degraded by certain microbes. For example, the ability of certain pathogens, like Mycobacterium tuberculosis, to degrade cholesterol contributes to their virulence. The side chains of cholesterol can be easily removed enzymatically, but degradation of the remaining fused rings is more problematic. The four fused rings are sequentially broken in a multistep process facilitated by specific enzymes, and the resulting products, including pyruvate, can be further catabolized in the Krebs cycle.
Exercise \(1\)
How can lipases and phospholipases contribute to virulence in microbes?
Protein Catabolism
Proteins are degraded through the concerted action of a variety of microbial protease enzymes. Extracellular proteases cut proteins internally at specific amino acid sequences, breaking them down into smaller peptides that can then be taken up by cells. Some clinically important pathogens can be identified by their ability to produce a specific type of extracellular protease. For example, the production of the extracellular protease gelatinase by members of the genera Proteus and Serratia can be used to distinguish them from other gram-negative enteric bacteria. Following inoculation and growth of microbes in gelatin broth, degradation of the gelatin protein due to gelatinase production prevents solidification of gelatin when refrigerated. Other pathogens can be distinguished by their ability to degrade casein, the main protein found in milk. When grown on skim milk agar, production of the extracellular protease caseinase causes degradation of casein, which appears as a zone of clearing around the microbial growth. Caseinase production by the opportunist pathogen Pseudomonas aeruginosa can be used to distinguish it from other related gram-negative bacteria.
After extracellular protease degradation and uptake of peptides in the cell, the peptides can then be broken down further into individual amino acids by additional intracellular proteases, and each amino acid can be enzymatically deaminated to remove the amino group. The remaining molecules can then enter the transition reaction or the Krebs cycle.
Exercise \(2\)
How can protein catabolism help identify microbes?
Clinical Focus: Part 3
Because bacterial meningitis progresses so rapidly, Hannah’s doctors had decided to treat her aggressively with antibiotics, based on empirical observation of her symptoms. However, laboratory testing to confirm the cause of Hannah’s meningitis was still important for several reasons. N. meningitidis is an infectious pathogen that can be spread from person to person through close contact; therefore, if tests confirm N. meningitidis as the cause of Hannah’s symptoms, Hannah’s parents and others who came into close contact with her might need to be vaccinated or receive prophylactic antibiotics to lower their risk of contracting the disease. On the other hand, if it turns out that N. meningitidis is not the cause, Hannah’s doctors might need to change her treatment.
The clinical laboratory performed a Gram stain on Hannah’s blood and CSF samples. The Gram stain showed the presence of a bean-shaped gram-negative diplococcus. The technician in the hospital lab cultured Hannah’s blood sample on both blood agar and chocolate agar, and the bacterium that grew on both media formed gray, nonhemolytic colonies. Next, he performed an oxidase test on this bacterium and determined that it was oxidase positive. Last, he examined the repertoire of sugars that the bacterium could use as a carbon source and found that the bacterium was positive for glucose and maltose use but negative for lactose and sucrose use. All of these test results are consistent with characteristics of N. meningitidis.
Exercise \(3\)
1. What do these test results tell us about the metabolic pathways of N. meningitidis?
2. Why do you think that the hospital used these biochemical tests for identification in lieu of molecular analysis by DNA testing?
Key Concepts and Summary
• Collectively, microbes have the ability to degrade a wide variety of carbon sources besides carbohydrates, including lipids and proteins. The catabolic pathways for all of these molecules eventually connect into glycolysis and the Krebs cycle.
• Several types of lipids can be microbially degraded. Triglycerides are degraded by extracellular lipases, releasing fatty acids from the glycerol backbone. Phospholipids are degraded by phospholipases, releasing fatty acids and the phosphorylated head group from the glycerol backbone. Lipases and phospholipases act as virulence factors for certain pathogenic microbes.
• Fatty acids can be further degraded inside the cell through β-oxidation, which sequentially removes two-carbon acetyl groups from the ends of fatty acid chains.
• Protein degradation involves extracellular proteases that degrade large proteins into smaller peptides. Detection of the extracellular proteases gelatinase and caseinase can be used to differentiate clinically relevant bacteria. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/08%3A_Microbial_Metabolism/8.05%3A_Catabolism_of_Lipids_and_Proteins.txt |
Learning Objectives
• Describe the function and locations of photosynthetic pigments in eukaryotes and prokaryotes
• Describe the major products of the light-dependent and light-independent reactions
• Describe the reactions that produce glucose in a photosynthetic cell
• Compare and contrast cyclic and noncyclic photophosphorylation
Heterotrophic organisms ranging from E. coli to humans rely on the chemical energy found mainly in carbohydrate molecules. Many of these carbohydrates are produced by photosynthesis, the biochemical process by which phototrophic organisms convert solar energy (sunlight) into chemical energy. Although photosynthesis is most commonly associated with plants, microbial photosynthesis is also a significant supplier of chemical energy, fueling many diverse ecosystems. In this section, we will focus on microbial photosynthesis.
Photosynthesis takes place in two sequential stages: the light-dependent reactions and the light-independent reactions (Figure \(1\)). In the light-dependent reactions, energy from sunlight is absorbed by pigment molecules in photosynthetic membranes and converted into stored chemical energy. In the light-independent reactions, the chemical ener gy produced by the light-dependent reactions is used to drive the assembly of sugar molecules using CO2; however, these reactions are still light dependent because the products of the light-dependent reactions necessary for driving them are short-lived. The light-dependent reactions produce ATP and either NADPH or NADH to temporarily store energy. These energy carriers are used in the light-independent reactions to drive the energetically unfavorable process of “fixing” inorganic CO2 in an organic form, sugar.
Photosynthetic Structures in Eukaryotes and Prokaryotes
In all phototrophic eukaryotes, photosynthesis takes place inside a chloroplast, an organelle that arose in eukaryotes by endosymbiosis of a photosynthetic bacterium (see Unique Characteristics of Eukaryotic Cells). These chloroplasts are enclosed by a double membrane with inner and outer layers. Within the chloroplast is a third membrane that forms stacked, disc-shaped photosynthetic structures called thylakoids (Figure \(2\)). A stack of thylakoids is called a granum, and the space surrounding the granum within the chloroplast is called stroma.
Photosynthetic membranes in prokaryotes, by contrast, are not organized into distinct membrane-enclosed organelles; rather, they are infolded regions of the plasma membrane. In cyanobacteria, for example, these infolded regions are also referred to as thylakoids. In either case, embedded within the thylakoid membranes or other photosynthetic bacterial membranes are photosynthetic pigment molecules organized into one or more photosystems, where light energy is actually converted into chemical energy.
Photosynthetic pigments within the photosynthetic membranes are organized into photosystems, each of which is composed of a light-harvesting (antennae) complex and a reaction center. The light-harvesting complex consists of multiple proteins and associated pigments that each may absorb light energy and, thus, become excited. This energy is transferred from one pigment molecule to another until eventually (after about a millionth of a second) it is delivered to the reaction center. Up to this point, only energy—not electrons—has been transferred between molecules. The reaction center contains a pigment molecule that can undergo oxidation upon excitation, actually giving up an electron. It is at this step in photosynthesis that light energy is converted into an excited electron.
Different kinds of light-harvesting pigments absorb unique patterns of wavelengths (colors) of visible light. Pigments reflect or transmit the wavelengths they cannot absorb, making them appear the corresponding color. Examples of photosynthetic pigments (molecules used to absorb solar energy) are bacteriochlorophylls (green, purple, or red), carotenoids (orange, red, or yellow), chlorophylls (green), phycocyanins (blue), and phycoerythrins (red). By having mixtures of pigments, an organism can absorb energy from more wavelengths. Because photosynthetic bacteria commonly grow in competition for sunlight, each type of photosynthetic bacteria is optimized for harvesting the wavelengths of light to which it is commonly exposed, leading to stratification of microbial communities in aquatic and soil ecosystems by light quality and penetration.
Once the light harvesting complex transfers the energy to the reaction center, the reaction center delivers its high-energy electrons, one by one, to an electron carrier in an electron transport system, and electron transfer through the ETS is initiated. The ETS is similar to that used in cellular respiration and is embedded within the photosynthetic membrane. Ultimately, the electron is used to produce NADH or NADPH. The electrochemical gradient that forms across the photosynthetic membrane is used to generate ATP by chemiosmosis through the process of photophosphorylation, another example of oxidative phosphorylation (Figure \(3\)).
Exercise \(1\)
In a phototrophic eukaryote, where does photosynthesis take place?
Oxygenic and Anoxygenic Photosynthesis
For photosynthesis to continue, the electron lost from the reaction center pigment must be replaced. The source of this electron (H2A) differentiates the oxygenic photosynthesis of plants and cyanobacteria from anoxygenic photosynthesis carried out by other types of bacterial phototrophs (Figure \(4\)). In oxygenic photosynthesis, H2O is split and supplies the electron to the reaction center. Because oxygen is generated as a byproduct and is released, this type of photosynthesis is referred to as oxygenic photosynthesis. However, when other reduced compounds serve as the electron donor, oxygen is not generated; these types of photosynthesis are called anoxygenic photosynthesis. Hydrogen sulfide (H2S) or thiosulfate \(\ce{(S2O3^{2-})}\) can serve as the electron donor, generating elemental sulfur and sulfate \(\ce{(SO4^{2-})}\) ions, respectively, as a result.
Photosystems have been classified into two types: photosystem I (PSI) and photosystem II (PSII) (Figure \(5\)). Cyanobacteria and plant chloroplasts have both photosystems, whereas anoxygenic photosynthetic bacteria use only one of the photosystems. Both photosystems are excited by light energy simultaneously. If the cell requires both ATP and NADPH for biosynthesis, then it will carry out noncyclic photophosphorylation. Upon passing of the PSII reaction center electron to the ETS that connects PSII and PSI, the lost electron from the PSII reaction center is replaced by the splitting of water. The excited PSI reaction center electron is used to reduce NADP+ to NADPH and is replaced by the electron exiting the ETS. The flow of electrons in this way is called the Z-scheme.
If a cell’s need for ATP is significantly greater than its need for NADPH, it may bypass the production of reducing power through cyclic photophosphorylation. Only PSI is used during cyclic photophosphorylation; the high-energy electron of the PSI reaction center is passed to an ETS carrier and then ultimately returns to the oxidized PSI reaction center pigment, thereby reducing it.
Exercise \(2\)
Why would a photosynthetic bacterium have different pigments?
Light-Independent Reactions
After the energy from the sun is converted into chemical energy and temporarily stored in ATP and NADPH molecules (having lifespans of millionths of a second), photoautotrophs have the fuel needed to build multicarbon carbohydrate molecules, which can survive for hundreds of millions of years, for long-term energy storage. The carbon comes from CO2, the gas that is a waste product of cellular respiration.
The Calvin-Benson cycle (named for Melvin Calvin [1911–1997] and Andrew Benson [1917–2015]), the biochemical pathway used for fixation of CO2, is located within the cytoplasm of photosynthetic bacteria and in the stroma of eukaryotic chloroplasts. The light-independent reactions of the Calvin cycle can be organized into three basic stages: fixation, reduction, and regeneration (see Appendix C for a detailed illustration of the Calvin cycle).
• Fixation: The enzyme ribulose bisphosphate carboxylase (RuBisCO) catalyzes the addition of a CO2 to ribulose bisphosphate (RuBP). This results in the production of 3-phosphoglycerate (3-PGA).
• Reduction: Six molecules of both ATP and NADPH (from the light-dependent reactions) are used to convert 3-PGA into glyceraldehyde 3-phosphate (G3P). Some G3P is then used to build glucose.
• Regeneration: The remaining G3P not used to synthesize glucose is used to regenerate RuBP, enabling the system to continue CO2 fixation. Three more molecules of ATP are used in these regeneration reactions.
The Calvin cycle is used extensively by plants and photoautotrophic bacteria, and the enzyme RuBisCO is said to be the most plentiful enzyme on earth, composing 30%–50% of the total soluble protein in plant chloroplasts.1 However, besides its prevalent use in photoautotrophs, the Calvin cycle is also used by many nonphotosynthetic chemoautotrophs to fix CO2. Additionally, other bacteria and archaea use alternative systems for CO2 fixation. Although most bacteria using Calvin cycle alternatives are chemoautotrophic, certain green sulfur photoautotrophic bacteria have been also shown to use an alternative CO2 fixation pathway.
Exercise \(3\)
Describe the three stages of the Calvin cycle.
Key Concepts and Summary
• Heterotrophs depend on the carbohydrates produced by autotrophs, many of which are photosynthetic, converting solar energy into chemical energy.
• Different photosynthetic organisms use different mixtures of photosynthetic pigments, which increase the range of the wavelengths of light an organism can absorb.
• Photosystems (PSI and PSII) each contain a light-harvesting complex, composed of multiple proteins and associated pigments that absorb light energy. The light-dependent reactions of photosynthesis convert solar energy into chemical energy, producing ATP and NADPH or NADH to temporarily store this energy.
• In oxygenic photosynthesis, H2O serves as the electron donor to replace the reaction center electron, and oxygen is formed as a byproduct. In anoxygenic photosynthesis, other reduced molecules like H2S or thiosulfate may be used as the electron donor; as such, oxygen is not formed as a byproduct.
• Noncyclic photophosphorylation is used in oxygenic photosynthesis when there is a need for both ATP and NADPH production. If a cell’s needs for ATP outweigh its needs for NADPH, then it may carry out cyclic photophosphorylation instead, producing only ATP.
• The light-independent reactions of photosynthesis use the ATP and NADPH from the light-dependent reactions to fix CO2 into organic sugar molecules.
Footnotes
1. 1 A. Dhingra et al. “Enhanced Translation of a Chloroplast-Expressed RbcS Gene Restores Small Subunit Levels and Photosynthesis in Nuclear RbcS Antisense Plants.” Proceedings of the National Academy of Sciences of the United States of America 101 no. 16 (2004):6315–6320. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/08%3A_Microbial_Metabolism/8.06%3A_Photosynthesis_and_the_Importance_of_Light.txt |
Learning Objectives
• Define and describe the importance of microorganisms in the biogeochemical cycles of carbon, nitrogen, and sulfur
• Define and give an example of bioremediation
Energy flows directionally through ecosystems, entering as sunlight for phototrophs or as inorganic molecules for chemoautotrophs. The six most common elements associated with organic molecules—carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur—take a variety of chemical forms and may exist for long periods in the atmosphere, on land, in water, or beneath earth’s surface. Geologic processes, such as erosion, water drainage, the movement of the continental plates, and weathering, all are involved in the cycling of elements on earth. Because geology and chemistry have major roles in the study of this process, the recycling of inorganic matter between living organisms and their nonliving environment is called a biogeochemical cycle. Here, we will focus on the function of microorganisms in these cycles, which play roles at each step, most frequently interconverting oxidized versions of molecules with reduced ones.
Carbon Cycle
Carbon is one of the most important elements to living organisms, as shown by its abundance and presence in all organic molecules. The carbon cycle exemplifies the connection between organisms in various ecosystems. Carbon is exchanged between heterotrophs and autotrophs within and between ecosystems primarily by way of atmospheric CO2, a fully oxidized version of carbon that serves as the basic building block that autotrophs use to build multicarbon, high-energy organic molecules such as glucose. Photoautotrophs and chemoautotrophs harness energy from the sun and from inorganic chemical compounds, respectively, to covalently bond carbon atoms together into reduced organic compounds whose energy can be later accessed through the processes of respiration and fermentation (Figure \(1\)).
Overall, there is a constant exchange of CO2 between the heterotrophs (which produce CO2 as a result of respiration or fermentation) and the autotrophs (which use the CO2 for fixation). Autotrophs also respire or ferment, consuming the organic molecules they form; they do not fix carbon for heterotrophs, but rather use it for their own metabolic needs.
Bacteria and archaea that use methane as their carbon source are called methanotrophs. Reduced one-carbon compounds like methane accumulate in certain anaerobic environments when CO2 is used as a terminal electron acceptor in anaerobic respiration by archaea called methanogens. Some methanogens also ferment acetate (carbons) to produce methane and CO2. Methane accumulation due to methanogenesis occurs in both natural anaerobic soil and aquatic environments; methane accumulation also occurs as a result of animal husbandry because methanogens are members of the normal microbiota of ruminants. Environmental methane accumulation due to methanogenesis is of consequence because it is a strong greenhouse gas, and methanotrophs help to reduce atmospheric methane levels.
Exercise \(1\)
Describe the interaction between heterotrophs and autotrophs in the carbon cycle.
Nitrogen Cycle
Many biological macromolecules, including proteins and nucleic acids, contain nitrogen; however, getting nitrogen into living organisms is difficult. Prokaryotes play essential roles in the nitrogen cycle (Figure \(2\)), transforming nitrogen between various forms for their own needs, benefiting other organisms indirectly. Plants and phytoplankton cannot incorporate nitrogen from the atmosphere (where it exists as tightly bonded, triple covalent N2), even though this molecule composes approximately 78% of the atmosphere. Nitrogen enters the living world through free-living and symbiotic bacteria, which incorporate nitrogen into their macromolecules through specialized biochemical pathways called nitrogen fixation. Cyanobacteria in aquatic ecosystems fix inorganic nitrogen (from nitrogen gas) into ammonia (NH3) that can be easily incorporated into biological macromolecules. Rhizobium bacteria (Figure 8.1) also fix nitrogen and live symbiotically in the root nodules of legumes (such as beans, peanuts, and peas), providing them with needed organic nitrogen while receiving fixed carbon as sugar in exchange. Free-living bacteria, such as members of the genus Azotobacter, are also able to fix nitrogen.
The nitrogen that enters living systems by nitrogen fixation is eventually converted from organic nitrogen back into nitrogen gas by microbes through three steps: ammonification, nitrification, and denitrification. In terrestrial systems, the first step is the ammonification process, in which certain bacteria and fungi convert nitrogenous waste from living animals or from the remains of dead organisms into ammonia (NH3). This ammonia is then oxidized to nitrite \((\ce{NO2-})\), then to nitrate \((\ce{NO3-})\), by nitrifying soil bacteria such as members of the genus Nitrosomonas, through the process of nitrification. Last, the process of denitrification occurs, whereby soil bacteria, such as members of the genera Pseudomonas and Clostridium, use nitrate as a terminal electron acceptor in anaerobic respiration, converting it into nitrogen gas that reenters the atmosphere. A similar process occurs in the marine nitrogen cycle, where these three processes are performed by marine bacteria and archaea.
Human activity releases nitrogen into the environment by the use of artificial fertilizers that contain nitrogen and phosphorus compounds, which are then washed into lakes, rivers, and streams by surface runoff. A major effect from fertilizer runoff is saltwater and freshwater eutrophication, in which nutrient runoff causes the overgrowth and subsequent death of aquatic algae, making water sources anaerobic and inhospitable for the survival of aquatic organisms.
Exercise \(2\)
What are the three steps of the nitrogen cycle?
Link to Learning
To learn more about the nitrogen cycle, visit the PBS website.
Sulfur Cycle
Sulfur is an essential element for the macromolecules of living organisms. As part of the amino acids cysteine and methionine, it is involved in the formation of proteins. It is also found in several vitamins necessary for the synthesis of important biological molecules like coenzyme A. Several groups of microbes are responsible for carrying out processes involved in the sulfur cycle (Figure \(3\)). Anoxygenic photosynthetic bacteria as well as chemoautotrophic archaea and bacteria use hydrogen sulfide as an electron donor, oxidizing it first to elemental sulfur (S0), then to sulfate \((\ce{SO4^2-})\).This leads to stratification of hydrogen sulfide in soil, with levels increasing at deeper, more anaerobic depths.
Many bacteria and plants can use sulfate as a sulfur source. Decomposition dead organisms by fungi and bacteria remove sulfur groups from amino acids, producing hydrogen sulfide, returning inorganic sulfur to the environment.
Exercise \(3\)
Which groups of microbes carry out the sulfur cycle?
Other Biogeochemical Cycles
Beyond their involvement in the carbon, nitrogen, and sulfur cycles, prokaryotes are involved in other biogeochemical cycles as well. Like the carbon, nitrogen, and sulfur cycles, several of these additional biogeochemical cycles, such as the iron (Fe), manganese (Mn), and chromium (Cr) cycles, also involve redox chemistry, with prokaryotes playing roles in both oxidation and reduction. Several other elements undergo chemical cycles that do not involve redox chemistry. Examples of these are phosphorus (P), calcium (Ca), and silica (Si) cycles. The cycling of these elements is particularly important in oceans because large quantities of these elements are incorporated into the exoskeletons of marine organisms. These biogeochemical cycles do not involve redox chemistry but instead involve fluctuations in the solubility of compounds containing calcium, phosphorous, and silica. The overgrowth of naturally occurring microbial communities is typically limited by the availability of nitrogen (as previously mentioned), phosphorus, and iron. Human activities introducing excessive amounts of iron, nitrogen, or phosphorus (typically from detergents) may lead to eutrophication.
Bioremediation
Microbial bioremediation leverages microbial metabolism to remove xenobiotics or other pollutants. Xenobiotics are compounds synthesized by humans and introduced into the environment in much higher concentrations than would naturally occur. Such environmental contamination may involve adhesives, dyes, flame retardants, lubricants, oil and petroleum products, organic solvents, pesticides, and products of the combustion of gasoline and oil. Many xenobiotics resist breakdown, and some accumulate in the food chain after being consumed or absorbed by fish and wildlife, which, in turn, may be eaten by humans. Of particular concern are contaminants like polycyclic aromatic hydrocarbon (PAH), a carcinogenic xenobiotic found in crude oil, and trichloroethylene (TCE), a common groundwater contaminant.
Bioremediation processes can be categorized as in situ or ex situ. Bioremediation conducted at the site of contamination is called in situ bioremediation and does not involve movement of contaminated material. In contrast, ex situ bioremediation involves the removal of contaminated material from the original site so that it can be treated elsewhere, typically in a large, lined pit where conditions are optimized for degradation of the contaminant.
Some bioremediation processes rely on microorganisms that are indigenous to the contaminated site or material. Enhanced bioremediation techniques, which may be applied to either in situ or ex situ processing, involve the addition of nutrients and/or air to encourage the growth of pollution-degrading microbes; they may also involve the addition of non-native microbes known for their ability to degrade contaminants. For example, certain bacteria of the genera Rhodococcus and Pseudomonas are known for their ability to degrade many environmental contaminants, including aromatic compounds like those found in oil, down to CO2. The genes encoding their degradatory enzymes are commonly found on plasmids. Others, like Alcanivorax borkumensis, produce surfactants that are useful in the solubilization of the hydrophobic molecules found in oil, making them more accessible to other microbes for degradation.
Exercise \(4\)
Compare and contrast the benefits of in situ and ex situ bioremediation.
Clinical Focus: Resolution
Although there is a DNA test specific for Neisseria meningitidis, it is not practical for use in some developing countries because it requires expensive equipment and a high level of expertise to perform. The hospital in Banjul was not equipped to perform DNA testing. Biochemical testing, however, is much less expensive and is still effective for microbial identification.
Fortunately for Hannah, her symptoms began to resolve with antibiotic therapy. Patients who survive bacterial meningitis often suffer from long-term complications such as brain damage, hearing loss, and seizures, but after several weeks of recovery, Hannah did not seem to be exhibiting any long-term effects and her behavior returned to normal. Because of her age, her parents were advised to monitor her closely for any signs of developmental issues and have her regularly evaluated by her pediatrician.
N. meningitidis is found in the normal respiratory microbiota in 10%–20% of the human population.1 In most cases, it does not cause disease, but for reasons not fully understood, the bacterium can sometimes invade the bloodstream and cause infections in other areas of the body, including the brain. The disease is more common in infants and children, like Hannah.
The prevalence of meningitis caused by N. meningitidis is particularly high in the so-called meningitis belt, a region of sub-Saharan African that includes 26 countries stretching from Senegal to Ethiopia (Figure \(4\)). The reasons for this high prevalence are not clear, but several factors may contribute to higher rates of transmission, such as the dry, dusty climate; overcrowding and low standards of living; and the relatively low immunocompetence and nutritional status of the population.2 A vaccine against four bacterial strains of N. meningitidis is available. Vaccination is recommended for 11- and 12-year-old children, with a booster at age 16 years. Vaccination is also recommended for young people who live in close quarters with others (e.g., college dormitories, military barracks), where the disease is more easily transmitted. Travelers visiting the “meningitis belt” should also be vaccinated, especially during the dry season (December through June) when the prevalence is highest.34
Key Concepts and Summary
• The recycling of inorganic matter between living organisms and their nonliving environment is called a biogeochemical cycle. Microbes play significant roles in these cycles.
• In the carbon cycle, heterotrophs degrade reduced organic molecule to produce carbon dioxide, whereas autotrophs fix carbon dioxide to produce organics. Methanogens typically form methane by using CO2 as a final electron acceptor during anaerobic respiration; methanotrophs oxidize the methane, using it as their carbon source.
• In the nitrogen cycle, nitrogen-fixing bacteria convert atmospheric nitrogen into ammonia (ammonification). The ammonia can then be oxidized to nitrite and nitrate (nitrification). Nitrates can then be assimilated by plants. Soil bacteria convert nitrate back to nitrogen gas (denitrification).
• In sulfur cycling, many anoxygenic photosynthesizers and chemoautotrophs use hydrogen sulfide as an electron donor, producing elemental sulfur and then sulfate; sulfate-reducing bacteria and archaea then use sulfate as a final electron acceptor in anaerobic respiration, converting it back to hydrogen sulfide.
• Human activities that introduce excessive amounts of naturally limited nutrients (like iron, nitrogen, or phosphorus) to aquatic systems may lead to eutrophication.
• Microbial bioremediation is the use of microbial metabolism to remove or degrade xenobiotics and other environmental contaminants and pollutants. Enhanced bioremediation techniques may involve the introduction of non-native microbes specifically chosen or engineered for their ability to degrade contaminants.
Footnotes
1. 1 Centers for Disease Control and Prevention. “Meningococcal Disease: Causes and Transmission.” www.cdc.gov/meningococcal/abo...nsmission.html. Accessed September 12, 2016.
2. 2 Centers for Disease Control and Prevention. “Meningococcal Disease in Other Countries.” http://www.cdc.gov/meningococcal/global.html. Accessed September 12, 2016.
3. 3 Centers for Disease Control and Prevention. “Health Information for Travelers to the Gambia: Traveler View.” wwwnc.cdc.gov/travel/destinat...one/the-gambia. Accessed September 12, 2016.
4. 4 Centers for Disease Control and Prevention. “Meningococcal: Who Needs to Be Vaccinated?” www.cdc.gov/vaccines/vpd-vac/...-vaccinate.htm. Accessed September 12, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/08%3A_Microbial_Metabolism/8.07%3A_Biogeochemical_Cycles.txt |
8.1: Energy, Matter, and Enzymes
Cellular processes such as the building or breaking down of complex molecules occur through series of stepwise, interconnected chemical reactions called metabolic pathways. The term anabolism refers to those endergonic metabolic pathways involved in biosynthesis, converting simple molecular building blocks into more complex molecules, and fueled by the use of cellular energy.
Multiple Choice
Which of the following is an organism that obtains its energy from the transfer of electrons originating from chemical compounds and its carbon from an inorganic source?
1. chemoautotroph
2. chemoheterotroph
3. photoheterotroph
4. photoautotroph
Answer
A
Which of the following molecules is reduced?
1. NAD+
2. FAD
3. O2
4. NADPH
Answer
D
Enzymes work by which of the following?
1. increasing the activation energy
2. reducing the activation energy
3. making exergonic reactions endergonic
4. making endergonic reactions exergonic
Answer
B
To which of the following does a competitive inhibitor most structurally resemble?
1. the active site
2. the allosteric site
3. the substrate
4. a coenzyme
Answer
C
Which of the following are organic molecules that help enzymes work correctly?
1. cofactors
2. coenzymes
3. holoenzymes
4. apoenzymes
Answer
B
Fill in the Blank
Processes in which cellular energy is used to make complex molecules from simpler ones are described as ________.
Answer
anabolic
The loss of an electron from a molecule is called ________.
Answer
oxidation
The part of an enzyme to which a substrate binds is called the ________.
Answer
active site
True/False
Competitive inhibitors bind to allosteric sites.
Answer
False
Short Answer
In cells, can an oxidation reaction happen in the absence of a reduction reaction? Explain.
What is the function of molecules like NAD+/NADH and FAD/FADH2 in cells?
8.2: Catabolism of Carbohydrates
Glycolysis is the first step in the breakdown of glucose, resulting in the formation of ATP, which is produced by substrate-level phosphorylation; NADH; and two pyruvate molecules. Glycolysis does not use oxygen and is not oxygen dependent. After glycolysis, a three-carbon pyruvate is decarboxylated to form a two-carbon acetyl group, coupled with the formation of NADH. The acetyl group is attached to a large carrier compound called coenzyme A.
Multiple Choice
During which of the following is ATP not made by substrate-level phosphorylation?
1. Embden-Meyerhof pathway
2. Transition reaction
3. Krebs cycle
4. Entner-Doudoroff pathway
Answer
B
Which of the following products is made during Embden-Meyerhof glycolysis?
1. NAD+
2. pyruvate
3. CO2
4. two-carbon acetyl
Answer
B
During the catabolism of glucose, which of the following is produced only in the Krebs cycle?
1. ATP
2. NADH
3. NADPH
4. FADH2
Answer
D
Which of the following is not a name for the cycle resulting in the conversion of a two-carbon acetyl to one ATP, two CO2, one FADH2, and three NADH molecules?
1. Krebs cycle
2. tricarboxylic acid cycle
3. Calvin cycle
4. citric acid cycle
Answer
C
True/False
Glycolysis requires oxygen or another inorganic final electron acceptor to proceed.
Answer
False
Fill in the Blank
Per turn of the Krebs cycle, one acetyl is oxidized, forming ____ CO2, ____ ATP, ____ NADH, and ____ FADH2molecules.
Answer
2; 1; 3; 1
Most commonly, glycolysis occurs by the ________ pathway.
Answer
Embden-Meyerhof
Short Answer
What is substrate-level phosphorylation? When does it occur during the breakdown of glucose to CO2?
Why is the Krebs cycle important in both catabolism and anabolism?
Critical Thinking
What would be the consequences to a cell of having a mutation that knocks out coenzyme A synthesis?
8.3: Cellular Respiration
Cellular respiration begins when electrons are transferred from NADH and FADH2—through a series of chemical reactions to a final inorganic electron acceptor (either oxygen in aerobic respiration or non-oxygen inorganic molecules in anaerobic respiration). These electron transfers take place on the inner part of the cell membrane of prokaryotic cells or in specialized protein complexes in the inner membrane of the mitochondria of eukaryotic cells.
Multiple Choice
Which is the location of electron transports systems in prokaryotes?
1. the outer mitochondrial membrane
2. the cytoplasm
3. the inner mitochondrial membrane
4. the cytoplasmic membrane
Answer
D
Which is the source of the energy used to make ATP by oxidative phosphorylation?
1. oxygen
2. high-energy phosphate bonds
3. the proton motive force
4. Pi
Answer
C
A cell might perform anaerobic respiration for which of the following reasons?
1. It lacks glucose for degradation.
2. It lacks the transition reaction to convert pyruvate to acetyl-CoA.
3. It lacks Krebs cycle enzymes for processing acetyl-CoA to CO2.
4. It lacks a cytochrome oxidase for passing electrons to oxygen.
Answer
D
In prokaryotes, which of the following is true?
1. As electrons are transferred through an ETS, H+ is pumped out of the cell.
2. As electrons are transferred through an ETS, H+ is pumped into the cell.
3. As protons are transferred through an ETS, electrons are pumped out of the cell.
4. As protons are transferred through an ETS, electrons are pumped into the cell.
Answer
A
Which of the following is not an electron carrier within an electron transport system?
1. flavoprotein
2. ATP synthase
3. ubiquinone
4. cytochrome oxidase
Answer
B
Fill in the Blank
The final ETS complex used in aerobic respiration that transfers energy-depleted electrons to oxygen to form H2O is called ________.
Answer
cytochrome oxidase
The passage of hydrogen ions through ________ down their electrochemical gradient harnesses the energy needed for ATP synthesis by oxidative phosphorylation.
Answer
ATP synthase
True/False
All organisms that use aerobic cellular respiration have cytochrome oxidase.
Answer
True
Short Answer
What is the relationship between chemiosmosis and the proton motive force?
How does oxidative phosphorylation differ from substrate-level phosphorylation?
How does the location of ATP synthase differ between prokaryotes and eukaryotes? Where do protons accumulate as a result of the ETS in each cell type?
8.4: Fermentation
Fermentation uses an organic molecule as a final electron acceptor to regenerate NAD+ from NADH so that glycolysis can continue. Fermentation does not involve an electron transport system, and no ATP is made by the fermentation process directly. Fermenters make very little ATP—only two ATP molecules per glucose molecule during glycolysis. Microbial fermentation processes have been used for the production of foods and pharmaceuticals, and for the identification of microbes.
Multiple Choice
Which of the following is the purpose of fermentation?
1. to make ATP
2. to make carbon molecule intermediates for anabolism
3. to make NADH
4. to make NAD+
Answer
D
Which molecule typically serves as the final electron acceptor during fermentation?
1. oxygen
2. NAD+
3. pyruvate
4. CO2
Answer
C
Which fermentation product is important for making bread rise?
1. ethanol
2. CO2
3. lactic acid
4. hydrogen gas
Answer
B
Which of the following is not a commercially important fermentation product?
1. ethanol
2. pyruvate
3. butanol
4. penicillin
Answer
B
Fill in the Blank
The microbe responsible for ethanol fermentation for the purpose of producing alcoholic beverages is ________.
Answer
yeast (Saccharomyces cerevisiae)
________ results in the production of a mixture of fermentation products, including lactic acid, ethanol and/or acetic acid, and CO2.
Answer
Heterolactic fermentation
Fermenting organisms make ATP through the process of ________.
Answer
glycolysis
Matching
Match the fermentation pathway with the correct commercial product it is used to produce:
___acetone-butanol-ethanol fermentation a. bread
___alcohol fermentation b. pharmaceuticals
___lactic acid fermentation c. Swiss cheese
___mixed acid fermentation d. yogurt
___propionic acid fermentation e. industrial solvents
Answer
e; 2. a; 3. d; 4. b; 5. c
Short Answer
Why are some microbes, including Streptococcus spp., unable to perform aerobic respiration, even in the presence of oxygen?
How can fermentation be used to differentiate various types of microbes?
Critical Thinking
The bacterium E. coli is capable of performing aerobic respiration, anaerobic respiration, and fermentation. When would it perform each process and why? How is ATP made in each case?
8.5: Catabolism of Lipids and Proteins
Collectively, microbes have the ability to degrade a wide variety of carbon sources besides carbohydrates, including lipids and proteins. The catabolic pathways for all of these molecules eventually connect into glycolysis and the Krebs cycle. Several types of lipids can be microbially degraded. Triglycerides are degraded by extracellular lipases, releasing fatty acids from the glycerol backbone. Phospholipids are degraded by phospholipases, releasing fatty acids and phosphorylated head groups.
Multiple Choice
Which of the following molecules is not produced during the breakdown of phospholipids?
1. glucose
2. glycerol
3. acetyl groups
4. fatty acids
Answer
A
Caseinase is which type of enzyme?
1. phospholipase
2. lipase
3. extracellular protease
4. intracellular protease
Answer
C
Which of the following is the first step in triglyceride degradation?
1. removal of fatty acids
2. β-oxidation
3. breakage of fused rings
4. formation of smaller peptides
Answer
A
Fill in the Blank
The process by which two-carbon units are sequentially removed from fatty acids, producing acetyl-CoA, FADH2, and NADH is called ________.
Answer
β-oxidation
The NADH and FADH2 produced during β-oxidation are used to make ________.
Answer
ATP by oxidative phosphorylation
________ is a type of medium used to detect the production of an extracellular protease called caseinase.
Answer
Skim milk agar
Short Answer
How are the products of lipid and protein degradation connected to glucose metabolism pathways?
What is the general strategy used by microbes for the degradation of macromolecules?
Critical Thinking
Do you think that β-oxidation can occur in an organism incapable of cellular respiration? Why or why not?
8.6: Photosynthesis and the Importance of Light
Heterotrophic organisms ranging from E. coli to humans rely on the chemical energy found mainly in carbohydrate molecules. Many of these carbohydrates are produced by photosynthesis, the biochemical process by which phototrophic organisms convert solar energy (sunlight) into chemical energy. Although photosynthesis is most commonly associated with plants, microbial photosynthesis is also a significant supplier of chemical energy, fueling many diverse ecosystems.
Multiple Choice
During the light-dependent reactions, which molecule loses an electron?
1. a light-harvesting pigment molecule
2. a reaction center pigment molecule
3. NADPH
4. 3-phosphoglycerate
Answer
B
In prokaryotes, in which direction are hydrogen ions pumped by the electron transport system of photosynthetic membranes?
1. to the outside of the plasma membrane
2. to the inside (cytoplasm) of the cell
3. to the stroma
4. to the intermembrane space of the chloroplast
Answer
A
Which of the following does not occur during cyclic photophosphorylation in cyanobacteria?
1. electron transport through an ETS
2. photosystem I use
3. ATP synthesis
4. NADPH formation
Answer
D
Which are two products of the light-dependent reactions are ________.
1. glucose and NADPH
2. NADPH and ATP
3. glyceraldehyde 3-phosphate and CO2
4. glucose and oxygen
Answer
B
True/False
Photosynthesis always results in the formation of oxygen.
Answer
False
Fill in the Blank
The enzyme responsible for CO2 fixation during the Calvin cycle is called ________.
Answer
ribulose bisphosphate carboxylase (RuBisCO)
The types of pigment molecules found in plants, algae, and cyanobacteria are ________ and ________.
Answer
chlorophylls and carotenoids
Short Answer
Why would an organism perform cyclic phosphorylation instead of noncyclic phosphorylation?
What is the function of photosynthetic pigments in the light-harvesting complex?
Critical Thinking
Is life dependent on the carbon fixation that occurs during the light-independent reactions of photosynthesis? Explain.
8.7: Biogeochemical Cycles
Energy flows directionally through ecosystems, entering as sunlight for phototrophs or as inorganic molecules for chemoautotrophs. The six most common elements associated with organic molecules—carbon, hydrogen, nitrogen, oxygen, phosphorus, and sulfur—take a variety of chemical forms and may exist for long periods in the atmosphere, on land, in water, or beneath earth’s surface.
Multiple Choice
Which of the following is the group of archaea that can use CO2 as their final electron acceptor during anaerobic respiration, producing CH4?
1. methylotrophs
2. methanotrophs
3. methanogens
4. anoxygenic photosynthesizers
Answer
C
Which of the following processes is not involved in the conversion of organic nitrogen to nitrogen gas?
1. nitrogen fixation
2. ammonification
3. nitrification
4. denitrification
Answer
A
Which of the following processes produces hydrogen sulfide?
1. anoxygenic photosynthesis
2. oxygenic photosynthesis
3. anaerobic respiration
4. chemoautrophy
Answer
C
The biogeochemical cycle of which of the following elements is based on changes in solubility rather than redox chemistry?
1. carbon
2. sulfur
3. nitrogen
4. phosphorus
Answer
D
Fill in the Blank
The molecule central to the carbon cycle that is exchanged within and between ecosystems, being produced by heterotrophs and used by autotrophs, is ________.
Answer
carbon dioxide
The use of microbes to remove pollutants from a contaminated system is called ________.
Answer
bioremediation
True/False
There are many naturally occurring microbes that have the ability to degrade several of the compounds found in oil.
Answer
True
Short Answer
Why must autotrophic organisms also respire or ferment in addition to fixing CO2?
How can human activity lead to eutrophication?
Critical Thinking
In considering the symbiotic relationship between Rhizobium species and their plant hosts, what metabolic activity does each organism perform that benefits the other member of the pair? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/08%3A_Microbial_Metabolism/8.E%3A_Microbial_Metabolism_%28Exercises%29.txt |
We are all familiar with the slimy layer on a pond surface or that makes rocks slippery. These are examples of biofilms—microorganisms embedded in thin layers of matrix material (Figure \(1\)). Biofilms were long considered random assemblages of cells and had little attention from researchers. Recently, progress in visualization and biochemical methods has revealed that biofilms are an organized ecosystem within which many cells, usually of different species of bacteria, fungi, and algae, interact through cell signaling and coordinated responses. The biofilm provides a protected environment in harsh conditions and aids colonization by microorganisms. Biofilms also have clinical importance. They form on medical devices, resist routine cleaning and sterilization, and cause health-acquired infections. Within the body, biofilms form on the teeth as plaque, in the lungs of patients with cystic fibrosis, and on the cardiac tissue of patients with endocarditis. The slime layer helps protect the cells from host immune defenses and antibiotic treatments.
Studying biofilms requires new approaches. Because of the cells’ adhesion properties, many of the methods for culturing and counting cells that are explored in this chapter are not easily applied to biofilms. This is the beginning of a new era of challenges and rewarding insight into the ways that microorganisms grow and thrive in nature.
• 9.1: How Microbes Grow
The bacterial cell cycle involves the formation of new cells through the replication of DNA and partitioning of cellular components into two daughter cells. In prokaryotes, reproduction is always asexual, although extensive genetic recombination in the form of horizontal gene transfer takes place, as will be explored in a different chapter. Most bacteria have a single circular chromosome; however, some exceptions exist.
• 9.2: Oxygen Requirements for Microbial Growth
Ask most people “What are the major requirements for life?” and the answers are likely to include water and oxygen. Few would argue about the need for water, but what about oxygen? Can there be life without oxygen? The answer is that molecular oxygen is not always needed. The earliest signs of life are dated to a period when conditions on earth were highly reducing and free oxygen gas was essentially nonexistent.
• 9.3: The Effects of pH on Microbial Growth
Bacteria are generally neutrophiles. They grow best at neutral pH close to 7.0. Acidophiles grow optimally at a pH near 3.0. Alkaliphiles are organisms that grow optimally between a pH of 8 and 10.5. Extreme acidophiles and alkaliphiles grow slowly or not at all near neutral pH. Microorganisms grow best at their optimum growth pH. Growth occurs slowly or not at all below the minimum growth pH and above the maximum growth pH.
• 9.4: Temperature and Microbial Growth
Microorganisms thrive at a wide range of temperatures; they have colonized different natural environments and have adapted to extreme temperatures. Both extreme cold and hot temperatures require evolutionary adjustments to macromolecules and biological processes. Psychrophiles grow best in the temperature range of 0–15 °C whereas psychrotrophs thrive between 4 °C and 25 °C. Mesophiles grow best at moderate temperatures in the range of 20 °C to about 45 °C. Pathogens are usually mesophiles.
• 9.5: Other Environmental Conditions that Affect Growth
Microorganisms interact with their environment along more dimensions than pH, temperature, and free oxygen levels, although these factors require significant adaptations. We also find microorganisms adapted to varying levels of salinity, barometric pressure, humidity, and light.
• 9.6: Media Used for Bacterial Growth
The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive under laboratory conditions. Culturing many microorganisms is challenging because of highly specific nutritional and environmental requirements and the diversity of these requirements among different species.
• 9.E: Microbial Growth (Exercises)
Thumbnail: Heavy rains cause runoff of fertilizers into Lake Erie, triggering extensive algal blooms, which can be observed along the shoreline. Notice the brown unplanted and green planted agricultural land on the shore. (credit: NASA)
09: Microbial Growth
Learning Objectives
• Define the generation time for growth based on binary fission
• Identify and describe the activities of microorganisms undergoing typical phases of binary fission (simple cell division) in a growth curve
• Explain several laboratory methods used to determine viable and total cell counts in populations undergoing exponential growth
• Describe examples of cell division not involving binary fission, such as budding or fragmentation
• Describe the formation and characteristics of biofilms
• Identify health risks associated with biofilms and how they are addressed
• Describe quorum sensing and its role in cell-to-cell communication and coordination of cellular activities
Clinical Focus: Part 1
Jeni, a 24-year-old pregnant woman in her second trimester, visits a clinic with complaints of high fever, 38.9 °C (102 °F), fatigue, and muscle aches—typical flu-like signs and symptoms. Jeni exercises regularly and follows a nutritious diet with emphasis on organic foods, including raw milk that she purchases from a local farmer’s market. All of her immunizations are up to date. However, the health-care provider who sees Jeni is concerned and orders a blood sample to be sent for testing by the microbiology laboratory.
Exercise \(1\)
Why is the health-care provider concerned about Jeni’s signs and symptoms?
The bacterial cell cycle involves the formation of new cells through the replication of DNA and partitioning of cellular components into two daughter cells. In prokaryotes, reproduction is always asexual, although extensive genetic recombination in the form of horizontal gene transfer takes place, as will be explored in a different chapter. Most bacteria have a single circular chromosome; however, some exceptions exist. For example, Borrelia burgdorferi, the causative agent of Lyme disease, has a linear chromosome.
Binary Fission
The most common mechanism of cell replication in bacteria is a process called binary fission, which is depicted in Figure \(1\):. Before dividing, the cell grows and increases its number of cellular components. Next, the replication of DNA starts at a location on the circular chromosome called the origin of replication, where the chromosome is attached to the inner cell membrane. Replication continues in opposite directions along the chromosome until the terminus is reached.
The center of the enlarged cell constricts until two daughter cells are formed, each offspring receiving a complete copy of the parental genome and a division of the cytoplasm (cytokinesis). This process of cytokinesis and cell division is directed by a protein called FtsZ. FtsZ assembles into a Z ring on the cytoplasmic membrane (Figure \(2\)). The Z ring is anchored by FtsZ-binding proteins and defines the division plane between the two daughter cells. Additional proteins required for cell division are added to the Z ring to form a structure called the divisome. The divisome activates to produce a peptidoglycan cell wall and build a septum that divides the two daughter cells. The daughter cells are separated by the division septum, where all of the cells’ outer layers (the cell wall and outer membranes, if present) must be remodeled to complete division. For example, we know that specific enzymes break bonds between the monomers in peptidoglycans and allow addition of new subunits along the division septum.
Exercise \(2\)
What is the name of the protein that assembles into a Z ring to initiate cytokinesis and cell division?
Generation Time
In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two successive generations. For example, the typical generation time for the human population is 25 years. This definition is not practical for bacteria, which may reproduce rapidly or remain dormant for thousands of years. In prokaryotes (Bacteria and Archaea), the generation time is also called the doubling time and is defined as the time it takes for the population to double through one round of binary fission. Bacterial doubling times vary enormously. Whereas Escherichia coli can double in as little as 20 minutes under optimal growth conditions in the laboratory, bacteria of the same species may need several days to double in especially harsh environments. Most pathogens grow rapidly, like E. coli, but there are exceptions. For example, Mycobacterium tuberculosis, the causative agent of tuberculosis, has a generation time of between 15 and 20 hours. On the other hand, M. leprae, which causes Hansen’s disease (leprosy), grows much more slowly, with a doubling time of 14 days.
Calculating Number of Cells
It is possible to predict the number of cells in a population when they divide by binary fission at a constant rate. As an example, consider what happens if a single cell divides every 30 minutes for 24 hours. The diagram in Figure \(3\) shows the increase in cell numbers for the first three generations.
The number of cells increases exponentially and can be expressed as 2n, where n is the number of generations. If cells divide every 30 minutes, after 24 hours, 48 divisions would have taken place. If we apply the formula 2n, where n is equal to 48, the single cell would give rise to 248 or 281,474,976,710,656 cells at 48 generations (24 hours). When dealing with such huge numbers, it is more practical to use scientific notation. Therefore, we express the number of cells as 2.8 × 1014 cells.
In our example, we used one cell as the initial number of cells. For any number of starting cells, the formula is adapted as follows:
\[N_n = N_02^n\]
Nn is the number of cells at any generation n, N0 is the initial number of cells, and n is the number of generations.
Exercise \(3\)
With a doubling time of 30 minutes and a starting population size of 1 × 105 cells, how many cells will be present after 2 hours, assuming no cell death?
The Growth Curve
Microorganisms grown in closed culture (also known as a batch culture), in which no nutrients are added and most waste is not removed, follow a reproducible growth pattern referred to as the growth curve. An example of a batch culture in nature is a pond in which a small number of cells grow in a closed environment. The culture density is defined as the number of cells per unit volume. In a closed environment, the culture density is also a measure of the number of cells in the population. Infections of the body do not always follow the growth curve, but correlations can exist depending upon the site and type of infection. When the number of live cells is plotted against time, distinct phases can be observed in the curve (Figure \(4\)).
The Lag Phase
The beginning of the growth curve represents a small number of cells, referred to as an inoculum, that are added to a fresh culture medium, a nutritional broth that supports growth. The initial phase of the growth curve is called the lag phase, during which cells are gearing up for the next phase of growth. The number of cells does not change during the lag phase; however, cells grow larger and are metabolically active, synthesizing proteins needed to grow within the medium. If any cells were damaged or shocked during the transfer to the new medium, repair takes place during the lag phase. The duration of the lag phase is determined by many factors, including the species and genetic make-up of the cells, the composition of the medium, and the size of the original inoculum.
The Log Phase
In the logarithmic (log) growth phase, sometimes called exponential growth phase, the cells are actively dividing by binary fission and their number increases exponentially. For any given bacterial species, the generation time under specific growth conditions (nutrients, temperature, pH, and so forth) is genetically determined, and this generation time is called the intrinsic growth rate. During the log phase, the relationship between time and number of cells is not linear but exponential; however, the growth curve is often plotted on a semilogarithmic graph, as shown in Figure \(5\), which gives the appearance of a linear relationship.
Cells in the log phase show constant growth rate and uniform metabolic activity. For this reason, cells in the log phase are preferentially used for industrial applications and research work. The log phase is also the stage where bacteria are the most susceptible to the action of disinfectants and common antibiotics that affect protein, DNA, and cell-wall synthesis.
Stationary Phase
As the number of cells increases through the log phase, several factors contribute to a slowing of the growth rate. Waste products accumulate and nutrients are gradually used up. In addition, gradual depletion of oxygen begins to limit aerobic cell growth. This combination of unfavorable conditions slows and finally stalls population growth. The total number of live cells reaches a plateau referred to as the stationary phase (Figure \(4\)). In this phase, the number of new cells created by cell division is now equivalent to the number of cells dying; thus, the total population of living cells is relatively stagnant. The culture density in a stationary culture is constant. The culture’s carrying capacity, or maximum culture density, depends on the types of microorganisms in the culture and the specific conditions of the culture; however, carrying capacity is constant for a given organism grown under the same conditions.
During the stationary phase, cells switch to a survival mode of metabolism. As growth slows, so too does the synthesis of peptidoglycans, proteins, and nucleic-acids; thus, stationary cultures are less susceptible to antibiotics that disrupt these processes. In bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase. Secondary metabolites, including antibiotics, are synthesized in the stationary phase. In certain pathogenic bacteria, the stationary phase is also associated with the expression of virulence factors, products that contribute to a microbe’s ability to survive, reproduce, and cause disease in a host organism. For example, quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful.
The Death Phase
As a culture medium accumulates toxic waste and nutrients are exhausted, cells die in greater and greater numbers. Soon, the number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells (Figure \(4\)). This is the aptly named death phase, sometimes called the decline phase. Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores. A few cells, the so-called persisters, are characterized by a slow metabolic rate. Persister cells are medically important because they are associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic treatment.
Sustaining Microbial Growth
The growth pattern shown in Figure \(4\) takes place in a closed environment; nutrients are not added and waste and dead cells are not removed. In many cases, though, it is advantageous to maintain cells in the logarithmic phase of growth. One example is in industries that harvest microbial products. A chemostat (Figure \(6\)) is used to maintain a continuous culture in which nutrients are supplied at a steady rate. A controlled amount of air is mixed in for aerobic processes. Bacterial suspension is removed at the same rate as nutrients flow in to maintain an optimal growth environment.
Exercise \(4\)
1. During which phase does growth occur at the fastest rate?
2. Name two factors that limit microbial growth.
Measurement of Bacterial Growth
Estimating the number of bacterial cells in a sample, known as a bacterial count, is a common task performed by microbiologists. The number of bacteria in a clinical sample serves as an indication of the extent of an infection. Quality control of drinking water, food, medication, and even cosmetics relies on estimates of bacterial counts to detect contamination and prevent the spread of disease. Two major approaches are used to measure cell number. The direct methods involve counting cells, whereas the indirect methods depend on the measurement of cell presence or activity without actually counting individual cells. Both direct and indirect methods have advantages and disadvantages for specific applications.
Direct Cell Count
Direct cell count refers to counting the cells in a liquid culture or colonies on a plate. It is a direct way of estimating how many organisms are present in a sample. Let’s look first at a simple and fast method that requires only a specialized slide and a compound microscope.
The simplest way to count bacteria is called the direct microscopic cell count, which involves transferring a known volume of a culture to a calibrated slide and counting the cells under a light microscope. The calibrated slide is called a Petroff-Hausser chamber (Figure \(7\)) and is similar to a hemocytometer used to count red blood cells. The central area of the counting chamber is etched into squares of various sizes. A sample of the culture suspension is added to the chamber under a coverslip that is placed at a specific height from the surface of the grid. It is possible to estimate the concentration of cells in the original sample by counting individual cells in a number of squares and determining the volume of the sample observed. The area of the squares and the height at which the coverslip is positioned are specified for the chamber. The concentration must be corrected for dilution if the sample was diluted before enumeration.
Cells in several small squares must be counted and the average taken to obtain a reliable measurement. The advantages of the chamber are that the method is easy to use, relatively fast, and inexpensive. On the downside, the counting chamber does not work well with dilute cultures because there may not be enough cells to count.
Using a counting chamber does not necessarily yield an accurate count of the number of live cells because it is not always possible to distinguish between live cells, dead cells, and debris of the same size under the microscope. However, newly developed fluorescence staining techniques make it possible to distinguish viable and dead bacteria. These viability stains (or live stains) bind to nucleic acids, but the primary and secondary stains differ in their ability to cross the cytoplasmic membrane. The primary stain, which fluoresces green, can penetrate intact cytoplasmic membranes, staining both live and dead cells. The secondary stain, which fluoresces red, can stain a cell only if the cytoplasmic membrane is considerably damaged. Thus, live cells fluoresce green because they only absorb the green stain, whereas dead cells appear red because the red stain displaces the green stain on their nucleic acids (Figure \(8\)).
Another technique uses an electronic cell counting device (Coulter counter) to detect and count the changes in electrical resistance in a saline solution. A glass tube with a small opening is immersed in an electrolyte solution. A first electrode is suspended in the glass tube. A second electrode is located outside of the tube. As cells are drawn through the small aperture in the glass tube, they briefly change the resistance measured between the two electrodes and the change is recorded by an electronic sensor (Figure \(9\)); each resistance change represents a cell. The method is rapid and accurate within a range of concentrations; however, if the culture is too concentrated, more than one cell may pass through the aperture at any given time and skew the results. This method also does not differentiate between live and dead cells.
Direct counts provide an estimate of the total number of cells in a sample. However, in many situations, it is important to know the number of live, or viable, cells. Counts of live cells are needed when assessing the extent of an infection, the effectiveness of antimicrobial compounds and medication, or contamination of food and water.
Exercise \(5\)
1. Why would you count the number of cells in more than one square in the Petroff-Hausser chamber to estimate cell numbers?
2. In the viability staining method, why do dead cells appear red?
Plate Count
The viable plate count, or simply plate count, is a count of viable or live cells. It is based on the principle that viable cells replicate and give rise to visible colonies when incubated under suitable conditions for the specimen. The results are usually expressed as colony-forming units per milliliter (CFU/mL) rather than cells per milliliter because more than one cell may have landed on the same spot to give rise to a single colony. Furthermore, samples of bacteria that grow in clusters or chains are difficult to disperse and a single colony may represent several cells. Some cells are described as viable but nonculturable and will not form colonies on solid media. For all these reasons, the viable plate count is considered a low estimate of the actual number of live cells. These limitations do not detract from the usefulness of the method, which provides estimates of live bacterial numbers.
Microbiologists typically count plates with 30–300 colonies. Samples with too few colonies (<30) do not give statistically reliable numbers, and overcrowded plates (>300 colonies) make it difficult to accurately count individual colonies. Also, counts in this range minimize occurrences of more than one bacterial cell forming a single colony. Thus, the calculated CFU is closer to the true number of live bacteria in the population.
There are two common approaches to inoculating plates for viable counts: the pour plate and the spread plate methods. Although the final inoculation procedure differs between these two methods, they both start with a serial dilution of the culture.
Serial Dilution
The serial dilution of a culture is an important first step before proceeding to either the pour plate or spread plate method. The goal of the serial dilution process is to obtain plates with CFUs in the range of 30–300, and the process usually involves several dilutions in multiples of 10 to simplify calculation. The number of serial dilutions is chosen according to a preliminary estimate of the culture density. Figure \(10\) illustrates the serial dilution method.
A fixed volume of the original culture, 1.0 mL, is added to and thoroughly mixed with the first dilution tube solution, which contains 9.0 mL of sterile broth. This step represents a dilution factor of 10, or 1:10, compared with the original culture. From this first dilution, the same volume, 1.0 mL, is withdrawn and mixed with a fresh tube of 9.0 mL of dilution solution. The dilution factor is now 1:100 compared with the original culture. This process continues until a series of dilutions is produced that will bracket the desired cell concentration for accurate counting. From each tube, a sample is plated on solid medium using either the pour plate method (Figure \(11\)) or the spread plate method (Figure \(12\)). The plates are incubated until colonies appear. Two to three plates are usually prepared from each dilution and the numbers of colonies counted on each plate are averaged. In all cases, thorough mixing of samples with the dilution medium (to ensure the cell distribution in the tube is random) is paramount to obtaining reliable results.
The dilution factor is used to calculate the number of cells in the original cell culture. In our example, an average of 50 colonies was counted on the plates obtained from the 1:10,000 dilution. Because only 0.1 mL of suspension was pipetted on the plate, the multiplier required to reconstitute the original concentration is 10 × 10,000. The number of CFU per mL is equal to 50 × 100 × 10,000 = 5,000,000. The number of bacteria in the culture is estimated as 5 million cells/mL. The colony count obtained from the 1:1000 dilution was 389, well below the expected 500 for a 10-fold difference in dilutions. This highlights the issue of inaccuracy when colony counts are greater than 300 and more than one bacterial cell grows into a single colony.
A very dilute sample—drinking water, for example—may not contain enough organisms to use either of the plate count methods described. In such cases, the original sample must be concentrated rather than diluted before plating. This can be accomplished using a modification of the plate count technique called the membrane filtration technique. Known volumes are vacuum-filtered aseptically through a membrane with a pore size small enough to trap microorganisms. The membrane is transferred to a Petri plate containing an appropriate growth medium. Colonies are counted after incubation. Calculation of the cell density is made by dividing the cell count by the volume of filtered liquid.
Link to Learning
Watch this video for demonstrations of serial dilutions and spread plate techniques.
The Most Probable Number
The number of microorganisms in dilute samples is usually too low to be detected by the plate count methods described thus far. For these specimens, microbiologists routinely use the most probable number (MPN) method, a statistical procedure for estimating of the number of viable microorganisms in a sample. Often used for water and food samples, the MPN method evaluates detectable growth by observing changes in turbidity or color due to metabolic activity.
A typical application of MPN method is the estimation of the number of coliforms in a sample of pond water. Coliforms are gram-negative rod bacteria that ferment lactose. The presence of coliforms in water is considered a sign of contamination by fecal matter. For the method illustrated in Figure \(13\), a series of three dilutions of the water sample is tested by inoculating five lactose broth tubes with 10 mL of sample, five lactose broth tubes with 1 mL of sample, and five lactose broth tubes with 0.1 mL of sample. The lactose broth tubes contain a pH indicator that changes color from red to yellow when the lactose is fermented. After inoculation and incubation, the tubes are examined for an indication of coliform growth by a color change in media from red to yellow. The first set of tubes (10-mL sample) showed growth in all the tubes; the second set of tubes (1 mL) showed growth in two tubes out of five; in the third set of tubes, no growth is observed in any of the tubes (0.1-mL dilution). The numbers 5, 2, and 0 are compared with Figure B1 in Appendix B, which has been constructed using a probability model of the sampling procedure. From our reading of the table, we conclude that 49 is the most probable number of bacteria per 100 mL of pond water.
Exercise \(6\)
1. What is a colony-forming unit?
2. What two methods are frequently used to estimate bacterial numbers in water samples?
Indirect Cell Counts
Besides direct methods of counting cells, other methods, based on an indirect detection of cell density, are commonly used to estimate and compare cell densities in a culture. The foremost approach is to measure the turbidity (cloudiness) of a sample of bacteria in a liquid suspension. The laboratory instrument used to measure turbidity is called a spectrophotometer (Figure \(14\)). In a spectrophotometer, a light beam is transmitted through a bacterial suspension, the light passing through the suspension is measured by a detector, and the amount of light passing through the sample and reaching the detector is converted to either percent transmission or a logarithmic value called absorbance (optical density). As the numbers of bacteria in a suspension increase, the turbidity also increases and causes less light to reach the detector. The decrease in light passing through the sample and reaching the detector is associated with a decrease in percent transmission and increase in absorbance measured by the spectrophotometer.
Measuring turbidity is a fast method to estimate cell density as long as there are enough cells in a sample to produce turbidity. It is possible to correlate turbidity readings to the actual number of cells by performing a viable plate count of samples taken from cultures having a range of absorbance values. Using these values, a calibration curve is generated by plotting turbidity as a function of cell density. Once the calibration curve has been produced, it can be used to estimate cell counts for all samples obtained or cultured under similar conditions and with densities within the range of values used to construct the curve.
Measuring dry weight of a culture sample is another indirect method of evaluating culture density without directly measuring cell counts. The cell suspension used for weighing must be concentrated by filtration or centrifugation, washed, and then dried before the measurements are taken. The degree of drying must be standardized to account for residual water content. This method is especially useful for filamentous microorganisms, which are difficult to enumerate by direct or viable plate count.
As we have seen, methods to estimate viable cell numbers can be labor intensive and take time because cells must be grown. Recently, indirect ways of measuring live cells have been developed that are both fast and easy to implement. These methods measure cell activity by following the production of metabolic products or disappearance of reactants. Adenosine triphosphate (ATP) formation, biosynthesis of proteins and nucleic acids, and consumption of oxygen can all be monitored to estimate the number of cells.
Exercise \(7\)
1. What is the purpose of a calibration curve when estimating cell count from turbidity measurements?
2. What are the newer indirect methods of counting live cells?
Alternative Patterns of Cell Division
Binary fission is the most common pattern of cell division in prokaryotes, but it is not the only one. Other mechanisms usually involve asymmetrical division (as in budding) or production of spores in aerial filaments.
In some cyanobacteria, many nucleoids may accumulate in an enlarged round cell or along a filament, leading to the generation of many new cells at once. The new cells often split from the parent filament and float away in a process called fragmentation (Figure \(15\)). Fragmentation is commonly observed in the Actinomycetes, a group of gram-positive, anaerobic bacteria commonly found in soil. Another curious example of cell division in prokaryotes, reminiscent of live birth in animals, is exhibited by the giant bacterium Epulopiscium. Several daughter cells grow fully in the parent cell, which eventually disintegrates, releasing the new cells to the environment. Other species may form a long narrow extension at one pole in a process called budding. The tip of the extension swells and forms a smaller cell, the bud that eventually detaches from the parent cell. Budding is most common in yeast (Figure \(15\)), but it is also observed in prosthecate bacteria and some cyanobacteria.
The soil bacteria Actinomyces grow in long filaments divided by septa, similar to the mycelia seen in fungi, resulting in long cells with multiple nucleoids. Environmental signals, probably related to low nutrient availability, lead to the formation of aerial filaments. Within these aerial filaments, elongated cells divide simultaneously. The new cells, which contain a single nucleoid, develop into spores that give rise to new colonies.
Exercise \(8\)
Identify at least one difference between fragmentation and budding.
Biofilms
In nature, microorganisms grow mainly in biofilms, complex and dynamic ecosystems that form on a variety of environmental surfaces, from industrial conduits and water treatment pipelines to rocks in river beds. Biofilms are not restricted to solid surface substrates, however. Almost any surface in a liquid environment containing some minimal nutrients will eventually develop a biofilm. Microbial mats that float on water, for example, are biofilms that contain large populations of photosynthetic microorganisms. Biofilms found in the human mouth may contain hundreds of bacterial species. Regardless of the environment where they occur, biofilms are not random collections of microorganisms; rather, they are highly structured communities that provide a selective advantage to their constituent microorganisms.
Biofilm Structure
Observations using confocal microscopy have shown that environmental conditions influence the overall structure of biofilms. Filamentous biofilms called streamers form in rapidly flowing water, such as freshwater streams, eddies, and specially designed laboratory flow cells that replicate growth conditions in fast-moving fluids. The streamers are anchored to the substrate by a “head” and the “tail” floats downstream in the current. In still or slow-moving water, biofilms mainly assume a mushroom-like shape. The structure of biofilms may also change with other environmental conditions such as nutrient availability.
Detailed observations of biofilms under confocal laser and scanning electron microscopes reveal clusters of microorganisms embedded in a matrix interspersed with open water channels. The extracellular matrix consists of extracellular polymeric substances (EPS) secreted by the organisms in the biofilm. The extracellular matrix represents a large fraction of the biofilm, accounting for 50%–90% of the total dry mass. The properties of the EPS vary according to the resident organisms and environmental conditions.
EPS is a hydrated gel composed primarily of polysaccharides and containing other macromolecules such as proteins, nucleic acids, and lipids. It plays a key role in maintaining the integrity and function of the biofilm. Channels in the EPS allow movement of nutrients, waste, and gases throughout the biofilm. This keeps the cells hydrated, preventing desiccation. EPS also shelters organisms in the biofilm from predation by other microbes or cells (e.g., protozoans, white blood cells in the human body).
Biofilm Formation
Free-floating microbial cells that live in an aquatic environment are called planktonic cells. The formation of a biofilm essentially involves the attachment of planktonic cells to a substrate, where they become sessile (attached to a surface). This occurs in stages, as depicted in Figure \(16\). The first stage involves the attachment of planktonic cells to a surface coated with a conditioning film of organic material. At this point, attachment to the substrate is reversible, but as cells express new phenotypes that facilitate the formation of EPS, they transition from a planktonic to a sessile lifestyle. The biofilm develops characteristic structures, including an extensive matrix and water channels. Appendages such as fimbriae, pili, and flagella interact with the EPS, and microscopy and genetic analysis suggest that such structures are required for the establishment of a mature biofilm. In the last stage of the biofilm life cycle, cells on the periphery of the biofilm revert to a planktonic lifestyle, sloughing off the mature biofilm to colonize new sites. This stage is referred to as dispersal.
Within a biofilm, different species of microorganisms establish metabolic collaborations in which the waste product of one organism becomes the nutrient for another. For example, aerobic microorganisms consume oxygen, creating anaerobic regions that promote the growth of anaerobes. This occurs in many polymicrobial infections that involve both aerobic and anaerobic pathogens.
The mechanism by which cells in a biofilm coordinate their activities in response to environmental stimuli is called quorum sensing. Quorum sensing—which can occur between cells of different species within a biofilm—enables microorganisms to detect their cell density through the release and binding of small, diffusible molecules called autoinducers. When the cell population reaches a critical threshold (a quorum), these autoinducers initiate a cascade of reactions that activate genes associated with cellular functions that are beneficial only when the population reaches a critical density. For example, in some pathogens, synthesis of virulence factors only begins when enough cells are present to overwhelm the immune defenses of the host. Although mostly studied in bacterial populations, quorum sensing takes place between bacteria and eukaryotes and between eukaryotic cells such as the fungus Candida albicans, a common member of the human microbiota that can cause infections in immunocompromised individuals.
The signaling molecules in quorum sensing belong to two major classes. Gram-negative bacteria communicate mainly using N-acylated homoserine lactones, whereas gram-positive bacteria mostly use small peptides (Figure \(17\)). In all cases, the first step in quorum sensing consists of the binding of the autoinducer to its specific receptor only when a threshold concentration of signaling molecules is reached. Once binding to the receptor takes place, a cascade of signaling events leads to changes in gene expression. The result is the activation of biological responses linked to quorum sensing, notably an increase in the production of signaling molecules themselves, hence the term autoinducer.
Biofilms and Human Health
The human body harbors many types of biofilms, some beneficial and some harmful. For example, the layers of normal microbiota lining the intestinal and respiratory mucosa play a role in warding off infections by pathogens. However, other biofilms in the body can have a detrimental effect on health. For example, the plaque that forms on teeth is a biofilm that can contribute to dental and periodontal disease. Biofilms can also form in wounds, sometimes causing serious infections that can spread. The bacterium Pseudomonas aeruginosa often colonizes biofilms in the airways of patients with cystic fibrosis, causing chronic and sometimes fatal infections of the lungs. Biofilms can also form on medical devices used in or on the body, causing infections in patients with in-dwelling catheters, artificial joints, or contact lenses.
Pathogens embedded within biofilms exhibit a higher resistance to antibiotics than their free-floating counterparts. Several hypotheses have been proposed to explain why. Cells in the deep layers of a biofilm are metabolically inactive and may be less susceptible to the action of antibiotics that disrupt metabolic activities. The EPS may also slow the diffusion of antibiotics and antiseptics, preventing them from reaching cells in the deeper layers of the biofilm. Phenotypic changes may also contribute to the increased resistance exhibited by bacterial cells in biofilms. For example, the increased production of efflux pumps, membrane-embedded proteins that actively extrude antibiotics out of bacterial cells, have been shown to be an important mechanism of antibiotic resistance among biofilm-associated bacteria. Finally, biofilms provide an ideal environment for the exchange of extrachromosomal DNA, which often includes genes that confer antibiotic resistance.
Exercise \(9\)
1. What is the matrix of a biofilm composed of?
2. What is the role of quorum sensing in a biofilm?
Key Concepts and Summary
• Most bacterial cells divide by binary fission. Generation time in bacterial growth is defined as the doubling timeof the population.
• Cells in a closed system follow a pattern of growth with four phases: lag, logarithmic (exponential), stationary, and death.
• Cells can be counted by direct viable cell count. The pour plate and spread plate methods are used to plate serial dilutions into or onto, respectively, agar to allow counting of viable cells that give rise to colony-forming units. Membrane filtration is used to count live cells in dilute solutions. The most probable cell number (MPN)method allows estimation of cell numbers in cultures without using solid media.
• Indirect methods can be used to estimate culture density by measuring turbidity of a culture or live cell density by measuring metabolic activity.
• Other patterns of cell division include multiple nucleoid formation in cells; asymmetric division, as in budding; and the formation of hyphae and terminal spores.
• Biofilms are communities of microorganisms enmeshed in a matrix of extracellular polymeric substance. The formation of a biofilm occurs when planktonic cells attach to a substrate and become sessile. Cells in biofilms coordinate their activity by communicating through quorum sensing.
• Biofilms are commonly found on surfaces in nature and in the human body, where they may be beneficial or cause severe infections. Pathogens associated with biofilms are often more resistant to antibiotics and disinfectants. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/09%3A_Microbial_Growth/9.01%3A_How_Microbes_Grow.txt |
Learning Objectives
• Interpret visual data demonstrating minimum, optimum, and maximum oxygen or carbon dioxide requirements for growth
• Identify and describe different categories of microbes with requirements for growth with or without oxygen: obligate aerobe, obligate anaerobe, facultative anaerobe, aerotolerant anaerobe, microaerophile, and capnophile
• Give examples of microorganisms for each category of growth requirements
Ask most people “What are the major requirements for life?” and the answers are likely to include water and oxygen. Few would argue about the need for water, but what about oxygen? Can there be life without oxygen?
The answer is that molecular oxygen (O2) is not always needed. The earliest signs of life are dated to a period when conditions on earth were highly reducing and free oxygen gas was essentially nonexistent. Only after cyanobacteria started releasing oxygen as a byproduct of photosynthesis and the capacity of iron in the oceans for taking up oxygen was exhausted did oxygen levels increase in the atmosphere. This event, often referred to as the Great Oxygenation Event or the Oxygen Revolution, caused a massive extinction. Most organisms could not survive the powerful oxidative properties of reactive oxygen species (ROS), highly unstable ions and molecules derived from partial reduction of oxygen that can damage virtually any macromolecule or structure with which they come in contact. Singlet oxygen (O2•), superoxide $(\ce{O2-})$, peroxides (H2O2), hydroxyl radical (OH•), and hypochlorite ion (OCl), the active ingredient of household bleach, are all examples of ROS. The organisms that were able to detoxify reactive oxygen species harnessed the high electronegativity of oxygen to produce free energy for their metabolism and thrived in the new environment.
Oxygen Requirements of Microorganisms
Many ecosystems are still free of molecular oxygen. Some are found in extreme locations, such as deep in the ocean or in earth’s crust; others are part of our everyday landscape, such as marshes, bogs, and sewers. Within the bodies of humans and other animals, regions with little or no oxygen provide an anaerobic environment for microorganisms. (Figure $1$).
We can easily observe different requirements for molecular oxygen by growing bacteria in thioglycolate tube cultures. A test-tube culture starts with autoclaved thioglycolate medium containing a low percentage of agar to allow motile bacteria to move throughout the medium. Thioglycolate has strong reducing properties and autoclaving flushes out most of the oxygen. The tubes are inoculated with the bacterial cultures to be tested and incubated at an appropriate temperature. Over time, oxygen slowly diffuses throughout the thioglycolate tube culture from the top. Bacterial density increases in the area where oxygen concentration is best suited for the growth of that particular organism.
The growth of bacteria with varying oxygen requirements in thioglycolate tubes is illustrated in Figure $2$. In tube A, all the growth is seen at the top of the tube. The bacteria are obligate (strict) aerobes that cannot grow without an abundant supply of oxygen. Tube B looks like the opposite of tube A. Bacteria grow at the bottom of tube B. Those are obligate anaerobes, which are killed by oxygen. Tube C shows heavy growth at the top of the tube and growth throughout the tube, a typical result with facultative anaerobes. Facultative anaerobes are organisms that thrive in the presence of oxygen but also grow in its absence by relying on fermentation or anaerobic respiration, if there is a suitable electron acceptor other than oxygen and the organism is able to perform anaerobic respiration. The aerotolerant anaerobes in tube D are indifferent to the presence of oxygen. They do not use oxygen because they usually have a fermentative metabolism, but they are not harmed by the presence of oxygen as obligate anaerobes are. Tube E on the right shows a “Goldilocks” culture. The oxygen level has to be just right for growth, not too much and not too little. These microaerophiles are bacteria that require a minimum level of oxygen for growth, about 1%–10%, well below the 21% found in the atmosphere.
Examples of obligate aerobes are Mycobacterium tuberculosis, the causative agent of tuberculosis and Micrococcus luteus, a gram-positive bacterium that colonizes the skin. Neisseria meningitidis, the causative agent of severe bacterial meningitis, and N. gonorrheae, the causative agent of sexually transmitted gonorrhea, are also obligate aerobes.
Many obligate anaerobes are found in the environment where anaerobic conditions exist, such as in deep sediments of soil, still waters, and at the bottom of the deep ocean where there is no photosynthetic life. Anaerobic conditions also exist naturally in the intestinal tract of animals. Obligate anaerobes, mainly Bacteroidetes, represent a large fraction of the microbes in the human gut. Transient anaerobic conditions exist when tissues are not supplied with blood circulation; they die and become an ideal breeding ground for obligate anaerobes. Another type of obligate anaerobe encountered in the human body is the gram-positive, rod-shaped Clostridium spp. Their ability to form endospores allows them to survive in the presence of oxygen. One of the major causes of health-acquired infections is C. difficile, known as C. diff. Prolonged use of antibiotics for other infections increases the probability of a patient developing a secondary C. difficile infection. Antibiotic treatment disrupts the balance of microorganisms in the intestine and allows the colonization of the gut by C. difficile, causing a significant inflammation of the colon.
Other clostridia responsible for serious infections include C. tetani, the agent of tetanus, and C. perfringens, which causes gas gangrene. In both cases, the infection starts in necrotic tissue (dead tissue that is not supplied with oxygen by blood circulation). This is the reason that deep puncture wounds are associated with tetanus. When tissue death is accompanied by lack of circulation, gangrene is always a danger.
The study of obligate anaerobes requires special equipment. Obligate anaerobic bacteria must be grown under conditions devoid of oxygen. The most common approach is culture in an anaerobic jar (Figure $3$). Anaerobic jars include chemical packs that remove oxygen and release carbon dioxide (CO2). An anaerobic chamber is an enclosed box from which all oxygen is removed. Gloves sealed to openings in the box allow handling of the cultures without exposing the culture to air (Figure $3$).
Staphylococci and Enterobacteriaceae are examples of facultative anaerobes. Staphylococci are found on the skin and upper respiratory tract. Enterobacteriaceae are found primarily in the gut and upper respiratory tract but can sometimes spread to the urinary tract, where they are capable of causing infections. It is not unusual to see mixed bacterial infections in which the facultative anaerobes use up the oxygen, creating an environment for the obligate anaerobes to flourish.
Examples of aerotolerant anaerobes include lactobacilli and streptococci, both found in the oral microbiota. Campylobacter jejuni, which causes gastrointestinal infections, is an example of a microaerophile and is grown under low-oxygen conditions.
The optimum oxygen concentration, as the name implies, is the ideal concentration of oxygen for a particular microorganism. The lowest concentration of oxygen that allows growth is called the minimum permissive oxygen concentration. The highest tolerated concentration of oxygen is the maximum permissive oxygen concentration. The organism will not grow outside the range of oxygen levels found between the minimum and maximum permissive oxygen concentrations.
Exercise $1$
1. Would you expect the oldest bacterial lineages to be aerobic or anaerobic?
2. Which bacteria grow at the top of a thioglycolate tube, and which grow at the bottom of the tube?
An Unwelcome Anaerobe
Charles is a retired bus driver who developed type 2 diabetes over 10 years ago. Since his retirement, his lifestyle has become very sedentary and he has put on a substantial amount of weight. Although he has felt tingling and numbness in his left foot for a while, he has not been worried because he thought his foot was simply “falling asleep.” Recently, a scratch on his foot does not seem to be healing and is becoming increasingly ugly. Because the sore did not bother him much, Charles figured it could not be serious until his daughter noticed a purplish discoloration spreading on the skin and oozing (Figure $4$). When he was finally seen by his physician, Charles was rushed to the operating room. His open sore, or ulcer, is the result of a diabetic foot.
The concern here is that gas gangrene may have taken hold in the dead tissue. The most likely agent of gas gangrene is Clostridium perfringens, an endospore-forming, gram-positive bacterium. It is an obligate anaerobe that grows in tissue devoid of oxygen. Since dead tissue is no longer supplied with oxygen by the circulatory system, the dead tissue provides pockets of ideal environment for the growth of C. perfringens.
A surgeon examines the ulcer and radiographs of Charles’s foot and determines that the bone is not yet infected. The wound will have to be surgically debrided (debridement refers to the removal of dead and infected tissue) and a sample sent for microbiological lab analysis, but Charles will not have to have his foot amputated. Many diabetic patients are not so lucky. In 2008, nearly 70,000 diabetic patients in the United States lost a foot or limb to amputation, according to statistics from the Centers for Disease Control and Prevention.
Exercise $2$
Which growth conditions would you recommend for the detection of C. perfringens?
Detoxification of Reactive Oxygen Species
Aerobic respiration constantly generates reactive oxygen species (ROS), byproducts that must be detoxified. Even organisms that do not use aerobic respiration need some way to break down some of the ROS that may form from atmospheric oxygen. Three main enzymes break down those toxic byproducts: superoxide dismutase, peroxidase, and catalase. Each one catalyzes a different reaction. Reactions of type seen in Reaction 1 are catalyzed by peroxidases.
$\mathrm{X-(2H^+)+H_2O_2 \rightarrow \text{oxidized-}X+2H_2O}$
In these reactions, an electron donor (reduced compound; e.g., reduced nicotinamide adenine dinucleotide [NADH]) oxidizes hydrogen peroxide, or other peroxides, to water. The enzymes play an important role by limiting the damage caused by peroxidation of membrane lipids. Reaction 2 is mediated by the enzyme superoxide dismutase (SOD) and breaks down the powerful superoxide anions generated by aerobic metabolism:
$\mathrm{2O_2^- + 2H^+ \rightarrow H_2O_2+O_2}$
The enzyme catalase converts hydrogen peroxide to water and oxygen as shown in Reaction 3.
$\mathrm{2H_2O_2 \rightarrow 2H_2O+O_2}$
Obligate anaerobes usually lack all three enzymes. Aerotolerant anaerobes do have SOD but no catalase. Reaction 3, shown occurring in Figure $5$, is the basis of a useful and rapid test to distinguish streptococci, which are aerotolerant and do not possess catalase, from staphylococci, which are facultative anaerobes. A sample of culture rapidly mixed in a drop of 3% hydrogen peroxide will release bubbles if the culture is catalase positive.
Bacteria that grow best in a higher concentration of CO2 and a lower concentration of oxygen than present in the atmosphere are called capnophiles. One common approach to grow capnophiles is to use a candle jar. A candle jar consists of a jar with a tight-fitting lid that can accommodate the cultures and a candle. After the cultures are added to the jar, the candle is lit and the lid closed. As the candle burns, it consumes most of the oxygen present and releases CO2.
Exercise $3$
1. What substance is added to a sample to detect catalase?
2. What is the function of the candle in a candle jar?
Clinical Focus: Part 2
The health-care provider who saw Jeni was concerned primarily because of her pregnancy. Her condition enhances the risk for infections and makes her more vulnerable to those infections. The immune system is downregulated during pregnancy, and pathogens that cross the placenta can be very dangerous for the fetus. A note on the provider’s order to the microbiology lab mentions a suspicion of infection by Listeria monocytogenes, based on the signs and symptoms exhibited by the patient.
Jeni’s blood samples are streaked directly on sheep blood agar, a medium containing tryptic soy agar enriched with 5% sheep blood. (Blood is considered sterile; therefore, competing microorganisms are not expected in the medium.) The inoculated plates are incubated at 37 °C for 24 to 48 hours. Small grayish colonies surrounded by a clear zone emerge. Such colonies are typical of Listeria and other pathogens such as streptococci; the clear zone surrounding the colonies indicates complete lysis of blood in the medium, referred to as beta-hemolysis (Figure $6$). When tested for the presence of catalase, the colonies give a positive response, eliminating Streptococcus as a possible cause. Furthermore, a Gram stain shows short gram-positive bacilli. Cells from a broth culture grown at room temperature displayed the tumbling motility characteristic of Listeria (Figure $6$). All of these clues lead the lab to positively confirm the presence of Listeria in Jeni’s blood samples.
Exercise $4$
How serious is Jeni’s condition and what is the appropriate treatment?
Key Concepts and Summary
• Aerobic and anaerobic environments can be found in diverse niches throughout nature, including different sites within and on the human body.
• Microorganisms vary in their requirements for molecular oxygen. Obligate aerobes depend on aerobic respiration and use oxygen as a terminal electron acceptor. They cannot grow without oxygen.
• Obligate anaerobes cannot grow in the presence of oxygen. They depend on fermentation and anaerobic respiration using a final electron acceptor other than oxygen.
• Facultative anaerobes show better growth in the presence of oxygen but will also grow without it.
• Although aerotolerant anaerobes do not perform aerobic respiration, they can grow in the presence of oxygen. Most aerotolerant anaerobes test negative for the enzyme catalase.
• Microaerophiles need oxygen to grow, albeit at a lower concentration than 21% oxygen in air.
• Optimum oxygen concentration for an organism is the oxygen level that promotes the fastest growth rate. The minimum permissive oxygen concentration and the maximum permissive oxygen concentration are, respectively, the lowest and the highest oxygen levels that the organism will tolerate.
• Peroxidase, superoxide dismutase, and catalase are the main enzymes involved in the detoxification of the reactive oxygen species. Superoxide dismutase is usually present in a cell that can tolerate oxygen. All three enzymes are usually detectable in cells that perform aerobic respiration and produce more ROS.
• A capnophile is an organism that requires a higher than atmospheric concentration of CO2 to grow.
Footnotes
1. 1 Centers for Disease Control and Prevention. “Living With Diabetes: Keep Your Feet Healthy.” http://www.cdc.gov/Features/DiabetesFootHealth/ | textbooks/bio/Microbiology/Microbiology_(OpenStax)/09%3A_Microbial_Growth/9.02%3A_Oxygen_Requirements_for_Microbial_Growth.txt |
Learning Objectives
• Illustrate and briefly describe minimum, optimum, and maximum pH requirements for growth
• Identify and describe the different categories of microbes with pH requirements for growth: acidophiles, neutrophiles, and alkaliphiles
• Give examples of microorganisms for each category of pH requirement
Yogurt, pickles, sauerkraut, and lime-seasoned dishes all owe their tangy taste to a high acid content (Figure \(1\)). Recall that acidity is a function of the concentration of hydrogen ions [H+] and is measured as pH. Environments with pH values below 7.0 are considered acidic, whereas those with pH values above 7.0 are considered basic. Extreme pH affects the structure of all macromolecules. The hydrogen bonds holding together strands of DNA break up at high pH. Lipids are hydrolyzed by an extremely basic pH. The proton motive force responsible for production of ATP in cellular respiration depends on the concentration gradient of H+ across the plasma membrane (see Cellular Respiration). If H+ ions are neutralized by hydroxide ions, the concentration gradient collapses and impairs energy production. But the component most sensitive to pH in the cell is its workhorse, the protein. Moderate changes in pH modify the ionization of amino-acid functional groups and disrupt hydrogen bonding, which, in turn, promotes changes in the folding of the molecule, promoting denaturation and destroying activity.
The optimum growth pH is the most favorable pH for the growth of an organism. The lowest pH value that an organism can tolerate is called the minimum growth pH and the highest pH is the maximum growth pH. These values can cover a wide range, which is important for the preservation of food and to microorganisms’ survival in the stomach. For example, the optimum growth pH of Salmonella spp. is 7.0–7.5, but the minimum growth pH is closer to 4.2.
Most bacteria are neutrophiles, meaning they grow optimally at a pH within one or two pH units of the neutral pH of 7 (see Figure \(2\)). Most familiar bacteria, like Escherichia coli, staphylococci, and Salmonella spp. are neutrophiles and do not fare well in the acidic pH of the stomach. However, there are pathogenic strains of E. coli, S. typhi, and other species of intestinal pathogens that are much more resistant to stomach acid. In comparison, fungi thrive at slightly acidic pH values of 5.0–6.0.
Microorganisms that grow optimally at pH less than 5.55 are called acidophiles. For example, the sulfur-oxidizing Sulfolobus spp. isolated from sulfur mud fields and hot springs in Yellowstone National Park are extreme acidophiles. These archaea survive at pH values of 2.5–3.5. Species of the archaean genus Ferroplasma live in acid mine drainage at pH values of 0–2.9. Lactobacillus bacteria, which are an important part of the normal microbiota of the vagina, can tolerate acidic environments at pH values 3.5–6.8 and also contribute to the acidity of the vagina (pH of 4, except at the onset of menstruation) through their metabolic production of lactic acid. The vagina’s acidity plays an important role in inhibiting other microbes that are less tolerant of acidity. Acidophilic microorganisms display a number of adaptations to survive in strong acidic environments. For example, proteins show increased negative surface charge that stabilizes them at low pH. Pumps actively eject H+ ions out of the cells. The changes in the composition of membrane phospholipids probably reflect the need to maintain membrane fluidity at low pH.
At the other end of the spectrum are alkaliphiles, microorganisms that grow best at pH between 8.0 and 10.5. Vibrio cholerae, the pathogenic agent of cholera, grows best at the slightly basic pH of 8.0; it can survive pH values of 11.0 but is inactivated by the acid of the stomach. When it comes to survival at high pH, the bright pink archaean Natronobacterium, found in the soda lakes of the African Rift Valley, may hold the record at a pH of 10.5 (Figure \(3\)). Extreme alkaliphiles have adapted to their harsh environment through evolutionary modification of lipid and protein structure and compensatory mechanisms to maintain the proton motive force in an alkaline environment. For example, the alkaliphile Bacillus firmus derives the energy for transport reactions and motility from a Na+ ion gradient rather than a proton motive force. Many enzymes from alkaliphiles have a higher isoelectric point, due to an increase in the number of basic amino acids, than homologous enzymes from neutrophiles.
Survival at the Low pH of the Stomach
Peptic ulcers (or stomach ulcers) are painful sores on the stomach lining. Until the 1980s, they were believed to be caused by spicy foods, stress, or a combination of both. Patients were typically advised to eat bland foods, take anti-acid medications, and avoid stress. These remedies were not particularly effective, and the condition often recurred. This all changed dramatically when the real cause of most peptic ulcers was discovered to be a slim, corkscrew-shaped bacterium, Helicobacter pylori. This organism was identified and isolated by Barry Marshall and Robin Warren, whose discovery earned them the Nobel Prize in Medicine in 2005.
The ability of H. pylori to survive the low pH of the stomach would seem to suggest that it is an extreme acidophile. As it turns out, this is not the case. In fact, H. pylori is a neutrophile. So, how does it survive in the stomach? Remarkably, H. pylori creates a microenvironment in which the pH is nearly neutral. It achieves this by producing large amounts of the enzyme urease, which breaks down urea to form NH4+ and CO2. The ammonium ion raises the pH of the immediate environment.
This metabolic capability of H. pylori is the basis of an accurate, noninvasive test for infection. The patient is given a solution of urea containing radioactively labeled carbon atoms. If H. pylori is present in the stomach, it will rapidly break down the urea, producing radioactive CO2 that can be detected in the patient’s breath. Because peptic ulcers may lead to gastric cancer, patients who are determined to have H. pylori infections are treated with antibiotics.
Exercise \(1\)
1. What effect do extremes of pH have on proteins?
2. What pH-adaptive type of bacteria would most human pathogens be?
Key Concepts and Summary
• Bacteria are generally neutrophiles. They grow best at neutral pH close to 7.0.
• Acidophiles grow optimally at a pH near 3.0. Alkaliphiles are organisms that grow optimally between a pH of 8 and 10.5. Extreme acidophiles and alkaliphiles grow slowly or not at all near neutral pH.
• Microorganisms grow best at their optimum growth pH. Growth occurs slowly or not at all below the minimum growth pH and above the maximum growth pH. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/09%3A_Microbial_Growth/9.03%3A_The_Effects_of_pH_on_Microbial_Growth.txt |
Learning Objectives
• Illustrate and briefly describe minimum, optimum, and maximum temperature requirements for growth
• Identify and describe different categories of microbes with temperature requirements for growth: psychrophile, psychrotrophs, mesophile, thermophile, hyperthermophile
• Give examples of microorganisms in each category of temperature tolerance
When the exploration of Lake Whillans started in Antarctica, researchers did not expect to find much life. Constant subzero temperatures and lack of obvious sources of nutrients did not seem to be conditions that would support a thriving ecosystem. To their surprise, the samples retrieved from the lake showed abundant microbial life. In a different but equally harsh setting, bacteria grow at the bottom of the ocean in sea vents (Figure \(1\)), where temperatures can reach 340 °C (700 °F).
Microbes can be roughly classified according to the range of temperature at which they can grow. The growth rates are the highest at the optimum growth temperature for the organism. The lowest temperature at which the organism can survive and replicate is its minimum growth temperature. The highest temperature at which growth can occur is its maximum growth temperature. The following ranges of permissive growth temperatures are approximate only and can vary according to other environmental factors.
Organisms categorized as mesophiles (“middle loving”) are adapted to moderate temperatures, with optimal growth temperatures ranging from room temperature (about 20 °C) to about 45 °C. As would be expected from the core temperature of the human body, 37 °C (98.6 °F), normal human microbiota and pathogens (e.g., E. coli, Salmonella spp., and Lactobacillus spp.) are mesophiles.
Organisms called psychrotrophs, also known as psychrotolerant, prefer cooler environments, from a high temperature of 25 °C to refrigeration temperature about 4 °C. They are found in many natural environments in temperate climates. They are also responsible for the spoilage of refrigerated food.
Clinical Focus: Resolution
The presence of Listeria in Jeni’s blood suggests that her symptoms are due to listeriosis, an infection caused by L. monocytogenes. Listeriosis is a serious infection with a 20% mortality rate and is a particular risk to Jeni’s fetus. A sample from the amniotic fluid cultured for the presence of Listeria gave negative results. Because the absence of organisms does not rule out the possibility of infection, a molecular test based on the nucleic acid amplification of the 16S ribosomal RNA of Listeria was performed to confirm that no bacteria crossed the placenta. Fortunately, the results from the molecular test were also negative.
Jeni was admitted to the hospital for treatment and recovery. She received a high dose of two antibiotics intravenously for 2 weeks. The preferred drugs for the treatment of listeriosis are ampicillin or penicillin G with an aminoglycoside antibiotic. Resistance to common antibiotics is still rare in Listeria and antibiotic treatment is usually successful. She was released to home care after a week and fully recovered from her infection.
L. monocytogenes is a gram-positive short rod found in soil, water, and food. It is classified as a psychrophile and is halotolerant. Its ability to multiply at refrigeration temperatures (4–10 °C) and its tolerance for high concentrations of salt (up to 10% sodium chloride [NaCl]) make it a frequent source of food poisoning. Because Listeria can infect animals, it often contaminates food such as meat, fish, or dairy products. Contamination of commercial foods can often be traced to persistent biofilms that form on manufacturing equipment that is not sufficiently cleaned.
Listeria infection is relatively common among pregnant women because the elevated levels of progesterone downregulate the immune system, making them more vulnerable to infection. The pathogen can cross the placenta and infect the fetus, often resulting in miscarriage, stillbirth, or fatal neonatal infection. Pregnant women are thus advised to avoid consumption of soft cheeses, refrigerated cold cuts, smoked seafood, and unpasteurized dairy products. Because Listeria bacteria can easily be confused with diphtheroids, another common group of gram-positive rods, it is important to alert the laboratory when listeriosis is suspected.
The organisms retrieved from arctic lakes such as Lake Whillans are considered extreme psychrophiles (cold loving). Psychrophiles are microorganisms that can grow at 0 °C and below, have an optimum growth temperature close to 15 °C, and usually do not survive at temperatures above 20 °C. They are found in permanently cold environments such as the deep waters of the oceans. Because they are active at low temperature, psychrophiles and psychrotrophs are important decomposers in cold climates.
Organisms that grow at optimum temperatures of 50 °C to a maximum of 80 °C are called thermophiles (“heat loving”). They do not multiply at room temperature. Thermophiles are widely distributed in hot springs, geothermal soils, and manmade environments such as garden compost piles where the microbes break down kitchen scraps and vegetal material. Examples of thermophiles include Thermus aquaticus and Geobacillus spp. Higher up on the extreme temperature scale we find the hyperthermophiles, which are characterized by growth ranges from 80 °C to a maximum of 110 °C, with some extreme examples that survive temperatures above 121 °C, the average temperature of an autoclave. The hydrothermal vents at the bottom of the ocean are a prime example of extreme environments, with temperatures reaching an estimated 340 °C (Figure \(1\)). Microbes isolated from the vents achieve optimal growth at temperatures higher than 100 °C. Noteworthy examples are Pyrobolus and Pyrodictium, archaea that grow at 105 °C and survive autoclaving. Figure \(2\) shows the typical skewed curves of temperature-dependent growth for the categories of microorganisms we have discussed.
Life in extreme environments raises fascinating questions about the adaptation of macromolecules and metabolic processes. Very low temperatures affect cells in many ways. Membranes lose their fluidity and are damaged by ice crystal formation. Chemical reactions and diffusion slow considerably. Proteins become too rigid to catalyze reactions and may undergo denaturation. At the opposite end of the temperature spectrum, heat denatures proteins and nucleic acids. Increased fluidity impairs metabolic processes in membranes. Some of the practical applications of the destructive effects of heat on microbes are sterilization by steam, pasteurization, and incineration of inoculating loops. Proteins in psychrophiles are, in general, rich in hydrophobic residues, display an increase in flexibility, and have a lower number of secondary stabilizing bonds when compared with homologous proteins from mesophiles. Antifreeze proteins and solutes that decrease the freezing temperature of the cytoplasm are common. The lipids in the membranes tend to be unsaturated to increase fluidity. Growth rates are much slower than those encountered at moderate temperatures. Under appropriate conditions, mesophiles and even thermophiles can survive freezing. Liquid cultures of bacteria are mixed with sterile glycerol solutions and frozen to −80 °C for long-term storage as stocks. Cultures can withstand freeze drying (lyophilization) and then be stored as powders in sealed ampules to be reconstituted with broth when needed.
Macromolecules in thermophiles and hyperthermophiles show some notable structural differences from what is observed in the mesophiles. The ratio of saturated to polyunsaturated lipids increases to limit the fluidity of the cell membranes. Their DNA sequences show a higher proportion of guanine–cytosine nitrogenous bases, which are held together by three hydrogen bonds in contrast to adenine and thymine, which are connected in the double helix by two hydrogen bonds. Additional secondary ionic and covalent bonds, as well as the replacement of key amino acids to stabilize folding, contribute to the resistance of proteins to denaturation. The so-called thermoenzymes purified from thermophiles have important practical applications. For example, amplification of nucleic acids in the polymerase chain reaction (PCR) depends on the thermal stability of Taq polymerase, an enzyme isolated from T. aquaticus. Degradation enzymes from thermophiles are added as ingredients in hot-water detergents, increasing their effectiveness.
Exercise \(1\)
1. What temperature requirements do most bacterial human pathogens have?
2. What DNA adaptation do thermophiles exhibit?
Feeding the World…and the World’s Algae
Artificial fertilizers have become an important tool in food production around the world. They are responsible for many of the gains of the so-called green revolution of the 20th century, which has allowed the planet to feed many of its more than 7 billion people. Artificial fertilizers provide nitrogen and phosphorus, key limiting nutrients, to crop plants, removing the normal barriers that would otherwise limit the rate of growth. Thus, fertilized crops grow much faster, and farms that use fertilizer produce higher crop yields.
However, careless use and overuse of artificial fertilizers have been demonstrated to have significant negative impacts on aquatic ecosystems, both freshwater and marine. Fertilizers that are applied at inappropriate times or in too-large quantities allow nitrogen and phosphorus compounds to escape use by crop plants and enter drainage systems. Inappropriate use of fertilizers in residential settings can also contribute to nutrient loads, which find their way to lakes and coastal marine ecosystems. As water warms and nutrients are plentiful, microscopic algae bloom, often changing the color of the water because of the high cell density.
Most algal blooms are not directly harmful to humans or wildlife; however, they can cause harm indirectly. As the algal population expands and then dies, it provides a large increase in organic matter to the bacteria that live in deep water. With this large supply of nutrients, the population of nonphotosynthetic microorganisms explodes, consuming available oxygen and creating “dead zones” where animal life has virtually disappeared.
Depletion of oxygen in the water is not the only damaging consequence of some algal blooms. The algae that produce red tides in the Gulf of Mexico, Karenia brevis, secrete potent toxins that can kill fish and other organisms and also accumulate in shellfish. Consumption of contaminated shellfish can cause severe neurological and gastrointestinal symptoms in humans. Shellfish beds must be regularly monitored for the presence of the toxins, and harvests are often shut down when it is present, incurring economic costs to the fishery. Cyanobacteria, which can form blooms in marine and freshwater ecosystems, produce toxins called microcystins, which can cause allergic reactions and liver damage when ingested in drinking water or during swimming. Recurring cyanobacterial algal blooms in Lake Erie (Figure \(3\)) have forced municipalities to issue drinking water bans for days at a time because of unacceptable toxin levels.
This is just a small sampling of the negative consequences of algal blooms, red tides, and dead zones. Yet the benefits of crop fertilizer—the main cause of such blooms—are difficult to dispute. There is no easy solution to this dilemma, as a ban on fertilizers is not politically or economically feasible. In lieu of this, we must advocate for responsible use and regulation in agricultural and residential contexts, as well as the restoration of wetlands, which can absorb excess fertilizers before they reach lakes and oceans.
Link to Learning
This video discusses algal blooms and dead zones in more depth.
Key Concepts and Summary
• Microorganisms thrive at a wide range of temperatures; they have colonized different natural environments and have adapted to extreme temperatures. Both extreme cold and hot temperatures require evolutionary adjustments to macromolecules and biological processes.
• Psychrophiles grow best in the temperature range of 0–15 °C whereas psychrotrophs thrive between 4 °C and 25 °C.
• Mesophiles grow best at moderate temperatures in the range of 20 °C to about 45 °C. Pathogens are usually mesophiles.
• Thermophiles and hyperthemophiles are adapted to life at temperatures above 50 °C.
• Adaptations to cold and hot temperatures require changes in the composition of membrane lipids and proteins. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/09%3A_Microbial_Growth/9.04%3A_Temperature_and_Microbial_Growth.txt |
Learning Objectives
• Identify and describe different categories of microbes with specific growth requirements other than oxygen, pH, and temperature, such as altered barometric pressure, osmotic pressure, humidity, and light
• Give at least one example microorganism for each category of growth requirement
Microorganisms interact with their environment along more dimensions than pH, temperature, and free oxygen levels, although these factors require significant adaptations. We also find microorganisms adapted to varying levels of salinity, barometric pressure, humidity, and light.
Osmotic and Barometric Pressure
Most natural environments tend to have lower solute concentrations than the cytoplasm of most microorganisms. Rigid cell walls protect the cells from bursting in a dilute environment. Not much protection is available against high osmotic pressure. In this case, water, following its concentration gradient, flows out of the cell. This results in plasmolysis (the shrinking of the protoplasm away from the intact cell wall) and cell death. This fact explains why brines and layering meat and fish in salt are time-honored methods of preserving food. Microorganisms called halophiles (“salt loving”) actually require high salt concentrations for growth. These organisms are found in marine environments where salt concentrations hover at 3.5%. Extreme halophilic microorganisms, such as the red alga Dunaliella salina and the archaeal species Halobacterium in Figure \(1\), grow in hypersaline lakes such as the Great Salt Lake, which is 3.5–8 times saltier than the ocean, and the Dead Sea, which is 10 times saltier than the ocean.
Dunaliella spp. counters the tremendous osmotic pressure of the environment with a high cytoplasmic concentration of glycerol and by actively pumping out salt ions. Halobacterium spp. accumulates large concentrations of K+ and other ions in its cytoplasm. Its proteins are designed for high salt concentrations and lose activity at salt concentrations below 1–2 M. Although most halotolerant organisms, for example Halomonas spp. in salt marshes, do not need high concentrations of salt for growth, they will survive and divide in the presence of high salt. Not surprisingly, the staphylococci, micrococci, and corynebacteria that colonize our skin tolerate salt in their environment. Halotolerant pathogens are an important cause of food-borne illnesses because they survive and multiply in salty food. For example, the halotolerant bacteria S. aureus, Bacillus cereus, and V. cholerae produce dangerous enterotoxins and are major causes of food poisoning.
Microorganisms depend on available water to grow. Available moisture is measured as water activity (aw), which is the ratio of the vapor pressure of the medium of interest to the vapor pressure of pure distilled water; therefore, the aw of water is equal to 1.0. Bacteria require high aw (0.97–0.99), whereas fungi can tolerate drier environments; for example, the range of aw for growth of Aspergillus spp. is 0.8–0.75. Decreasing the water content of foods by drying, as in jerky, or through freeze-drying or by increasing osmotic pressure, as in brine and jams, are common methods of preventing spoilage.
Microorganisms that require high atmospheric pressure for growth are called barophiles. The bacteria that live at the bottom of the ocean must be able to withstand great pressures. Because it is difficult to retrieve intact specimens and reproduce such growth conditions in the laboratory, the characteristics of these microorganisms are largely unknown.
Light
Photoautotrophs, such as cyanobacteria or green sulfur bacteria, and photoheterotrophs, such as purple nonsulfur bacteria, depend on sufficient light intensity at the wavelengths absorbed by their pigments to grow and multiply. Energy from light is captured by pigments and converted into chemical energy that drives carbon fixation and other metabolic processes. The portion of the electromagnetic spectrum that is absorbed by these organisms is defined as photosynthetically active radiation (PAR). It lies within the visible light spectrum ranging from 400 to 700 nanometers (nm) and extends in the near infrared for some photosynthetic bacteria. A number of accessory pigments, such as fucoxanthin in brown algae and phycobilins in cyanobacteria, widen the useful range of wavelengths for photosynthesis and compensate for the low light levels available at greater depths of water. Other microorganisms, such as the archaea of the class Halobacteria, use light energy to drive their proton and sodium pumps. The light is absorbed by a pigment protein complex called bacteriorhodopsin, which is similar to the eye pigment rhodopsin. Photosynthetic bacteria are present not only in aquatic environments but also in soil and in symbiosis with fungi in lichens. The peculiar watermelon snow is caused by a microalga Chlamydomonas nivalis, a green alga rich in a secondary red carotenoid pigment (astaxanthin) which gives the pink hue to the snow where the alga grows.
Exercise \(1\)
1. Which photosynthetic pigments were described in this section?
2. What is the fundamental stress of a hypersaline environment for a cell?
Key Concepts and Summary
• Halophiles require high salt concentration in the medium, whereas halotolerant organisms can grow and multiply in the presence of high salt but do not require it for growth.
• Halotolerant pathogens are an important source of foodborne illnesses because they contaminate foods preserved in salt.
• Photosynthetic bacteria depend on visible light for energy.
• Most bacteria, with few exceptions, require high moisture to grow. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/09%3A_Microbial_Growth/9.05%3A_Other_Environmental_Conditions_that_Affect_Growth.txt |
Learning Objectives
• Identify and describe culture media for the growth of bacteria, including examples of all-purpose media, enriched, selective, differential, defined, and enrichment media
The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive under laboratory conditions. Culturing many microorganisms is challenging because of highly specific nutritional and environmental requirements and the diversity of these requirements among different species.
Nutritional Requirements
The number of available media to grow bacteria is considerable. Some media are considered general all-purpose media and support growth of a large variety of organisms. A prime example of an all-purpose medium is tryptic soy broth (TSB). Specialized media are used in the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics. One type, enriched media, contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms, organisms that cannot make certain nutrients and require them to be added to the medium. When the complete chemical composition of a medium is known, it is called a chemically defined medium. For example, in EZ medium, all individual chemical components are identified and the exact amounts of each is known. In complex media, which contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known. Amounts of individual components are undetermined and variable. Nutrient broth, tryptic soy broth, and brain heart infusion, are all examples of complex media.
Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by supplying nutrients and reducing competition are called selective media. An example of a selective medium is MacConkey agar. It contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria, particularly the Enterobacteriaceae. These species are commonly named enterics, reside in the intestine, and are adapted to the presence of bile salts. The enrichment cultures foster the preferential growth of a desired microorganism that represents a fraction of the organisms present in an inoculum. For example, if we want to isolate bacteria that break down crude oil, hydrocarbonoclastic bacteria, sequential subculturing in a medium that supplies carbon only in the form of crude oil will enrich the cultures with oil-eating bacteria. The differential media make it easy to distinguish colonies of different bacteria by a change in the color of the colonies or the color of the medium. Color changes are the result of end products created by interaction of bacterial enzymes with differential substrates in the medium or, in the case of hemolytic reactions, the lysis of red blood cells in the medium. In Figure \(1\), the differential fermentation of lactose can be observed on MacConkey agar. The lactose fermenters produce acid, which turns the medium and the colonies of strong fermenters hot pink. The medium is supplemented with the pH indicator neutral red, which turns to hot pink at low pH. Selective and differential media can be combined and play an important role in the identification of bacteria by biochemical methods.
Exercise \(1\)
1. Distinguish complex and chemically defined media.
2. Distinguish selective and enrichment media.
Link to Learning
Compare the compositions of EZ medium and sheep blood agar.
The End-of-Year Picnic
The microbiology department is celebrating the end of the school year in May by holding its traditional picnic on the green. The speeches drag on for a couple of hours, but finally all the faculty and students can dig into the food: chicken salad, tomatoes, onions, salad, and custard pie. By evening, the whole department, except for two vegetarian students who did not eat the chicken salad, is stricken with nausea, vomiting, retching, and abdominal cramping. Several individuals complain of diarrhea. One patient shows signs of shock (low blood pressure). Blood and stool samples are collected from patients, and an analysis of all foods served at the meal is conducted.
Bacteria can cause gastroenteritis (inflammation of the stomach and intestinal tract) either by colonizing and replicating in the host, which is considered an infection, or by secreting toxins, which is considered intoxication. Signs and symptoms of infections are typically delayed, whereas intoxication manifests within hours, as happened after the picnic.
Blood samples from the patients showed no signs of bacterial infection, which further suggests that this was a case of intoxication. Since intoxication is due to secreted toxins, bacteria are not usually detected in blood or stool samples. MacConkey agar and sorbitol-MacConkey agar plates and xylose-lysine-deoxycholate (XLD) plates were inoculated with stool samples and did not reveal any unusually colored colonies, and no black colonies or white colonies were observed on XLD. All lactose fermenters on MacConkey agar also ferment sorbitol. These results ruled out common agents of food-borne illnesses: E. coli, Salmonella spp., and Shigella spp.
Analysis of the chicken salad revealed an abnormal number of gram-positive cocci arranged in clusters (Figure \(2\)). A culture of the gram-positive cocci releases bubbles when mixed with hydrogen peroxide. The culture turned mannitol salt agar yellow after a 24-hour incubation.
All the tests point to Staphylococcus aureus as the organism that secreted the toxin. Samples from the salad showed the presence of gram-positive cocci bacteria in clusters. The colonies were positive for catalase. The bacteria grew on mannitol salt agar fermenting mannitol, as shown by the change to yellow of the medium. The pH indicator in mannitol salt agar is phenol red, which turns to yellow when the medium is acidified by the products of fermentation.
The toxin secreted by S. aureus is known to cause severe gastroenteritis. The organism was probably introduced into the salad during preparation by the food handler and multiplied while the salad was kept in the warm ambient temperature during the speeches.
Exercise \(2\)
1. What are some other factors that might have contributed to rapid growth of S. aureus in the chicken salad?
2. Why would S. aureus not be inhibited by the presence of salt in the chicken salad?
Key Concepts and Summary
• Chemically defined media contain only chemically known components.
• Selective media favor the growth of some microorganisms while inhibiting others.
• Enriched media contain added essential nutrients a specific organism needs to grow
• Differential media help distinguish bacteria by the color of the colonies or the change in the medium. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/09%3A_Microbial_Growth/9.06%3A_Media_Used_for_Bacterial_Growth.txt |
9.1: How Microbes Grow
The bacterial cell cycle involves the formation of new cells through the replication of DNA and partitioning of cellular components into two daughter cells. In prokaryotes, reproduction is always asexual, although extensive genetic recombination in the form of horizontal gene transfer takes place, as will be explored in a different chapter. Most bacteria have a single circular chromosome; however, some exceptions exist.
Multiple Choice
Which of the following methods would be used to measure the concentration of bacterial contamination in processed peanut butter?
1. turbidity measurement
2. total plate count
3. dry weight measurement
4. direct counting of bacteria on a calibrated slide under the microscope
Answer
B
In which phase would you expect to observe the most endospores in a Bacillus cell culture?
1. death phase
2. lag phase
3. log phase
4. log, lag, and death phases would all have roughly the same number of endospores.
Answer
A
During which phase would penicillin, an antibiotic that inhibits cell-wall synthesis, be most effective?
1. death phase
2. lag phase
3. log phase
4. stationary phase
Answer
C
Which of the following is the best definition of generation time in a bacterium?
1. the length of time it takes to reach the log phase
2. the length of time it takes for a population of cells to double
3. the time it takes to reach stationary phase
4. the length of time of the exponential phase
Answer
B
What is the function of the Z ring in binary fission?
1. It controls the replication of DNA.
2. It forms a contractile ring at the septum.
3. It separates the newly synthesized DNA molecules.
4. It mediates the addition of new peptidoglycan subunits.
Answer
B
If a culture starts with 50 cells, how many cells will be present after five generations with no cell death?
1. 200
2. 400
3. 1600
4. 3200
Answer
C
Filamentous cyanobacteria often divide by which of the following?
1. budding
2. mitosis
3. fragmentation
4. formation of endospores
Answer
C
Which is a reason for antimicrobial resistance being higher in a biofilm than in free-floating bacterial cells?
1. The EPS allows faster diffusion of chemicals in the biofilm.
2. Cells are more metabolically active at the base of a biofilm.
3. Cells are metabolically inactive at the base of a biofilm.
4. The structure of a biofilm favors the survival of antibiotic resistant cells.
Answer
C
Quorum sensing is used by bacterial cells to determine which of the following?
1. the size of the population
2. the availability of nutrients
3. the speed of water flow
4. the density of the population
Answer
D
Which of the following statements about autoinducers is incorrect?
1. They bind directly to DNA to activate transcription.
2. They can activate the cell that secreted them.
3. N-acylated homoserine lactones are autoinducers in gram-negative cells.
4. Autoinducers may stimulate the production of virulence factors.
Answer
A
Fill in the Blank
Direct count of total cells can be performed using a ________ or a ________.
Answer
hemocytometer, Petroff-Hausser counting chamber
The ________ method allows direct count of total cells growing on solid medium.
Answer
plate count
A statistical estimate of the number of live cells in a liquid is usually done by ________.
Answer
most probable number
For this indirect method of estimating the growth of a culture, you measure ________ using a spectrophotometer.
Answer
turbidity
Active growth of a culture may be estimated indirectly by measuring the following products of cell metabolism: ________ or ________.
Answer
ATP, acid from fermentation
Matching
Match the definition with the name of the growth phase in the growth curve.
___Number of dying cells is higher than the number of cells dividing A. Lag phase
___Number of new cells equal to number of dying cells B. Log phase
___New enzymes to use available nutrients are induced C. Stationary phase
___Binary fission is occurring at maximum rate D. Death phase
Answer
D, C, A, B
Short Answer
Why is it important to measure the transmission of light through a control tube with only broth in it when making turbidity measures of bacterial cultures?
In terms of counting cells, what does a plating method accomplish that an electronic cell counting method does not?
Order the following stages of the development of a biofilm from the earliest to the last step.
1. secretion of EPS
2. reversible attachment
3. dispersal
4. formation of water channels
5. irreversible attachment
Infections among hospitalized patients are often related to the presence of a medical device in the patient. Which conditions favor the formation of biofilms on in-dwelling catheters and prostheses?
Critical Thinking
A patient in the hospital has an intravenous catheter inserted to allow for the delivery of medications, fluids, and electrolytes. Four days after the catheter is inserted, the patient develops a fever and an infection in the skin around the catheter. Blood cultures reveal that the patient has a blood-borne infection. Tests in the clinical laboratory identify the blood-borne pathogen as Staphylococcus epidermidis, and antibiotic susceptibility tests are performed to provide doctors with essential information for selecting the best drug for treatment of the infection. Antibacterial chemotherapy is initiated and delivered through the intravenous catheter that was originally inserted into the patient. Within 7 days, the skin infection is gone, blood cultures are negative for S. epidermidis, and the antibacterial chemotherapy is discontinued. However, 2 days after discontinuing the antibacterial chemotherapy, the patient develops another fever and skin infection and the blood cultures are positive for the same strain of S. epidermidis that had been isolated the previous week. This time, doctors remove the intravenous catheter and administer oral antibiotics, which successfully treat both the skin and blood-borne infection caused by S. epidermidis. Furthermore, the infection does not return after discontinuing the oral antibacterial chemotherapy. What are some possible reasons why intravenous chemotherapy failed to completely cure the patient despite laboratory tests showing the bacterial strain was susceptible to the prescribed antibiotic? Why might the second round of antibiotic therapy have been more successful? Justify your answers.
Why are autoinducers small molecules?
Refer to Figure B.1 in Appendix B. If the results from a pond water sample were recorded as 3, 2, 1, what would be the MPN of bacteria in 100 mL of pond water?
Refer to Figure 9.1.14. Why does turbidity lose reliability at high cell concentrations when the culture reaches the stationary phase?
9.2: Oxygen Requirements for Microbial Growth
Ask most people “What are the major requirements for life?” and the answers are likely to include water and oxygen. Few would argue about the need for water, but what about oxygen? Can there be life without oxygen? The answer is that molecular oxygen is not always needed. The earliest signs of life are dated to a period when conditions on earth were highly reducing and free oxygen gas was essentially nonexistent.
Multiple Choice
An inoculated thioglycolate medium culture tube shows dense growth at the surface and turbidity throughout the rest of the tube. What is your conclusion?
1. The organisms die in the presence of oxygen
2. The organisms are facultative anaerobes.
3. The organisms should be grown in an anaerobic chamber.
4. The organisms are obligate aerobes.
Answer
B
An inoculated thioglycolate medium culture tube is clear throughout the tube except for dense growth at the bottom of the tube. What is your conclusion?
1. The organisms are obligate anaerobes.
2. The organisms are facultative anaerobes.
3. The organisms are aerotolerant.
4. The organisms are obligate aerobes.
Answer
A
Pseudomonas aeruginosa is a common pathogen that infects the airways of patients with cystic fibrosis. It does not grow in the absence of oxygen. The bacterium is probably which of the following?
1. an aerotolerant anaerobe
2. an obligate aerobe
3. an obligate anaerobe
4. a facultative anaerobe
Answer
B
Streptococcus mutans is a major cause of cavities. It resides in the gum pockets, does not have catalase activity, and can be grown outside of an anaerobic chamber. The bacterium is probably which of the following?
1. a facultative anaerobe
2. an obligate aerobe
3. an obligate anaerobe
4. an aerotolerant anaerobe
Answer
D
Why do the instructions for the growth of Neisseria gonorrheae recommend a CO2-enriched atmosphere?
1. It uses CO2 as a final electron acceptor in respiration.
2. It is an obligate anaerobe.
3. It is a capnophile.
4. It fixes CO2 through photosynthesis.
Answer
C
Matching
Four tubes are illustrated with cultures grown in a medium that slows oxygen diffusion. Match the culture tube with the correct type of bacteria from the following list: facultative anaerobe, obligate anaerobe, microaerophile, aerotolerant anaerobe, obligate aerobe.
Answer
(a) obligate anaerobe, (b) obligate aerobe, (c) microaerophile, (d) facultative anaerobe
Short Answer
Why are some obligate anaerobes able to grow in tissues (e.g., gum pockets) that are not completely free of oxygen?
Why should Haemophilus influenzae be grown in a candle jar?
In terms of oxygen requirements, what type of organism would most likely be responsible for a foodborne illness associated with canned foods?
Critical Thinking
A microbiology instructor prepares cultures for a gram-staining practical laboratory by inoculating growth medium with a gram-positive coccus (nonmotile) and a gram-negative rod (motile). The goal is to demonstrate staining of a mixed culture. The flask is incubated at 35 °C for 24 hours without aeration. A sample is stained and reveals only gram-negative rods. Both cultures are known facultative anaerobes. Give a likely reason for success of the gram-negative rod. Assume that the cultures have comparable intrinsic growth rates.
9.3: The Effects of pH on Microbial Growth
Bacteria are generally neutrophiles. They grow best at neutral pH close to 7.0. Acidophiles grow optimally at a pH near 3.0. Alkaliphiles are organisms that grow optimally between a pH of 8 and 10.5. Extreme acidophiles and alkaliphiles grow slowly or not at all near neutral pH. Microorganisms grow best at their optimum growth pH. Growth occurs slowly or not at all below the minimum growth pH and above the maximum growth pH.
Multiple Choice
Bacteria that grow in mine drainage at pH 1–2 are probably which of the following?
1. alkaliphiles
2. acidophiles
3. neutrophiles
4. obligate anaerobes
Answer
B
Bacteria isolated from Lake Natron, where the water pH is close to 10, are which of the following?
1. alkaliphiles
2. facultative anaerobes
3. neutrophiles
4. obligate anaerobes
Answer
A
In which environment are you most likely to encounter an acidophile?
1. human blood at pH 7.2
2. a hot vent at pH 1.5
3. human intestine at pH 8.5
4. milk at pH 6.5
Answer
B
Fill in the Blank
A bacterium that thrives in a soda lake where the average pH is 10.5 can be classified as a(n) ________.
Answer
alkaliphile
Lactobacillus acidophilus grows best at pH 4.5. It is considered a(n) ________.
Answer
acidophile
Short Answer
Which macromolecule in the cell is most sensitive to changes in pH?
Which metabolic process in the bacterial cell is particularly challenging at high pH?
Critical Thinking
People who use proton pumps inhibitors or antacids are more prone to infections of the gastrointestinal tract. Can you explain the observation in light of what you have learned?
9.4: Temperature and Microbial Growth
Microorganisms thrive at a wide range of temperatures; they have colonized different natural environments and have adapted to extreme temperatures. Both extreme cold and hot temperatures require evolutionary adjustments to macromolecules and biological processes. Psychrophiles grow best in the temperature range of 0–15 °C whereas psychrotrophs thrive between 4 °C and 25 °C. Mesophiles grow best at moderate temperatures in the range of 20 °C to about 45 °C. Pathogens are usually mesophiles.
Multiple Choice
A soup container was forgotten in the refrigerator and shows contamination. The contaminants are probably which of the following?
1. thermophiles
2. acidophiles
3. mesophiles
4. psychrotrophs
Answer
D
Bacteria isolated from a hot tub at 39 °C are probably which of the following?
1. thermophiles
2. psychrotrophs
3. mesophiles
4. hyperthermophiles
Answer
C
In which environment are you most likely to encounter a hyperthermophile?
1. hot tub
2. warm ocean water in Florida
3. hydrothermal vent at the bottom of the ocean
4. human body
Answer
C
Which of the following environments would harbor psychrophiles?
1. mountain lake with a water temperature of 12 °C
2. contaminated plates left in a 35 °C incubator
3. yogurt cultured at room temperature
4. salt pond in the desert with a daytime temperature of 34 °C
Answer
A
Matching
Match the type of bacterium with its environment. Each choice may be used once, more than once, or not at all. Put the appropriate letter beside the environment.
___psychotroph A. water heater set at 50 °C
___mesophile B. hydrothermal vent
___thermophile C. deep ocean waters
___hyperthermophile D. human pathogen
___psychrophile E. soil bacteria in temperate forest
Answer
C, D, E, B, A
Short Answer
How are hyperthermophile’s proteins adapted to the high temperatures of their environment?
Why would NASA be funding microbiology research in Antarctica?
Critical Thinking
The bacterium that causes Hansen’s disease (leprosy), Mycobacterium leprae, infects mostly the extremities of the body: hands, feet, and nose. Can you make an educated guess as to its optimum temperature of growth?
Refer to Figure 9.4.2. Some hyperthermophiles can survive autoclaving temperatures. Are they a concern in health care?
9.5: Other Environmental Conditions that Affect Growth
Microorganisms interact with their environment along more dimensions than pH, temperature, and free oxygen levels, although these factors require significant adaptations. We also find microorganisms adapted to varying levels of salinity, barometric pressure, humidity, and light.
Multiple Choice
Which of the following is the reason jams and dried meats often do not require refrigeration to prevent spoilage?
1. low pH
2. toxic alkaline chemicals
3. naturally occurring antibiotics
4. low water activity
Answer
D
Bacteria living in salt marshes are most likely which of the following?
1. acidophiles
2. barophiles
3. halotolerant
4. thermophiles
Answer
C
Fill in the Blank
A bacterium that thrives in the Great Salt Lake but not in fresh water is probably a ________.
Answer
halophile
Bacteria isolated from the bottom of the ocean need high atmospheric pressures to survive. They are ________.
Answer
barophiles
Staphylococcus aureus can be grown on multipurpose growth medium or on mannitol salt agar that contains 7.5% NaCl. The bacterium is ________.
Answer
halotolerant
Short Answer
Fish sauce is a salty condiment produced using fermentation. What type of organism is likely responsible for the fermentation of the fish sauce?
9.6: Media Used for Bacterial Growth
The study of microorganisms is greatly facilitated if we are able to culture them, that is, to keep reproducing populations alive under laboratory conditions. Culturing many microorganisms is challenging because of highly specific nutritional and environmental requirements and the diversity of these requirements among different species.
Multiple Choice
EMB agar is a medium used in the identification and isolation of pathogenic bacteria. It contains digested meat proteins as a source of organic nutrients. Two indicator dyes, eosin and methylene blue, inhibit the growth of gram-positive bacteria and distinguish between lactose fermenting and nonlactose fermenting organisms. Lactose fermenters form metallic green or deep purple colonies, whereas the nonlactose fermenters form completely colorless colonies. EMB agar is an example of which of the following?
1. a selective medium only
2. a differential medium only
3. a selective medium and a chemically defined medium
4. a selective medium, a differential medium, and a complex medium
Answer
D
Haemophilus influenzae must be grown on chocolate agar, which is blood agar treated with heat to release growth factors in the medium. H. influenzae is described as ________.
1. an acidophile
2. a thermophile
3. an obligate anaerobe
4. fastidious
Answer
D
Fill in the Blank
Blood agar contains many unspecified nutrients, supports the growth of a large number of bacteria, and allows differentiation of bacteria according to hemolysis (breakdown of blood). The medium is ________ and ________.
Answer
complex, differential
Rogosa agar contains yeast extract. The pH is adjusted to 5.2 and discourages the growth of many microorganisms; however, all the colonies look similar. The medium is ________ and ________.
Answer
complex, selective
Short Answer
What is the major difference between an enrichment culture and a selective culture?
Critical Thinking
Haemophilus, influenzae grows best at 35–37 °C with ~5% CO2 (or in a candle-jar) and requires hemin (X factor) and nicotinamide-adenine-dinucleotide (NAD, also known as V factor) for growth.1 Using the vocabulary learned in this chapter, describe H. influenzae.
Footnotes
1. 1 Centers for Disease Control and Prevention, World Health Organization. “CDC Laboratory Methods for the Diagnosis of Meningitis Caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenza. WHO Manual, 2nd edition.” 2011. http://www.cdc.gov/meningitis/lab-ma...ull-manual.pdf | textbooks/bio/Microbiology/Microbiology_(OpenStax)/09%3A_Microbial_Growth/9.E%3A_Microbial_Growth_%28Exercises%29.txt |
Children inherit some characteristics from each parent. Siblings typically look similar to each other, but not exactly the same—except in the case of identical twins. How can we explain these phenomena? The answers lie in heredity (the transmission of traits from one generation to the next) and genetics (the science of heredity). Because humans reproduce sexually, 50% of a child’s genes come from the mother’s egg cell and the remaining 50% from the father’s sperm cell. Sperm and egg are formed through the process of meiosis, where DNA recombination occurs. Thus, there is no predictable pattern as to which 50% comes from which parent. Thus, siblings have only some genes, and their associated characteristics, in common. Identical twins are the exception, because they are genetically identical.
Genetic differences among related microbes also dictate many observed biochemical and virulence differences. For example, some strains of the bacterium Escherichia coli are harmless members of the normal microbiota in the human gastrointestinal tract. Other strains of the same species have genes that give them the ability to cause disease. In bacteria, such genes are not inherited via sexual reproduction, as in humans. Often, they are transferred via plasmids, small circular pieces of double-stranded DNA that can be exchanged between prokaryotes.
• 10.1: Using Microbiology to Discover the Secrets of Life
DNA was discovered and characterized long before its role in heredity was understood. Microbiologists played significant roles in demonstrating that DNA is the hereditary information found within cells. In the 1850s and 1860s, Gregor Mendel experimented with true-breeding garden peas to demonstrate the heritability of specific observable traits. In 1869, Friedrich Miescher isolated and purified a compound rich in phosphorus from the nuclei of white blood cells; he named the compound nuclein.
• 10.2: Structure and Function of DNA
Nucleic acids are composed of nucleotides, each of which contains a pentose sugar, a phosphate group, and a nitrogenous base. Deoxyribonucleotides within DNA contain deoxyribose as the pentose sugar. DNA contains the pyrimidines cytosine and thymine, and the purines adenine and guanine. Nucleotides are linked together by phosphodiester bonds between the 5ʹ phosphate group of one nucleotide and the 3ʹ hydroxyl group of another.
• 10.3: Structure and Function of RNA
Ribonucleic acid (RNA) is typically single stranded and contains ribose as its pentose sugar and the pyrimidine uracil instead of thymine. An RNA strand can undergo significant intramolecular base pairing to take on a three-dimensional structure. There are three main types of RNA, all involved in protein synthesis. Messenger RNA (mRNA) serves as the intermediary between DNA and the synthesis of protein products during translation.
• 10.4: The Structure and Function of Cellular Genomes
The entire genetic content of a cell is its genome. Genes code for proteins, or stable RNA molecules, each of which carries out a specific function in the cell. Although the genotype that a cell possesses remains constant, expression of genes is dependent on environmental conditions. A phenotype is the observable characteristics of a cell (or organism) at a given point in time and results from the complement of genes currently being used.
• 10.E: Biochemistry of the Genome (Exercises)
Thumbnail: In the laboratory, the double helix can be denatured to single-stranded DNA through exposure to heat or chemicals, and then renatured through cooling or removal of chemical denaturants to allow the DNA strands to reanneal. (credit: modification of work by Hernández-Lemus E, Nicasio-Collazo LA, Castañeda-Priego R)
10: Biochemistry of the Genome
Learning Objectives
• Describe the discovery of nucleic acid and nucleotides
• Explain the historical experiments that led to the characterization of DNA
• Describe how microbiology and microorganisms have been used to discover the biochemistry of genes
• Explain how scientists established the link between DNA and heredity
Clinical Focus: Part 1
Alex is a 22-year-old college student who vacationed in Puerta Vallarta, Mexico, for spring break. Unfortunately, two days after flying home to Ohio, he began to experience abdominal cramping and extensive watery diarrhea. Because of his discomfort, he sought medical attention at a large Cincinnati hospital nearby.
Exercise \(1\)
What types of infections or other conditions may be responsible?
Through the early 20th century, DNA was not yet recognized as the genetic material responsible for heredity, the passage of traits from one generation to the next. In fact, much of the research was dismissed until the mid-20th century. The scientific community believed, incorrectly, that the process of inheritance involved a blending of parental traits that produced an intermediate physical appearance in offspring; this hypothetical process appeared to be correct because of what we know now as continuous variation, which results from the action of many genes to determine a particular characteristic, like human height. Offspring appear to be a “blend” of their parents’ traits when we look at characteristics that exhibit continuous variation. The blending theory of inheritance asserted that the original parental traits were lost or absorbed by the blending in the offspring, but we now know that this is not the case.
Two separate lines of research, begun in the mid to late 1800s, ultimately led to the discovery and characterization of DNA and the foundations of genetics, the science of heredity. These lines of research began to converge in the 1920s, and research using microbial systems ultimately resulted in significant contributions to elucidating the molecular basis of genetics.
Discovery and Characterization of DNA
Modern understanding of DNA has evolved from the discovery of nucleic acid to the development of the double-helix model. In the 1860s, Friedrich Miescher (1844–1895), a physician by profession, was the first person to isolate phosphorus-rich chemicals from leukocytes (white blood cells) from the pus on used bandages from a local surgical clinic. He named these chemicals (which would eventually be known as RNA and DNA) “nuclein” because they were isolated from the nuclei of the cells. His student Richard Altmann (1852–1900) subsequently termed it “nucleic acid” 20 years later when he discovered the acidic nature of nuclein. In the last two decades of the 19th century, German biochemist Albrecht Kossel (1853–1927) isolated and characterized the five different nucleotide bases composing nucleic acid. These are adenine, guanine, cytosine, thymine (in DNA), and uracil (in RNA). Kossell received the Nobel Prize in Physiology or Medicine in 1910 for his work on nucleic acids and for his considerable work on proteins, including the discovery of histidine.
Foundations of Genetics
Despite the discovery of DNA in the late 1800s, scientists did not make the association with heredity for many more decades. To make this connection, scientists, including a number of microbiologists, performed many experiments on plants, animals, and bacteria.
Mendel’s Pea Plants
While Miescher was isolating and discovering DNA in the 1860s, Austrian monk and botanist Johann Gregor Mendel(1822–1884) was experimenting with garden peas, demonstrating and documenting basic patterns of inheritance, now known as Mendel’s laws.
In 1856, Mendel began his decade-long research into inheritance patterns. He used the diploid garden pea, Pisum sativum, as his primary model system because it naturally self-fertilizes and is highly inbred, producing “true-breeding” pea plant lines—plants that always produce offspring that look like the parent. By experimenting with true-breeding pea plants, Mendel avoided the appearance of unexpected traits in offspring that might occur if he used plants that were not true-breeding. Mendel performed hybridizations, which involve mating two true-breeding individuals (P generation) that have different traits, and examined the characteristics of their offspring (first filial generation, F1) as well as the offspring of self-fertilization of the F1 generation (second filial generation, F2) (Figure \(1\)).
In 1865, Mendel presented the results of his experiments with nearly 30,000 pea plants to the local natural history society. He demonstrated that traits are transmitted faithfully from parents to offspring independently of other traits. In 1866, he published his work, “Experiments in Plant Hybridization,”1 in the Proceedings of the Natural History Society of Brünn. Mendel’s work went virtually unnoticed by the scientific community, which believed, incorrectly, in the theory of blending of traits in continuous variation.
He was not recognized for his extraordinary scientific contributions during his lifetime. In fact, it was not until 1900 that his work was rediscovered, reproduced, and revitalized by scientists on the brink of discovering the chromosomal basis of heredity.
The Chromosomal Theory of Inheritance
Mendel carried out his experiments long before chromosomes were visualized under a microscope. However, with the improvement of microscopic techniques during the late 1800s, cell biologists could stain and visualize subcellular structures with dyes and observe their actions during meiosis. They were able to observe chromosomes replicating, condensing from an amorphous nuclear mass into distinct X-shaped bodies and migrating to separate cellular poles. The speculation that chromosomes might be the key to understanding heredity led several scientists to examine Mendel’s publications and re-evaluate his model in terms of the behavior of chromosomes during mitosis and meiosis.
In 1902, Theodor Boveri (1862–1915) observed that in sea urchins, nuclear components (chromosomes) determined proper embryonic development. That same year, Walter Sutton (1877–1916) observed the separation of chromosomes into daughter cells during meiosis. Together, these observations led to the development of the Chromosomal Theory of Inheritance, which identified chromosomes as the genetic material responsible for Mendelian inheritance.
Despite compelling correlations between the behavior of chromosomes during meiosis and Mendel’s observations, the Chromosomal Theory of Inheritance was proposed long before there was any direct evidence that traits were carried on chromosomes. Thomas Hunt Morgan (1866–1945) and his colleagues spent several years carrying out crosses with the fruit fly, Drosophila melanogaster. They performed meticulous microscopic observations of fly chromosomes and correlated these observations with resulting fly characteristics. Their work provided the first experimental evidence to support the Chromosomal Theory of Inheritance in the early 1900s. In 1915, Morgan and his “Fly Room” colleagues published The Mechanism of Mendelian Heredity, which identified chromosomes as the cellular structures responsible for heredity. For his many significant contributions to genetics, Morgan received the Nobel Prize in Physiology or Medicine in 1933.
In the late 1920s, Barbara McClintock (1902–1992) developed chromosomal staining techniques to visualize and differentiate between the different chromosomes of maize (corn). In the 1940s and 1950s, she identified a breakage event on chromosome 9, which she named the dissociation locus (Ds). Ds could change position within the chromosome. She also identified an activator locus (Ac). Ds chromosome breakage could be activated by an Ac element (transposase enzyme). At first, McClintock’s finding of these jumping genes, which we now call transposons, was not accepted by the scientific community. It wasn’t until the 1960s and later that transposons were discovered in bacteriophages, bacteria, and Drosophila. Today, we know that transposons are mobile segments of DNA that can move within the genome of an organism. They can regulate gene expression, protein expression, and virulence (ability to cause disease).
Microbes and Viruses in Genetic Research
Microbiologists have also played a crucial part in our understanding of genetics. Experimental organisms such as Mendel’s garden peas, Morgan’s fruit flies, and McClintock’s corn had already been used successfully to pave the way for an understanding of genetics. However, microbes and viruses were (and still are) excellent model systems for the study of genetics because, unlike peas, fruit flies, and corn, they are propagated more easily in the laboratory, growing to high population densities in a small amount of space and in a short time. In addition, because of their structural simplicity, microbes and viruses are more readily manipulated genetically.
Fortunately, despite significant differences in size, structure, reproduction strategies, and other biological characteristics, there is biochemical unity among all organisms; they have in common the same underlying molecules responsible for heredity and the use of genetic material to give cells their varying characteristics. In the words of French scientist Jacques Monod, “What is true for E. coli is also true for the elephant,” meaning that the biochemistry of life has been maintained throughout evolution and is shared in all forms of life, from simple unicellular organisms to large, complex organisms. This biochemical continuity makes microbes excellent models to use for genetic studies.
In a clever set of experiments in the 1930s and 1940s, German scientist Joachim Hämmerling (1901–1980), using the single-celled alga Acetabularia as a microbial model, established that the genetic information in a eukaryotic cell is housed within the nucleus. Acetabularia spp. are unusually large algal cells that grow asymmetrically, forming a “foot” containing the nucleus, which is used for substrate attachment; a stalk; and an umbrella-like cap—structures that can all be easily seen with the naked eye. In an early set of experiments, Hämmerling removed either the cap or the foot of the cells and observed whether new caps or feet were regenerated (Figure \(2\)). He found that when the foot of these cells was removed, new feet did not grow; however, when caps were removed from the cells, new caps were regenerated. This suggested that the hereditary information was located in the nucleus-containing foot of each cell.
In another set of experiments, Hämmerling used two species of Acetabularia that have different cap morphologies, A. crenulata and A. mediterranea (Figure \(3\)). He cut the caps from both types of cells and then grafted the stalk from an A. crenulata onto an A. mediterranea foot, and vice versa. Over time, he observed that the grafted cell with the A. crenulata foot and A. mediterranea stalk developed a cap with the A. crenulata morphology. Conversely, the grafted cell with the A. mediterranea foot and A. crenulata stalk developed a cap with the A. mediterranea morphology. He microscopically confirmed the presence of nuclei in the feet of these cells and attributed the development of these cap morphologies to the nucleus of each grafted cell. Thus, he showed experimentally that the nucleus was the location of genetic material that dictated a cell’s properties.
Another microbial model, the red bread mold Neurospora crassa, was used by George Beadle and Edward Tatum to demonstrate the relationship between genes and the proteins they encode. Beadle had worked with fruit flies in Morgan’s laboratory but found them too complex to perform certain types of experiments. N. crassa, on the other hand, is a simpler organism and has the ability to grow on a minimal medium because it contains enzymatic pathways that allow it to use the medium to produce its own vitamins and amino acids.
Beadle and Tatum irradiated the mold with X-rays to induce changes to a sequence of nucleic acids, called mutations. They mated the irradiated mold spores and attempted to grow them on both a complete medium and a minimal medium. They looked for mutants that grew on a complete medium, supplemented with vitamins and amino acids, but did not grow on the minimal medium lacking these supplements. Such molds theoretically contained mutations in the genes that encoded biosynthetic pathways. Upon finding such mutants, they systematically tested each to determine which vitamin or amino acid it was unable to produce (Figure \(4\)) and published this work in 1941.
Subsequent work by Beadle, Tatum, and colleagues showed that they could isolate different classes of mutants that required a particular supplement, like the amino acid arginine (Figure \(5\)). With some knowledge of the arginine biosynthesis pathway, they identified three classes of arginine mutants by supplementing the minimal medium with intermediates (citrulline or ornithine) in the pathway. The three mutants differed in their abilities to grow in each of the media, which led the group of scientists to propose, in 1945, that each type of mutant had a defect in a different gene in the arginine biosynthesis pathway. This led to the so-called one gene–one enzyme hypothesis, which suggested that each gene encodes one enzyme.
Subsequent knowledge about the processes of transcription and translation led scientists to revise this to the “one gene–one polypeptide” hypothesis. Although there are some genes that do not encode polypeptides (but rather encode for transfer RNAs [tRNAs] or ribosomal RNAs [rRNAs], which we will discuss later), the one gene–one enzyme hypothesis is true in many cases, especially in microbes. Beadle and Tatum’s discovery of the link between genes and corresponding characteristics earned them the 1958 Nobel Prize in Physiology and Medicine and has since become the basis for modern molecular genetics.
Link to Learning
To learn more about the experiments of Beadle and Tatum, visit this website from the DNA Learning Center.
Exercise \(2\)
1. What organism did Morgan and his colleagues use to develop the Chromosomal Theory of Inheritance? What traits did they track?
2. What did Hämmerling prove with his experiments on Acetabularia?
DNA as the Molecule Responsible for Heredity
By the beginning of the 20th century, a great deal of work had already been done on characterizing DNA and establishing the foundations of genetics, including attributing heredity to chromosomes found within the nucleus. Despite all of this research, it was not until well into the 20th century that these lines of research converged and scientists began to consider that DNA could be the genetic material that offspring inherited from their parents. DNA, containing only four different nucleotides, was thought to be structurally too simple to encode such complex genetic information. Instead, protein was thought to have the complexity required to serve as cellular genetic information because it is composed of 20 different amino acids that could be combined in a huge variety of combinations. Microbiologists played a pivotal role in the research that determined that DNA is the molecule responsible for heredity.
Griffith’s Transformation Experiments
British bacteriologist Frederick Griffith (1879–1941) was perhaps the first person to show that hereditary information could be transferred from one cell to another “horizontally” (between members of the same generation), rather than “vertically” (from parent to offspring). In 1928, he reported the first demonstration of bacterial transformation, a process in which external DNA is taken up by a cell, thereby changing its characteristics.3 He was working with two strains of Streptococcus pneumoniae, a bacterium that causes pneumonia: a rough (R) strain and a smooth (S) strain. The R strain is nonpathogenic and lacks a capsule on its outer surface; as a result, colonies from the R strain appear rough when grown on plates. The S strain is pathogenic and has a capsule outside its cell wall, allowing it to escape phagocytosis by the host immune system. The capsules cause colonies from the S strain to appear smooth when grown on plates.
In a series of experiments, Griffith analyzed the effects of live R, live S, and heat-killed S strains of S. pneumoniae on live mice (Figure \(6\)). When mice were injected with the live S strain, the mice died. When he injected the mice with the live R strain or the heat-killed S strain, the mice survived. But when he injected the mice with a mixture of live R strain and heat-killed S strain, the mice died. Upon isolating the live bacteria from the dead mouse, he only recovered the S strain of bacteria. When he then injected this isolated S strain into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and “transformed” it into the pathogenic S strain; he called this the “transforming principle.” These experiments are now famously known as Griffith’s transformation experiments.
In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty were interested in exploring Griffith’s transforming principle further. They isolated the S strain from infected dead mice, heat-killed it, and inactivated various components of the S extract, conducting a systematic elimination study (Figure \(7\)). They used enzymes that specifically degraded proteins, RNA, and DNA and mixed the S extract with each of these individual enzymes. Then, they tested each extract/enzyme combination’s resulting ability to transform the R strain, as observed by the diffuse growth of the S strain in culture media and confirmed visually by growth on plates. They found that when DNA was degraded, the resulting mixture was no longer able to transform the R strain bacteria, whereas no other enzymatic treatment was able to prevent transformation. This led them to conclude that DNA was the transforming principle. Despite their results, many scientists did not accept their conclusion, instead believing that there were protein contaminants within their extracts.
Exercise \(3\)
How did Avery, MacLeod, and McCarty’s experiments show that DNA was the transforming principle first described by Griffith?
Hershey and Chase’s Proof of DNA as Genetic Material
Alfred Hershey and Martha Chase performed their own experiments in 1952 and were able to provide confirmatory evidence that DNA, not protein, was the genetic material (Figure \(8\)).4 Hershey and Chase were studying a bacteriophage, a virus that infects bacteria. Viruses typically have a simple structure: a protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA (see Viruses). The particular bacteriophage they were studying was the T2 bacteriophage, which infects E. coli cells. As we now know today, T2 attaches to the surface of the bacterial cell and then it injects its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages.
Hershey and Chase labeled the protein coat in one batch of phage using radioactive sulfur, 35S, because sulfur is found in the amino acids methionine and cysteine but not in nucleic acids. They labeled the DNA in another batch using radioactive phosphorus, 32P, because phosphorus is found in DNA and RNA but not typically in protein.
Each batch of phage was allowed to infect the cells separately. After infection, Hershey and Chase put each phage bacterial suspension in a blender, which detached the phage coats from the host cell, and spun down the resulting suspension in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant. In the tube with the protein labeled, the radioactivity remained only in the supernatant. In the tube with the DNA labeled, the radioactivity was detected only in the bacterial cells. Hershey and Chase concluded that it was the phage DNA that was injected into the cell that carried the information to produce more phage particles, thus proving that DNA, not proteins, was the source of the genetic material. As a result of their work, the scientific community more broadly accepted DNA as the molecule responsible for heredity.
By the time Hershey and Chase published their experiment in the early 1950s, microbiologists and other scientists had been researching heredity for over 80 years. Building on one another’s research during that time culminated in the general agreement that DNA was the genetic material responsible for heredity (Figure \(9\)). This knowledge set the stage for the age of molecular biology to come and the significant advancements in biotechnology and systems biology that we are experiencing today.
Link to Learning
To learn more about the experiments involved in the history of genetics and the discovery of DNA as the genetic material of cells, visit this website from the DNA Learning Center.
Exercise \(4\)
How did Hershey and Chase use microbes to prove that DNA is genetic material?
Key Concepts and Summary
• DNA was discovered and characterized long before its role in heredity was understood. Microbiologists played significant roles in demonstrating that DNA is the hereditary information found within cells.
• In the 1850s and 1860s, Gregor Mendel experimented with true-breeding garden peas to demonstrate the heritability of specific observable traits.
• In 1869, Friedrich Miescher isolated and purified a compound rich in phosphorus from the nuclei of white blood cells; he named the compound nuclein. Miescher’s student Richard Altmann discovered its acidic nature, renaming it nucleic acid. Albrecht Kossell characterized the nucleotide bases found within nucleic acids.
• Although Walter Sutton and Theodor Boveri proposed the Chromosomal Theory of Inheritance in 1902, it was not scientifically demonstrated until the 1915 publication of the work of Thomas Hunt Morgan and his colleagues.
• Using Acetabularia, a large algal cell, as his model system, Joachim Hämmerling demonstrated in the 1930s and 1940s that the nucleus was the location of hereditary information in these cells.
• In the 1940s, George Beadle and Edward Tatum used the mold Neurospora crassa to show that each protein’s production was under the control of a single gene, demonstrating the “one gene–one enzyme” hypothesis.
• In 1928, Frederick Griffith showed that dead encapsulated bacteria could pass genetic information to live nonencapsulated bacteria and transform them into harmful strains. In 1944, Oswald Avery, Colin McLeod, and Maclyn McCarty identified the compound as DNA.
• The nature of DNA as the molecule that stores genetic information was unequivocally demonstrated in the experiment of Alfred Hershey and Martha Chase published in 1952. Labeled DNA from bacterial viruses entered and infected bacterial cells, giving rise to more viral particles. The labeled protein coats did not participate in the transmission of genetic information.
Footnotes
1. 1 J.G. Mendel. “Versuche über Pflanzenhybriden.” Verhandlungen des naturforschenden Vereines in Brünn, Bd. Abhandlungen 4 (1865):3–7. (For English translation, see http://www.mendelweb.org/Mendel.plain.html)
2. 2 G.W. Beadle, E.L. Tatum. “Genetic Control of Biochemical Reactions in Neurospora.” Proceedings of the National Academy of Sciences 27 no. 11 (1941):499–506.
3. 3 F. Griffith. “The Significance of Pneumococcal Types.” Journal of Hygiene 27 no. 2 (1928):8–159.
4. 4 A.D. Hershey, M. Chase. “Independent Functions of Viral Protein and Nucleic Acid in Growth of Bacteriophage.” Journal of General Physiology 36 no. 1 (1952):39–56. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/10%3A_Biochemistry_of_the_Genome/10.01%3A_Using_Microbiology_to_Discover_the_Secrets_of_Life.txt |
Learning Objectives
• Describe the biochemical structure of deoxyribonucleotides
• Identify the base pairs used in the synthesis of deoxyribonucleotides
• Explain why the double helix of DNA is described as antiparallel
In Microbial Metabolism, we discussed three classes of macromolecules: proteins, lipids, and carbohydrates. In this chapter, we will discuss a fourth class of macromolecules: nucleic acids. Like other macromolecules, nucleic acids are composed of monomers, called nucleotides, which are polymerized to form large strands. Each nucleic acid strand contains certain nucleotides that appear in a certain order within the strand, called its base sequence. The base sequence of deoxyribonucleic acid (DNA) is responsible for carrying and retaining the hereditary information in a cell. In Mechanisms of Microbial Genetics, we will discuss in detail the ways in which DNA uses its own base sequence to direct its own synthesis, as well as the synthesis of RNA and proteins, which, in turn, gives rise to products with diverse structure and function. In this section, we will discuss the basic structure and function of DNA.
DNA Nucleotides
The building blocks of nucleic acids are nucleotides. Nucleotides that compose DNA are called deoxyribonucleotides. The three components of a deoxyribonucleotide are a five-carbon sugar called deoxyribose, a phosphate group, and a nitrogenous base, a nitrogen-containing ring structure that is responsible for complementary base pairing between nucleic acid strands (Figure \(1\)). The carbon atoms of the five-carbon deoxyribose are numbered 1ʹ, 2ʹ, 3ʹ, 4ʹ, and 5ʹ (1ʹ is read as “one prime”). A nucleoside comprises the five-carbon sugar and nitrogenous base.
The deoxyribonucleotide is named according to the nitrogenous bases (Figure \(2\)). The nitrogenous bases adenine (A) and guanine (G) are the purines; they have a double-ring structure with a six-carbon ring fused to a five-carbon ring. The pyrimidines, cytosine (C) and thymine (T), are smaller nitrogenous bases that have only a six-carbon ring structure.
Individual nucleoside triphosphates combine with each other by covalent bonds known as 5ʹ-3ʹ phosphodiester bonds, or linkages whereby the phosphate group attached to the 5ʹ carbon of the sugar of one nucleotide bonds to the hydroxyl group of the 3ʹ carbon of the sugar of the next nucleotide. Phosphodiester bonding between nucleotides forms the sugar-phosphate backbone, the alternating sugar-phosphate structure composing the framework of a nucleic acid strand (Figure \(3\)). During the polymerization process, deoxynucleotide triphosphates (dNTP) are used. To construct the sugar-phosphate backbone, the two terminal phosphates are released from the dNTP as a pyrophosphate. The resulting strand of nucleic acid has a free phosphate group at the 5ʹ carbon end and a free hydroxyl group at the 3ʹ carbon end. The two unused phosphate groups from the nucleotide triphosphate are released as pyrophosphate during phosphodiester bond formation. Pyrophosphate is subsequently hydrolyzed, releasing the energy used to drive nucleotide polymerization.
Exercise \(1\)
What is meant by the 5ʹ and 3ʹ ends of a nucleic acid strand?
Discovering the Double Helix
By the early 1950s, considerable evidence had accumulated indicating that DNA was the genetic material of cells, and now the race was on to discover its three-dimensional structure. Around this time, Austrian biochemist Erwin Chargaff1(1905–2002) examined the content of DNA in different species and discovered that adenine, thymine, guanine, and cytosine were not found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine was very close to equaling the amount of thymine, and the amount of cytosine was very close to equaling the amount of guanine, or A = T and G = C. These relationships are also known as Chargaff’s rules.
Other scientists were also actively exploring this field during the mid-20th century. In 1952, American scientist Linus Pauling (1901–1994) was the world’s leading structural chemist and odds-on favorite to solve the structure of DNA. Pauling had earlier discovered the structure of protein α helices, using X-ray diffraction, and, based upon X-ray diffraction images of DNA made in his laboratory, he proposed a triple-stranded model of DNA.2 At the same time, British researchers Rosalind Franklin (1920–1958) and her graduate student R.G. Gosling were also using X-ray diffraction to understand the structure of DNA (Figure \(4\)). It was Franklin’s scientific expertise that resulted in the production of more well-defined X-ray diffraction images of DNA that would clearly show the overall double-helix structure of DNA.
James Watson (1928–), an American scientist, and Francis Crick (1916–2004), a British scientist, were working together in the 1950s to discover DNA’s structure. They used Chargaff’s rules and Franklin and Wilkins’ X-ray diffractionimages of DNA fibers to piece together the purine-pyrimidine pairing of the double helical DNA molecule (Figure \(5\)). In April 1953, Watson and Crick published their model of the DNA double helix in Nature.3 The same issue additionally included papers by Wilkins and colleagues,4 as well as by Franklin and Gosling,5 each describing different aspects of the molecular structure of DNA. In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Physiology and Medicine. Unfortunately, by then Franklin had died, and Nobel prizes at the time were not awarded posthumously. Work continued, however, on learning about the structure of DNA. In 1973, Alexander Rich(1924–2015) and colleagues were able to analyze DNA crystals to confirm and further elucidate DNA structure.6
Exercise \(2\)
Which scientists are given most of the credit for describing the molecular structure of DNA?
DNA Structure
Watson and Crick proposed that DNA is made up of two strands that are twisted around each other to form a right-handed helix. The two DNA strands are antiparallel, such that the 3ʹ end of one strand faces the 5ʹ end of the other (Figure \(6\)). The 3ʹ end of each strand has a free hydroxyl group, while the 5ʹ end of each strand has a free phosphate group. The sugar and phosphate of the polymerized nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside. These nitrogenous bases on the interior of the molecule interact with each other, base pairing.
Analysis of the diffraction patterns of DNA has determined that there are approximately 10 bases per turn in DNA. The asymmetrical spacing of the sugar-phosphate backbones generates major grooves (where the backbone is far apart) and minor grooves (where the backbone is close together) (Figure \(6\)). These grooves are locations where proteins can bind to DNA. The binding of these proteins can alter the structure of DNA, regulate replication, or regulate transcription of DNA into RNA.
Base pairing takes place between a purine and pyrimidine. In DNA, adenine (A) and thymine (T) are complementary base pairs, and cytosine (C) and guanine (G) are also complementary base pairs, explaining Chargaff’s rules (Figure \(7\)). The base pairs are stabilized by hydrogen bonds; adenine and thymine form two hydrogen bonds between them, whereas cytosine and guanine form three hydrogen bonds between them.
In the laboratory, exposing the two DNA strands of the double helix to high temperatures or to certain chemicals can break the hydrogen bonds between complementary bases, thus separating the strands into two separate single strands of DNA (single-stranded DNA [ssDNA]). This process is called DNA denaturation and is analogous to protein denaturation, as described in Proteins. The ssDNA strands can also be put back together as double-stranded DNA (dsDNA), through reannealing or renaturing by cooling or removing the chemical denaturants, allowing these hydrogen bonds to reform. The ability to artificially manipulate DNA in this way is the basis for several important techniques in biotechnology (Figure \(8\)). Because of the additional hydrogen bonding between the C = G base pair, DNA with a high GC content is more difficult to denature than DNA with a lower GC content.
Link to Learning
View an animation on DNA structure from the DNA Learning Center to learn more.
Exercise \(3\)
What are the two complementary base pairs of DNA and how are they bonded together?
DNA Function
DNA stores the information needed to build and control the cell. The transmission of this information from mother to daughter cells is called vertical gene transfer and it occurs through the process of DNA replication. DNA is replicated when a cell makes a duplicate copy of its DNA, then the cell divides, resulting in the correct distribution of one DNA copy to each resulting cell. DNA can also be enzymatically degraded and used as a source of nucleosides and nucleotides for the cell. Unlike other macromolecules, DNA does not serve a structural role in cells.
Exercise \(4\)
How does DNA transmit genetic information to offspring?
Paving the Way for Women in Science and Health Professions
Historically, women have been underrepresented in the sciences and in medicine, and often their pioneering contributions have gone relatively unnoticed. For example, although Rosalind Franklin performed the X-ray diffraction studies demonstrating the double helical structure of DNA, it is Watson and Crick who became famous for this discovery, building on her data. There still remains great controversy over whether their acquisition of her data was appropriate and whether personality conflicts and gender bias contributed to the delayed recognition of her significant contributions. Similarly, Barbara McClintock did pioneering work in maize (corn) genetics from the 1930s through 1950s, discovering transposons (jumping genes), but she was not recognized until much later, receiving a Nobel Prize in Physiology or Medicine in 1983 (Figure \(9\)).
Today, women still remain underrepresented in many fields of science and medicine. While more than half of the undergraduate degrees in science are awarded to women, only 46% of doctoral degrees in science are awarded to women. In academia, the number of women at each level of career advancement continues to decrease, with women holding less than one-third of the positions of Ph.D.-level scientists in tenure-track positions, and less than one-quarter of the full professorships at 4-year colleges and universities.7 Even in the health professions, like nearly all other fields, women are often underrepresented in many medical careers and earn significantly less than their male counterparts, as shown in a 2013 study published by the Journal of the American Medical Association.8
Why do such disparities continue to exist and how do we break these cycles? The situation is complex and likely results from the combination of various factors, including how society conditions the behaviors of girls from a young age and supports their interests, both professionally and personally. Some have suggested that women do not belong in the laboratory, including Nobel Prize winner Tim Hunt, whose 2015 public comments suggesting that women are too emotional for science9 were met with widespread condemnation.
Perhaps girls should be supported more from a young age in the areas of science and math (Figure \(9\)). Science, technology, engineering, and mathematics (STEM) programs sponsored by the American Association of University Women (AAUW)10 and National Aeronautics and Space Administration (NASA)11 are excellent examples of programs that offer such support. Contributions by women in science should be made known more widely to the public, and marketing targeted to young girls should include more images of historically and professionally successful female scientists and medical professionals, encouraging all bright young minds, including girls and women, to pursue careers in science and medicine.
Clinical Focus: Part 2
Based upon his symptoms, Alex’s physician suspects that he is suffering from a foodborne illness that he acquired during his travels. Possibilities include bacterial infection (e.g., enterotoxigenic E. coli, Vibrio cholerae, Campylobacter jejuni, Salmonella), viral infection (rotavirus or norovirus), or protozoan infection (Giardia lamblia, Cryptosporidium parvum, or Entamoeba histolytica).
His physician orders a stool sample to identify possible causative agents (e.g., bacteria, cysts) and to look for the presence of blood because certain types of infectious agents (like C. jejuni, Salmonella, and E. histolytica) are associated with the production of bloody stools.
Alex’s stool sample showed neither blood nor cysts. Following analysis of his stool sample and based upon his recent travel history, the hospital physician suspected that Alex was suffering from traveler’s diarrhea caused by enterotoxigenic E. coli (ETEC), the causative agent of most traveler’s diarrhea. To verify the diagnosis and rule out other possibilities, Alex’s physician ordered a diagnostic lab test of his stool sample to look for DNA sequences encoding specific virulence factors of ETEC. The physician instructed Alex to drink lots of fluids to replace what he was losing and discharged him from the hospital.
ETEC produces several plasmid-encoded virulence factors that make it pathogenic compared with typical E. coli. These include the secreted toxins heat-labile enterotoxin (LT) and heat-stabile enterotoxin (ST), as well as colonization factor (CF). Both LT and ST cause the excretion of chloride ions from intestinal cells to the intestinal lumen, causing a consequent loss of water from intestinal cells, resulting in diarrhea. CF encodes a bacterial protein that aids in allowing the bacterium to adhere to the lining of the small intestine.
Exercise \(5\)
Why did Alex’s physician use genetic analysis instead of either isolation of bacteria from the stool sample or direct Gram stain of the stool sample alone?
Key Concepts and Summary
• Nucleic acids are composed of nucleotides, each of which contains a pentose sugar, a phosphate group, and a nitrogenous base. Deoxyribonucleotides within DNA contain deoxyribose as the pentose sugar.
• DNA contains the pyrimidines cytosine and thymine, and the purines adenine and guanine.
• Nucleotides are linked together by phosphodiester bonds between the 5ʹ phosphate group of one nucleotide and the 3ʹ hydroxyl group of another. A nucleic acid strand has a free phosphate group at the 5ʹ end and a free hydroxyl group at the 3ʹ end.
• Chargaff discovered that the amount of adenine is approximately equal to the amount of thymine in DNA, and that the amount of the guanine is approximately equal to cytosine. These relationships were later determined to be due to complementary base pairing.
• Watson and Crick, building on the work of Chargaff, Franklin and Gosling, and Wilkins, proposed the double helix model and base pairing for DNA structure.
• DNA is composed of two complementary strands oriented antiparallel to each other with the phosphodiester backbones on the exterior of the molecule. The nitrogenous bases of each strand face each other and complementary bases hydrogen bond to each other, stabilizing the double helix.
• Heat or chemicals can break the hydrogen bonds between complementary bases, denaturing DNA. Cooling or removing chemicals can lead to renaturation or reannealing of DNA by allowing hydrogen bonds to reform between complementary bases.
• DNA stores the instructions needed to build and control the cell. This information is transmitted from parent to offspring through vertical gene transfer.
Footnotes
1. 1 N. Kresge et al. “Chargaff's Rules: The Work of Erwin Chargaff.” Journal of Biological Chemistry 280 (2005):e21.
2. 2 L. Pauling, “A Proposed Structure for the Nucleic Acids.” Proceedings of the National Academy of Science of the United States of America 39 no. 2 (1953):84–97.
3. 3 J.D. Watson, F.H.C. Crick. “A Structure for Deoxyribose Nucleic Acid.” Nature 171 no. 4356 (1953):737–738.
4. 4 M.H.F. Wilkins et al. “Molecular Structure of Deoxypentose Nucleic Acids.” Nature 171 no. 4356 (1953):738–740.
5. 5 R. Franklin, R.G. Gosling. “Molecular Configuration in Sodium Thymonucleate.” Nature 171 no. 4356 (1953):740–741.
6. 6 R.O. Day et al. “A Crystalline Fragment of the Double Helix: The Structure of the Dinucleoside Phosphate Guanylyl-3',5'-Cytidine.” Proceedings of the National Academy of Sciences of the United States of America 70 no. 3 (1973):849–853.
7. 7 N.H. Wolfinger “For Female Scientists, There's No Good Time to Have Children.” The Atlantic July 29, 2013. www.theatlantic.com/sexes/arc...ildren/278165/.
8. 8 S.A. Seabury et al. “Trends in the Earnings of Male and Female Health Care Professionals in the United States, 1987 to 2010.” Journal of the American Medical Association Internal Medicine 173 no. 18 (2013):1748–1750.
9. 9 E. Chung. “Tim Hunt, Sexism and Science: The Real 'Trouble With Girls' in Labs.” CBC News Technology and Science, June 12, 2015. http://www.cbc.ca/news/technology/ti...labs-1.3110133. Accessed 8/4/2016.
10. 10 American Association of University Women. “Building a STEM Pipeline for Girls and Women.” www.aauw.org/what-we-do/stem-education/. Accessed June 10, 2016.
11. 11 National Aeronautics and Space Administration. “Outreach Programs: Women and Girls Initiative.” http://women.nasa.gov/outreach-programs/. Accessed June 10, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/10%3A_Biochemistry_of_the_Genome/10.02%3A_Structure_and_Function_of_DNA.txt |
Learning Objectives
• Describe the biochemical structure of ribonucleotides
• Describe the similarities and differences between RNA and DNA
• Describe the functions of the three main types of RNA used in protein synthesis
• Explain how RNA can serve as hereditary information
Structurally speaking, ribonucleic acid (RNA), is quite similar to DNA. However, whereas DNA molecules are typically long and double stranded, RNA molecules are much shorter and are typically single stranded. RNA molecules perform a variety of roles in the cell but are mainly involved in the process of protein synthesis (translation) and its regulation.
RNA Structure
RNA is typically single stranded and is made of ribonucleotides that are linked by phosphodiester bonds. A ribonucleotide in the RNA chain contains ribose (the pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and a phosphate group. The subtle structural difference between the sugars gives DNA added stability, making DNA more suitable for storage of genetic information, whereas the relative instability of RNA makes it more suitable for its more short-term functions.
The RNA-specific pyrimidine uracil forms a complementary base pair with adenine and is used instead of the thymine used in DNA. Even though RNA is single stranded, most types of RNA molecules show extensive intramolecular base pairing between complementary sequences within the RNA strand, creating a predictable three-dimensional structure essential for their function (Figure \(1\) and Figure \(2\)).
Exercise \(1\)
How does the structure of RNA differ from the structure of DNA?
Functions of RNA in Protein Synthesis
Cells access the information stored in DNA by creating RNA to direct the synthesis of proteins through the process of translation. Proteins within a cell have many functions, including building cellular structures and serving as enzyme catalysts for cellular chemical reactions that give cells their specific characteristics. The three main types of RNA directly involved in protein synthesis are messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).
In 1961, French scientists François Jacob and Jacques Monod hypothesized the existence of an intermediary between DNA and its protein products, which they called messenger RNA.1 Evidence supporting their hypothesis was gathered soon afterwards showing that information from DNA is transmitted to the ribosome for protein synthesis using mRNA. If DNA serves as the complete library of cellular information, mRNA serves as a photocopy of specific information needed at a particular point in time that serves as the instructions to make a protein.
The mRNA carries the message from the DNA, which controls all of the cellular activities in a cell. If a cell requires a certain protein to be synthesized, the gene for this product is “turned on” and the mRNA is synthesized through the process of transcription (see RNA Transcription). The mRNA then interacts with ribosomes and other cellular machinery (Figure \(3\)) to direct the synthesis of the protein it encodes during the process of translation (see Protein Synthesis). mRNA is relatively unstable and short-lived in the cell, especially in prokaryotic cells, ensuring that proteins are only made when needed.
rRNA and tRNA are stable types of RNA. In prokaryotes and eukaryotes, tRNA and rRNA are encoded in the DNA, then copied into long RNA molecules that are cut to release smaller fragments containing the individual mature RNA species. In eukaryotes, synthesis, cutting, and assembly of rRNA into ribosomes takes place in the nucleolus region of the nucleus, but these activities occur in the cytoplasm of prokaryotes. Neither of these types of RNA carries instructions to direct the synthesis of a polypeptide, but they play other important roles in protein synthesis.
Ribosomes are composed of rRNA and protein. As its name suggests, rRNA is a major constituent of ribosomes, composing up to about 60% of the ribosome by mass and providing the location where the mRNA binds. The rRNA ensures the proper alignment of the mRNA, tRNA, and the ribosomes; the rRNA of the ribosome also has an enzymatic activity (peptidyl transferase) and catalyzes the formation of the peptide bonds between two aligned amino acids during protein synthesis. Although rRNA had long been thought to serve primarily a structural role, its catalytic role within the ribosome was proven in 2000.2 Scientists in the laboratories of Thomas Steitz (1940–) and Peter Moore(1939–) at Yale University were able to crystallize the ribosome structure from Haloarcula marismortui, a halophilic archaeon isolated from the Dead Sea. Because of the importance of this work, Steitz shared the 2009 Nobel Prize in Chemistry with other scientists who made significant contributions to the understanding of ribosome structure.
Transfer RNA is the third main type of RNA and one of the smallest, usually only 70–90 nucleotides long. It carries the correct amino acid to the site of protein synthesis in the ribosome. It is the base pairing between the tRNA and mRNA that allows for the correct amino acid to be inserted in the polypeptide chain being synthesized (Figure \(4\)). Any mutations in the tRNA or rRNA can result in global problems for the cell because both are necessary for proper protein synthesis (Table \(1\)).
Table \(1\): Structure and Function of RNA
mRNA rRNA tRNA
Structure Short, unstable, single-stranded RNAcorresponding to a gene encoded within DNA Longer, stable RNA molecules composing 60% of ribosome’s mass Short (70-90 nucleotides), stable RNA with extensive intramolecular base pairing; contains an amino acid binding site and an mRNA binding site
Function Serves as intermediary between DNA and protein; used by ribosome to direct synthesis of protein it encodes Ensures the proper alignment of mRNA, tRNA, and ribosome during protein synthesis; catalyzes peptide bond formation between amino acids Carries the correct amino acid to the site of protein synthesis in the ribosome
Exercise \(1\)
What are the functions of the three major types of RNA molecules involved in protein synthesis?
RNA as Hereditary Information
Although RNA does not serve as the hereditary information in most cells, RNA does hold this function for many viruses that do not contain DNA. Thus, RNA clearly does have the additional capacity to serve as genetic information. Although RNA is typically single stranded within cells, there is significant diversity in viruses. Rhinoviruses, which cause the common cold; influenza viruses; and the Ebola virus are single-stranded RNA viruses. Rotaviruses, which cause severe gastroenteritis in children and other immunocompromised individuals, are examples of double-stranded RNA viruses. Because double-stranded RNA is uncommon in eukaryotic cells, its presence serves as an indicator of viral infection. The implications for a virus having an RNA genome instead of a DNA genome are discussed in more detail in Viruses.
Key Concepts and Summary
• Ribonucleic acid (RNA) is typically single stranded and contains ribose as its pentose sugar and the pyrimidine uracil instead of thymine. An RNA strand can undergo significant intramolecular base pairing to take on a three-dimensional structure.
• There are three main types of RNA, all involved in protein synthesis.
• Messenger RNA (mRNA) serves as the intermediary between DNA and the synthesis of protein products during translation.
• Ribosomal RNA (rRNA) is a type of stable RNA that is a major constituent of ribosomes. It ensures the proper alignment of the mRNA and the ribosomes during protein synthesis and catalyzes the formation of the peptide bonds between two aligned amino acids during protein synthesis.
• Transfer RNA (tRNA) is a small type of stable RNA that carries an amino acid to the corresponding site of protein synthesis in the ribosome. It is the base pairing between the tRNA and mRNA that allows for the correct amino acid to be inserted in the polypeptide chain being synthesized.
• Although RNA is not used for long-term genetic information in cells, many viruses do use RNA as their genetic material.
Footnotes
1. 1 A. Rich. “The Era of RNA Awakening: Structural Biology of RNA in the Early Years.” Quarterly Reviews of Biophysics 42 no. 2 (2009):117–137.
2. 2 P. Nissen et al. “The Structural Basis of Ribosome Activity in Peptide Bond Synthesis.” Science 289 no. 5481 (2000):920–930. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/10%3A_Biochemistry_of_the_Genome/10.03%3A_Structure_and_Function_of_RNA.txt |
Learning Objectives
• Define gene and genotype and differentiate genotype from phenotype
• Describe chromosome structure and packaging
• Compare prokaryotic and eukaryotic chromosomes
• Explain why extrachromosomal DNA is important in a cell
Thus far, we have discussed the structure and function of individual pieces of DNA and RNA. In this section, we will discuss how all of an organism’s genetic material—collectively referred to as its genome—is organized inside of the cell. Since an organism’s genetics to a large extent dictate its characteristics, it should not be surprising that organisms differ in the arrangement of their DNA and RNA.
Genotype versus Phenotype
All cellular activities are encoded within a cell’s DNA. The sequence of bases within a DNA molecule represents the genetic information of the cell. Segments of DNA molecules are called genes, and individual genes contain the instructional code necessary for synthesizing various proteins, enzymes, or stable RNA molecules.
The full collection of genes that a cell contains within its genome is called its genotype. However, a cell does not express all of its genes simultaneously. Instead, it turns on (expresses) or turns off certain genes when necessary. The set of genes being expressed at any given point in time determines the cell’s activities and its observable characteristics, referred to as its phenotype. Genes that are always expressed are known as constitutive genes; some constitutive genes are known as housekeeping genes because they are necessary for the basic functions of the cell.
While the genotype of a cell remains constant, the phenotype may change in response to environmental signals (e.g., changes in temperature or nutrient availability) that affect which nonconstitutive genes are expressed. For example, the oral bacterium Streptococcus mutans produces a sticky slime layer that allows it to adhere to teeth, forming dental plaque; however, the genes that control the production of the slime layer are only expressed in the presence of sucrose (table sugar). Thus, while the genotype of S. mutans is constant, its phenotype changes depending on the presence and absence of sugar in its environment. Temperature can also regulate gene expression. For example, the gram-negative bacterium Serratia marcescens, a pathogen frequently associated with hospital-acquired infections, produces a red pigment at 28 °C but not at 37 °C, the normal internal temperature of the human body (Figure \(1\)).
Organization of Genetic Material
The vast majority of an organism’s genome is organized into the cell’s chromosomes, which are discrete DNA structures within cells that control cellular activity. Recall that while eukaryotic chromosomes are housed in the membrane-bound nucleus, most prokaryotes contain a single, circular chromosome that is found in an area of the cytoplasm called the nucleoid (see Unique Characteristics of Prokaryotic Cells). A chromosome may contain several thousand genes.
Organization of Eukaryotic Chromosome
Chromosome structure differs somewhat between eukaryotic and prokaryotic cells. Eukaryotic chromosomes are typically linear, and eukaryotic cells contain multiple distinct chromosomes. Many eukaryotic cells contain two copies of each chromosome and, therefore, are diploid.
The length of a chromosome greatly exceeds the length of the cell, so a chromosome needs to be packaged into a very small space to fit within the cell. For example, the combined length of all of the 3 billion base pairs1 of DNA of the human genome would measure approximately 2 meters if completely stretched out, and some eukaryotic genomes are many times larger than the human genome. DNA supercoiling refers to the process by which DNA is twisted to fit inside the cell. Supercoiling may result in DNA that is either underwound (less than one turn of the helix per 10 base pairs) or overwound (more than one turn per 10 base pairs) from its normal relaxed state. Proteins known to be involved in supercoiling include topoisomerases; these enzymes help maintain the structure of supercoiled chromosomes, preventing overwinding of DNA during certain cellular processes like DNA replication.
During DNA packaging, DNA-binding proteins called histones perform various levels of DNA wrapping and attachment to scaffolding proteins. The combination of DNA with these attached proteins is referred to as chromatin. In eukaryotes, the packaging of DNA by histones may be influenced by environmental factors that affect the presence of methyl groups on certain cytosine nucleotides of DNA. The influence of environmental factors on DNA packaging is called epigenetics. Epigenetics is another mechanism for regulating gene expression without altering the sequence of nucleotides. Epigenetic changes can be maintained through multiple rounds of cell division and, therefore, can be heritable.
Link to Learning
View this animation from the DNA Learning Center to learn more about on DNA packaging in eukaryotes.
Organization of Prokaryotic Chromosomes
Chromosomes in bacteria and archaea are usually circular, and a prokaryotic cell typically contains only a single chromosome within the nucleoid. Because the chromosome contains only one copy of each gene, prokaryotes are haploid. As in eukaryotic cells, DNA supercoiling is necessary for the genome to fit within the prokaryotic cell. The DNA in the bacterial chromosome is arranged in several supercoiled domains. As with eukaryotes, topoisomerases are involved in supercoiling DNA. DNA gyrase is a type of topoisomerase, found in bacteria and some archaea, that helps prevent the overwinding of DNA. (Some antibiotics kill bacteria by targeting DNA gyrase.) In addition, histone-like proteins bind DNA and aid in DNA packaging. Other proteins bind to the origin of replication, the location in the chromosome where DNA replication initiates. Because different regions of DNA are packaged differently, some regions of chromosomal DNA are more accessible to enzymes and thus may be used more readily as templates for gene expression. Interestingly, several bacteria, including Helicobacter pylori and Shigella flexneri, have been shown to induce epigenetic changes in their hosts upon infection, leading to chromatin remodeling that may cause long-term effects on host immunity.2
Exercise \(1\)
1. What is the difference between a cell’s genotype and its phenotype?
2. How does DNA fit inside cells?
Noncoding DNA
In addition to genes, a genome also contains many regions of noncoding DNA that do not encode proteins or stable RNA products. Noncoding DNA is commonly found in areas prior to the start of coding sequences of genes as well as in intergenic regions (i.e., DNA sequences located between genes) (Figure \(2\)).
Prokaryotes appear to use their genomes very efficiently, with only an average of 12% of the genome being taken up by noncoding sequences. In contrast, noncoding DNA can represent about 98% of the genome in eukaryotes, as seen in humans, but the percentage of noncoding DNA varies between species.3 These noncoding DNA regions were once referred to as “junk DNA”; however, this terminology is no longer widely accepted because scientists have since found roles for some of these regions, many of which contribute to the regulation of transcription or translation through the production of small noncoding RNA molecules, DNA packaging, and chromosomal stability. Although scientists may not fully understand the roles of all noncoding regions of DNA, it is generally believed that they do have purposes within the cell.
Exercise \(2\)
What is the role of noncoding DNA?
Extrachromosomal DNA
Although most DNA is contained within a cell’s chromosomes, many cells have additional molecules of DNA outside the chromosomes, called extrachromosomal DNA, that are also part of its genome. The genomes of eukaryotic cells would also include the chromosomes from any organelles such as mitochondria and/or chloroplasts that these cells maintain (Figure \(3\)). The maintenance of circular chromosomes in these organelles is a vestige of their prokaryotic origins and supports the endosymbiotic theory (see Foundations of Modern Cell Theory). In some cases, genomes of certain DNA viruses can also be maintained independently in host cells during latent viral infection. In these cases, these viruses are another form of extrachromosomal DNA. For example, the human papillomavirus (HPV) may be maintained in infected cells in this way.
Besides chromosomes, some prokaryotes also have smaller loops of DNA called plasmids that may contain one or a few genes not essential for normal growth (Figure 3.3.1). Bacteria can exchange these plasmids with other bacteria in a process known as horizontal gene transfer (HGT). The exchange of genetic material on plasmids sometimes provides microbes with new genes beneficial for growth and survival under special conditions. In some cases, genes obtained from plasmids may have clinical implications, encoding virulence factors that give a microbe the ability to cause disease or make a microbe resistant to certain antibiotics. Plasmids are also used heavily in genetic engineering and biotechnology as a way to move genes from one cell to another. The role of plasmids in horizontal gene transfer and biotechnology will be discussed further in Mechanisms of Microbial Genetics and Modern Applications of Microbial Genetics.
Exercise \(3\)
How are plasmids involved in antibiotic resistance?
Lethal Plasmids
Maria, a 20-year-old anthropology student from Texas, recently became ill in the African nation of Botswana, where she was conducting research as part of a study-abroad program. Maria’s research was focused on traditional African methods of tanning hides for the production of leather. Over a period of three weeks, she visited a tannery daily for several hours to observe and participate in the tanning process. One day, after returning from the tannery, Maria developed a fever, chills, and a headache, along with chest pain, muscle aches, nausea, and other flu-like symptoms. Initially, she was not concerned, but when her fever spiked and she began to cough up blood, her African host family became alarmed and rushed her to the hospital, where her condition continued to worsen.
After learning about her recent work at the tannery, the physician suspected that Maria had been exposed to anthrax. He ordered a chest X-ray, a blood sample, and a spinal tap, and immediately started her on a course of intravenous penicillin. Unfortunately, lab tests confirmed the physician’s presumptive diagnosis. Maria’s chest X-ray exhibited pleural effusion, the accumulation of fluid in the space between the pleural membranes, and a Gram stain of her blood revealed the presence of gram-positive, rod-shaped bacteria in short chains, consistent with Bacillus anthracis. Blood and bacteria were also shown to be present in her cerebrospinal fluid, indicating that the infection had progressed to meningitis. Despite supportive treatment and aggressive antibiotic therapy, Maria slipped into an unresponsive state and died three days later.
Anthrax is a disease caused by the introduction of endospores from the gram-positive bacterium B. anthracis into the body. Once infected, patients typically develop meningitis, often with fatal results. In Maria’s case, she inhaled the endospores while handling the hides of animals that had been infected.
The genome of B. anthracis illustrates how small structural differences can lead to major differences in virulence. In 2003, the genomes of B. anthracis and Bacillus cereus, a similar but less pathogenic bacterium of the same genus, were sequenced and compared.4 Researchers discovered that the 16S rRNA gene sequences of these bacteria are more than 99% identical, meaning that they are actually members of the same species despite their traditional classification as separate species. Although their chromosomal sequences also revealed a great deal of similarity, several virulence factors of B. anthracis were found to be encoded on two large plasmids not found in B. cereus. The plasmid pX01 encodes a three-part toxin that suppresses the host immune system, whereas the plasmid pX02 encodes a capsular polysaccharide that further protects the bacterium from the host immune system (Figure \(4\)). Since B. cereus lacks these plasmids, it does not produce these virulence factors, and although it is still pathogenic, it is typically associated with mild cases of diarrhea from which the body can quickly recover. Unfortunately for Maria, the presence of these toxin-encoding plasmids in B. anthracis gives it its lethal virulence.
Exercise \(4\)
What do you think would happen to the pathogenicity of B. anthracis if it lost one or both of its plasmids?
Clinical Focus: Resolution
Within 24 hours, the results of the diagnostic test analysis of Alex’s stool sample revealed that it was positive for heat-labile enterotoxin (LT), heat-stabile enterotoxin (ST), and colonization factor (CF), confirming the hospital physician’s suspicion of ETEC. During a follow-up with Alex’s family physician, this physician noted that Alex’s symptoms were not resolving quickly and he was experiencing discomfort that was preventing him from returning to classes. The family physician prescribed Alex a course of ciprofloxacin to resolve his symptoms. Fortunately, the ciprofloxacin resolved Alex’s symptoms within a few days.
Alex likely got his infection from ingesting contaminated food or water. Emerging industrialized countries like Mexico are still developing sanitation practices that prevent the contamination of water with fecal material. Travelers in such countries should avoid the ingestion of undercooked foods, especially meats, seafood, vegetables, and unpasteurized dairy products. They should also avoid use of water that has not been treated; this includes drinking water, ice cubes, and even water used for brushing teeth. Using bottled water for these purposes is a good alternative. Good hygiene (handwashing) can also aid the prevention of an ETEC infection. Alex had not been careful about his food or water consumption, which led to his illness.
Alex’s symptoms were very similar to those of cholera, caused by the gram-negative bacterium Vibrio cholerae, which also produces a toxin similar to ST and LT. At some point in the evolutionary history of ETEC, a nonpathogenic strain of E. coli similar to those typically found in the gut may have acquired the genes encoding the ST and LT toxins from V. cholerae. The fact that the genes encoding those toxins are encoded on extrachromosomal plasmids in ETEC supports the idea that these genes were acquired by E. coli and are likely maintained in bacterial populations through horizontal gene transfer.
Viral Genomes
Viral genomes exhibit significant diversity in structure. Some viruses have genomes that consist of DNA as their genetic material. This DNA may be single stranded, as exemplified by human parvoviruses, or double stranded, as seen in the herpesviruses and poxviruses. Additionally, although all cellular life uses DNA as its genetic material, some viral genomes are made of either single-stranded or double-stranded RNA molecules, as we have discussed. Viral genomes are typically smaller than most bacterial genomes, encoding only a few genes, because they rely on their hosts to carry out many of the functions required for their replication. The diversity of viral genome structures and their implications for viral replication life cycles are discussed in more detail in The Viral Life Cycle.
Exercise \(5\)
Why do viral genomes vary widely among viruses?
Genome Size Matters
There is great variation in size of genomes among different organisms. Most eukaryotes maintain multiple chromosomes; humans, for example have 23 pairs, giving them 46 chromosomes. Despite being large at 3 billion base pairs, the human genome is far from the largest genome. Plants often maintain very large genomes, up to 150 billion base pairs, and commonly are polyploid, having multiple copies of each chromosome.
The size of bacterial genomes also varies considerably, although they tend to be smaller than eukaryotic genomes (Figure \(5\)). Some bacterial genomes may be as small as only 112,000 base pairs. Often, the size of a bacterium’s genome directly relates to how much the bacterium depends on its host for survival. When a bacterium relies on the host cell to carry out certain functions, it loses the genes encoding the abilities to carry out those functions itself. These types of bacterial endosymbionts are reminiscent of the prokaryotic origins of mitochondria and chloroplasts.
From a clinical perspective, obligate intracellular pathogens also tend to have small genomes (some around 1 million base pairs). Because host cells supply most of their nutrients, they tend to have a reduced number of genes encoding metabolic functions. Due to their small sizes, the genomes of organisms like Mycoplasma genitalium (580,000 base pairs), Chlamydia trachomatis (1.0 million), Rickettsia prowazekii (1.1 million), and Treponema pallidum (1.1 million) were some of the earlier bacterial genomes sequenced. Respectively, these pathogens cause urethritis and pelvic inflammation, chlamydia, typhus, and syphilis.
Whereas obligate intracellular pathogens have unusually small genomes, other bacteria with a great variety of metabolic and enzymatic capabilities have unusually large bacterial genomes. Pseudomonas aeruginosa, for example, is a bacterium commonly found in the environment and is able to grow on a wide range of substrates. Its genome contains 6.3 million base pairs, giving it a high metabolic ability and the ability to produce virulence factors that cause several types of opportunistic infections.
Interestingly, there has been significant variability in genome size in viruses as well, ranging from 3,500 base pairs to 2.5 million base pairs, significantly exceeding the size of many bacterial genomes. The great variation observed in viral genome sizes further contributes to the great diversity of viral genome characteristics already discussed.
Link to Learning
Visit the genome database of the National Center for Biotechnology Information (NCBI) to see the genomes that have been sequenced and their sizes.
Key Concepts and Summary
• The entire genetic content of a cell is its genome.
• Genes code for proteins, or stable RNA molecules, each of which carries out a specific function in the cell.
• Although the genotype that a cell possesses remains constant, expression of genes is dependent on environmental conditions.
• A phenotype is the observable characteristics of a cell (or organism) at a given point in time and results from the complement of genes currently being used.
• The majority of genetic material is organized into chromosomes that contain the DNA that controls cellular activities.
• Prokaryotes are typically haploid, usually having a single circular chromosome found in the nucleoid. Eukaryotes are diploid; DNA is organized into multiple linear chromosomes found in the nucleus.
• Supercoiling and DNA packaging using DNA binding proteins allows lengthy molecules to fit inside a cell. Eukaryotes and archaea use histone proteins, and bacteria use different proteins with similar function.
• Prokaryotic and eukaryotic genomes both contain noncoding DNA, the function of which is not well understood. Some noncoding DNA appears to participate in the formation of small noncoding RNA molecules that influence gene expression; some appears to play a role in maintaining chromosomal structure and in DNA packaging.
• Extrachromosomal DNA in eukaryotes includes the chromosomes found within organelles of prokaryotic origin (mitochondria and chloroplasts) that evolved by endosymbiosis. Some viruses may also maintain themselves extrachromosomally.
• Extrachromosomal DNA in prokaryotes is commonly maintained as plasmids that encode a few nonessential genes that may be helpful under specific conditions. Plasmids can be spread through a bacterial community by horizontal gene transfer.
• Viral genomes show extensive variation and may be composed of either RNA or DNA, and may be either double or single stranded.
Footnotes
1. 1 National Human Genome Research Institute. “The Human Genome Project Completion: Frequently Asked Questions.” https://www.genome.gov/11006943. Accessed June 10, 2016
2. 2 H. Bierne et al. “Epigenetics and Bacterial Infections.” Cold Spring Harbor Perspectives in Medicine 2 no. 12 (2012):a010272.
3. 3 R.J. Taft et al. “The Relationship between Non-Protein-Coding DNA and Eukaryotic Complexity.” Bioessays 29 no. 3 (2007):288–299.
4. 4 N. Ivanova et al. “Genome Sequence of Bacillus cereus and Comparative Analysis with Bacillus anthracis.” Nature 423 no. 6935 (2003):87–91. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/10%3A_Biochemistry_of_the_Genome/10.04%3A_The_Structure_and_Function_of_Cellular_Genomes.txt |
10.1: Using Microbiology to Discover the Secrets of Life
DNA was discovered and characterized long before its role in heredity was understood. Microbiologists played significant roles in demonstrating that DNA is the hereditary information found within cells. In the 1850s and 1860s, Gregor Mendel experimented with true-breeding garden peas to demonstrate the heritability of specific observable traits. In 1869, Friedrich Miescher isolated and purified a compound rich in phosphorus from the nuclei of white blood cells; he named the compound nuclein.
Multiple Choice
Frederick Griffith infected mice with a combination of dead R and live S bacterial strains. What was the outcome, and why did it occur?
1. The mice will live. Transformation was not required.
2. The mice will die. Transformation of genetic material from R to S was required.
3. The mice will live. Transformation of genetic material from S to R was required.
4. The mice will die. Transformation was not required.
Answer
D
Why was the alga Acetabularia a good model organism for Joachim Hämmerling to use to identify the location of genetic material?
1. It lacks a nuclear membrane.
2. It self-fertilizes.
3. It is a large, asymmetrical, single cell easy to see with the naked eye.
4. It makes a protein capsid.
Answer
C
Which of the following best describes the results from Hershey and Chase’s experiment using bacterial viruses with 35S-labeled proteins or 32P-labeled DNA that are consistent with protein being the molecule responsible for hereditary?
1. After infection with the 35S-labeled viruses and centrifugation, only the pellet would be radioactive.
2. After infection with the 35S-labeled viruses and centrifugation, both the pellet and the supernatant would be radioactive.
3. After infection with the 32P-labeled viruses and centrifugation, only the pellet would be radioactive.
4. After infection with the 32P-labeled viruses and centrifugation, both the pellet and the supernatant would be radioactive.
Answer
A
Which method did Morgan and colleagues use to show that hereditary information was carried on chromosomes?
1. statistical predictions of the outcomes of crosses using true-breeding parents
2. correlations between microscopic observations of chromosomal movement and the characteristics of offspring
3. transformation of nonpathogenic bacteria to pathogenic bacteria
4. mutations resulting in distinct defects in metabolic enzymatic pathways
Answer
B
According to Beadle and Tatum’s “one gene–one enzyme” hypothesis, which of the following enzymes will eliminate the transformation of hereditary material from pathogenic bacteria to nonpathogenic bacteria?
1. carbohydrate-degrading enzymes
2. proteinases
3. ribonucleases
4. deoxyribonucleases
Answer
D
Fill in the Blank
The element ____________ is unique to nucleic acids compared with other macromolecules.
Answer
phosphorus
In the late 1800s and early 1900s, the macromolecule thought to be responsible for heredity was ______________.
Answer
protein
Short Answer
Why do bacteria and viruses make good model systems for various genetic studies?
Why was nucleic acid disregarded for so long as the molecule responsible for the transmission of hereditary information?
Bacteriophages inject their genetic material into host cells, whereas animal viruses enter host cells completely. Why was it important to use a bacteriophage in the Hershey–Chase experiment rather than an animal virus?
Critical Thinking
In the figure shown, if the nuclei were contained within the stalks of Acetabularia, what types of caps would you expect from the pictured grafts?
Why are Hershey and Chase credited with identifying DNA as the carrier of heredity even though DNA had been discovered many years before?
10.2: Structure and Function of DNA
Nucleic acids are composed of nucleotides, each of which contains a pentose sugar, a phosphate group, and a nitrogenous base. Deoxyribonucleotides within DNA contain deoxyribose as the pentose sugar. DNA contains the pyrimidines cytosine and thymine, and the purines adenine and guanine. Nucleotides are linked together by phosphodiester bonds between the 5ʹ phosphate group of one nucleotide and the 3ʹ hydroxyl group of another.
Multiple Choice
Which of the following is not found within DNA?
1. thymine
2. phosphodiester bonds
3. complementary base pairing
4. amino acids
Answer
D
If 30% of the bases within a DNA molecule are adenine, what is the percentage of thymine?
1. 20%
2. 25%
3. 30%
4. 35%
Answer
C
Which of the following statements about base pairing in DNA is incorrect?
1. Purines always base pairs with pyrimidines.
2. Adenine binds to guanine.
3. Base pairs are stabilized by hydrogen bonds.
4. Base pairing occurs at the interior of the double helix.
Answer
B
If a DNA strand contains the sequence 5ʹ-ATTCCGGATCGA-3ʹ, which of the following is the sequence of the complementary strand of DNA?
1. 5ʹ-TAAGGCCTAGCT-3ʹ
2. 5ʹ-ATTCCGGATCGA-3ʹ
3. 3ʹ-TAACCGGTACGT-5ʹ
4. 5ʹ-TCGATCCGGAAT-3ʹ
Answer
D
During denaturation of DNA, which of the following happens?
1. Hydrogen bonds between complementary bases break.
2. Phosphodiester bonds break within the sugar-phosphate backbone.
3. Hydrogen bonds within the sugar-phosphate backbone break.
4. Phosphodiester bonds between complementary bases break.
Answer
A.
Fill in the Blank
The end of a nucleic acid strand with a free phosphate group is called the ________.
Answer
5ʹ end
True/False
The work of Rosalind Franklin and R.G. Gosling was important in demonstrating the helical nature of DNA.
Answer
True
The A-T base pair has more hydrogen bonding than the C-G base pair.
Answer
False
Short Answer
What is the role of phosphodiester bonds within the sugar-phosphate backbone of DNA?
What is meant by the term “antiparallel?”
Why is DNA with a high GC content more difficult to denature than that with a low GC content?
Critical Thinking
A certain DNA sample is found to have a makeup consisting of 22% thymine. Use Chargaff’s rules to fill in the percentages for the other three nitrogenous bases.
In considering the structure of the DNA double helix, how would you expect the structure to differ if there was base pairing between two purines? Between two pyrimidines?
10.3: Structure and Function of RNA
Ribonucleic acid (RNA) is typically single stranded and contains ribose as its pentose sugar and the pyrimidine uracil instead of thymine. An RNA strand can undergo significant intramolecular base pairing to take on a three-dimensional structure. There are three main types of RNA, all involved in protein synthesis. Messenger RNA (mRNA) serves as the intermediary between DNA and the synthesis of protein products during translation.
Multiple Choice
Which of the following types of RNA codes for a protein?
1. dsRNA
2. mRNA
3. rRNA
4. tRNA
Answer
B
A nucleic acid is purified from a mixture. The molecules are relatively small, contain uracil, and most are covalently bound to an amino acid. Which of the following was purified?
1. DNA
2. mRNA
3. rRNA
4. tRNA
Answer
D
Which of the following types of RNA is known for its catalytic abilities?
1. dsRNA
2. mRNA
3. rRNA
4. tRNA
Answer
C
Ribosomes are composed of rRNA and what other component?
1. protein
2. polypeptides
3. DNA
4. mRNA
Answer
A
Which of the following may use RNA as its genome?
1. a bacterium
2. an archaeon
3. a virus
4. a eukaryote
Answer
C
Matching
Match the correct molecule with its description:
___tRNA
___rRNA
___mRNA
A. is a major component of ribosome
B. is a copy of the information in a gene
C. carries an amino acid to the ribosome
Answer
C, A, B
True/False
Ribosomes are composed mostly of RNA.
Answer
True
Double-stranded RNA is commonly found inside cells.
Answer
False
Short Answer
What are the differences between DNA nucleotides and RNA nucleotides?
How is the information stored within the base sequence of DNA used to determine a cell’s properties?
How do complementary base pairs contribute to intramolecular base pairing within an RNA molecule?
If an antisense RNA has the sequence 5ʹAUUCGAAUGC3ʹ, what is the sequence of the mRNA to which it will bind? Be sure to label the 5ʹ and 3ʹ ends of the molecule you draw.
Why does double-stranded RNA (dsRNA) stimulate RNA interference?
Critical Thinking
Identify the location of mRNA, rRNA, and tRNA in the figure.
Why does it make sense that tRNA and rRNA molecules are more stable than mRNA molecules?
10.4: The Structure and Function of Cellular Genomes
The entire genetic content of a cell is its genome. Genes code for proteins, or stable RNA molecules, each of which carries out a specific function in the cell. Although the genotype that a cell possesses remains constant, expression of genes is dependent on environmental conditions. A phenotype is the observable characteristics of a cell (or organism) at a given point in time and results from the complement of genes currently being used.
Multiple Choice
Which of the following correctly describes the structure of the typical eukaryotic genome?
1. diploid
2. linear
3. singular
4. double stranded
Answer
A
Which of the following is typically found as part of the prokaryotic genome?
1. chloroplast DNA
2. linear chromosomes
3. plasmids
4. mitochondrial DNA
Answer
C
Serratia marcescens cells produce a red pigment at room temperature. The red color of the colonies is an example of which of the following?
1. genotype
2. phenotype
3. change in DNA base composition
4. adaptation to the environment
Answer
B
Which of the following genes would not likely be encoded on a plasmid?
1. genes encoding toxins that damage host tissue
2. genes encoding antibacterial resistance
3. gene encoding enzymes for glycolysis
4. genes encoding enzymes for the degradation of an unusual substrate
Answer
C
Histones are DNA binding proteins that are important for DNA packaging in which of the following?
1. double-stranded and single-stranded DNA viruses
2. archaea and bacteria
3. bacteria and eukaryotes
4. eukaryotes and archaea
Answer
D
True/False
Within an organism, phenotypes may change while genotypes remain constant.
Answer
True
Noncoding DNA has no biological purpose.
Answer
False
Fill in the Blank
Plasmids are typically transferred among members of a bacterial community by ________ gene transfer.
Answer
horizontal
Short Answer
What are some differences in chromosomal structures between prokaryotes and eukaryotes?
How do prokaryotes and eukaryotes manage to fit their lengthy DNA inside of cells? Why is this necessary?
What are some functions of noncoding DNA?
In the chromatin of eukaryotic cells, which regions of the chromosome would you expect to be more compact: the regions that contain genes being actively copied into RNA or those that contain inactive genes?
Critical Thinking
A new type of bacteriophage has been isolated and you are in charge of characterizing its genome. The base composition of the bacteriophage is A (15%), C (20%), T (35%), and G (30%). What can you conclude about the genome of the virus? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/10%3A_Biochemistry_of_the_Genome/10.E%3A_Biochemistry_of_the_Genome_%28Exercises%29.txt |
In 1954, French scientist and future Nobel laureate Jacques Monod (1910–1976) famously said, “What is true in E. coli is true in the elephant,” suggesting that the biochemistry of life was maintained throughout evolution and is shared in all forms of known life. Since Monod’s famous statement, we have learned a great deal about the mechanisms of gene regulation, expression, and replication in living cells. All cells use DNA for information storage, share the same genetic code, and use similar mechanisms to replicate and express it. Although many aspects of genetics are universally shared, variations do exist among contemporary genetic systems. We now know that within the shared overall theme of the genetic mechanism, there are significant differences among the three domains of life: Eukarya, Archaea, and Bacteria. Additionally, viruses, cellular parasites but not themselves living cells, show dramatic variation in their genetic material and the replication and gene expression processes. Some of these differences have allowed us to engineer clinical tools such as antibiotics and antiviral drugs that specifically inhibit the reproduction of pathogens yet are harmless to their hosts.
• 11.1: What Are Genes?
A gene is composed of DNA that is “read” or transcribed to produce an RNA molecule during the process of transcription. One major type of RNA molecule, called messenger RNA (mRNA), provides the information for the ribosome to catalyze protein synthesis in a process called translation. The processes of transcription and translation are collectively referred to as gene expression.
• 11.2: DNA Replication
The DNA replication process is semiconservative, which results in two DNA molecules, each having one parental strand of DNA and one newly synthesized strand. In bacteria, the initiation of replication occurs at the origin of replication, where supercoiled DNA is unwound by DNA gyrase, made single-stranded by helicase, and bound by single-stranded binding protein to maintain its single-stranded state.
• 11.3: RNA Transcription
During the process of transcription, the information encoded within the DNA sequence of one or more genes is transcribed into a strand of RNA, also called an RNA transcript. The resulting single-stranded RNA molecule, composed of ribonucleotides containing the bases adenine, cytosine, guanine, and uracil, acts as a mobile molecular copy of the original DNA sequence. Transcription in prokaryotes and in eukaryotes requires the DNA double helix to partially unwind in the region of RNA synthesis.
• 11.4: Protein Synthesis (Translation)
The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other macromolecule of living organisms. They perform virtually every function of a cell, serving as both functional (e.g., enzymes) and structural elements. The process of translation, or protein synthesis, the second part of gene expression, involves the decoding by a ribosome of an mRNA message into a polypeptide product.
• 11.5: Mutations
A mutation is a heritable change in the DNA sequence of an organism. The resulting organism, called a mutant, may have a recognizable change in phenotype compared to the wild type, which is the phenotype most commonly observed in nature. A change in the DNA sequence is conferred to mRNA through transcription, and may lead to an altered amino acid sequence in a protein on translation.
• 11.6: How Asexual Prokaryotes Achieve Genetic Diversity
How do organisms whose dominant reproductive mode is asexual create genetic diversity? In prokaryotes, horizontal gene transfer (HGT), the introduction of genetic material from one organism to another organism within the same generation, is an important way to introduce genetic diversity. HGT allows even distantly related species to share genes, influencing their phenotypes.
• 11.7: Gene Regulation - Operon Theory
Genomic DNA contains both structural genes, which encode products that serve as cellular structures or enzymes, and regulatory genes, which encode products that regulate gene expression. The expression of a gene is a highly regulated process. Whereas regulating gene expression in multicellular organisms allows for cellular differentiation, in single-celled organisms like prokaryotes, it ensures that a cell’s resources are not wasted making proteins that the cell does not need at that time.
• 11.E: Mechanisms of Microbial Genetics (Exercises)
Thumbnail: DNA Double Helix. (Public Domain; Apers0n).
11: Mechanisms of Microbial Genetics
Learning Objectives
• Explain the two functions of the genome
• Explain the meaning of the central dogma of molecular biology
• Differentiate between genotype and phenotype and explain how environmental factors influence phenotype
Clinical Focus: Part 1
Mark is 60-year-old software engineer who suffers from type II diabetes, which he monitors and keeps under control largely through diet and exercise. One spring morning, while doing some gardening, he scraped his lower leg while walking through blackberry brambles. He continued working all day in the yard and did not bother to clean the wound and treat it with antibiotic ointment until later that evening. For the next 2 days, his leg became increasingly red, swollen, and warm to the touch. It was sore not only on the surface, but deep in the muscle. After 24 hours, Mark developed a fever and stiffness in the affected leg. Feeling increasingly weak, he called a neighbor, who drove him to the emergency department.
Exercise \(1\)
1. Did Mark wait too long to seek medical attention? At what point do his signs and symptoms warrant seeking medical attention?
2. What types of infections or other conditions might be responsible for Mark’s symptoms?
DNA serves two essential functions that deal with cellular information. First, DNA is the genetic material responsible for inheritance and is passed from parent to offspring for all life on earth. To preserve the integrity of this genetic information, DNA must be replicated with great accuracy, with minimal errors that introduce changes to the DNA sequence. A genome contains the full complement of DNA within a cell and is organized into smaller, discrete units called genes that are arranged on chromosomes and plasmids. The second function of DNA is to direct and regulate the construction of the proteins necessary to a cell for growth and reproduction in a particular cellular environment.
A gene is composed of DNA that is “read” or transcribed to produce an RNA molecule during the process of transcription. One major type of RNA molecule, called messenger RNA (mRNA), provides the information for the ribosome to catalyze protein synthesis in a process called translation. The processes of transcription and translation are collectively referred to as gene expression. Gene expression is the synthesis of a specific protein with a sequence of amino acids that is encoded in the gene. The flow of genetic information from DNA to RNA to protein is described by the central dogma (Figure \(1\)). This central dogma of molecular biology further elucidates the mechanism behind Beadle and Tatum’s “one gene-one enzyme” hypothesis (see Using Microbiology to Discover the Secrets of Life). Each of the processes of replication, transcription, and translation includes the stages of 1) initiation, 2) elongation (polymerization), and 3) termination. These stages will be described in more detail in this chapter.
A cell’s genotype is the full collection of genes it contains, whereas its phenotype is the set of observable characteristics that result from those genes. The phenotype is the product of the array of proteins being produced by the cell at a given time, which is influenced by the cell’s genotype as well as interactions with the cell’s environment. Genes code for proteins that have functions in the cell. Production of a specific protein encoded by an individual gene often results in a distinct phenotype for the cell compared with the phenotype without that protein. For this reason, it is also common to refer to the genotype of an individual gene and its phenotype. Although a cell’s genotype remains constant, not all genes are used to direct the production of their proteins simultaneously. Cells carefully regulate expression of their genes, only using genes to make specific proteins when those proteins are needed (Figure \(2\)).
Exercise \(2\)
1. What are the two functions of DNA?
2. Distinguish between the genotype and phenotype of a cell.
3. How can cells have the same genotype but differ in their phenotype?
Use and Abuse of Genome Data
Why can some humans harbor opportunistic pathogens like Haemophilus influenzae, Staphylococcus aureus, or Streptococcus pyogenes, in their upper respiratory tracts but remain asymptomatic carriers, while other individuals become seriously ill when infected? There is evidence suggesting that differences in susceptibility to infection between patients may be a result, at least in part, of genetic differences between human hosts. For example, genetic differences in human leukocyte antigens (HLAs) and red blood cell antigens among hosts have been implicated in different immune responses and resulting disease progression from infection with H. influenzae.
Because the genetic interplay between pathogen and host may contribute to disease outcomes, understanding differences in genetic makeup between individuals may be an important clinical tool. Ecological genomics is a relatively new field that seeks to understand how the genotypes of different organisms interact with each other in nature. The field answers questions about how gene expression of one organism affects gene expression of another. Medical applications of ecological genomics will focus on how pathogens interact with specific individuals, as opposed to humans in general. Such analyses would allow medical professionals to use knowledge of an individual’s genotype to apply more individualized plans for treatment and prevention of disease.
With the advent of next-generation sequencing, it is relatively easy to obtain the entire genomic sequences of pathogens; a bacterial genome can be sequenced in as little as a day.1 The speed and cost of sequencing the human genome has also been greatly reduced and, already, individuals can submit samples to receive extensive reports on their personal genetic traits, including ancestry and carrier status for various genetic diseases. As sequencing technologies progress further, such services will continue to become less expensive, more extensive, and quicker.
However, as this day quickly approaches, there are many ethical concerns with which society must grapple. For example, should genome sequencing be a standard practice for everybody? Should it be required by law or by employers if it will lower health-care costs? If one refuses genome sequencing, does he or she forfeit his or her right to health insurance coverage? For what purposes should the data be used? Who should oversee proper use of these data? If genome sequencing reveals predisposition to a particular disease, do insurance companies have the right to increase rates? Will employers treat an employee differently? Knowing that environmental influences also affect disease development, how should the data on the presence of a particular disease-causing allele in an individual be used ethically? The Genetic Information Nondiscrimination Act of 2008 (GINA) currently prohibits discriminatory practices based on genetic information by both health insurance companies and employers. However, GINA does not cover life, disability, or long-term care insurance policies. Clearly, all members of society must continue to engage in conversations about these issues so that such genomic data can be used to improve health care while simultaneously protecting an individual’s rights.
Key Concepts and Summary
• DNA serves two important cellular functions: It is the genetic material passed from parent to offspring and it serves as the information to direct and regulate the construction of the proteins necessary for the cell to perform all of its functions.
• The central dogma states that DNA organized into genes specifies the sequences of messenger RNA (mRNA), which, in turn, specifies the amino acid sequence of proteins.
• The genotype of a cell is the full collection of genes a cell contains. Not all genes are used to make proteins simultaneously. The phenotype is a cell’s observable characteristics resulting from the proteins it is producing at a given time under specific environmental conditions.
Footnotes
1. 1 D.J. Edwards, K.E. Holt. “Beginner’s Guide to Comparative Bacterial Genome Analysis Using Next-Generation Sequence Data.” Microbial Informatics and Experimentation 3 no. 1 (2013):2. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/11%3A_Mechanisms_of_Microbial_Genetics/11.01%3A_What_Are_Genes.txt |
Learning Objectives
• Explain the meaning of semiconservative DNA replication
• Explain why DNA replication is bidirectional and includes both a leading and lagging strand
• Explain why Okazaki fragments are formed
• Describe the process of DNA replication and the functions of the enzymes involved
• Identify the differences between DNA replication in bacteria and eukaryotes
• Explain the process of rolling circle replication
The elucidation of the structure of the double helix by James Watson and Francis Crick in 1953 provided a hint as to how DNA is copied during the process of replication. Separating the strands of the double helix would provide two templates for the synthesis of new complementary strands, but exactly how new DNA molecules were constructed was still unclear. In one model, semiconservative replication, the two strands of the double helix separate during DNA replication, and each strand serves as a template from which the new complementary strand is copied; after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. There were two competing models also suggested: conservative and dispersive, which are shown in Figure \(1\).
Matthew Meselson (1930–) and Franklin Stahl (1929–) devised an experiment in 1958 to test which of these models correctly represents DNA replication (Figure \(2\)). They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen (15N) that was incorporated into nitrogenous bases and, eventually, into the DNA. This labeled the parental DNA. The E. coli culture was then shifted into a medium containing 14N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was separated by ultracentrifugation, during which the DNA formed bands according to its density. DNA grown in 15N would be expected to form a band at a higher density position than that grown in 14N. Meselson and Stahl noted that after one generation of growth in 14N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15N or 14N. This suggested either a semiconservative or dispersive mode of replication. Some cells were allowed to grow for one more generation in 14N and spun again. The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N, and the other corresponded to the band of 14N DNA. These results could only be explained if DNA replicates in a semiconservative manner. Therefore, the other two models were ruled out. As a result of this experiment, we now know that during DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or “old” strand. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Exercise \(1\)
What would have been the conclusion of Meselson and Stahl’s experiment if, after the first generation, they had found two bands of DNA?
DNA Replication in Bacteria
DNA replication has been well studied in bacteria primarily because of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs (Mbp) in a single circular chromosome and all of it is replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle bidirectionally (i.e., in both directions). This means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs with few errors.
DNA replication uses a large number of proteins and enzymes (Table \(1\)). One of the key players is the enzyme DNA polymerase, also known as DNA pol. In bacteria, three main types of DNA polymerases are known: DNA pol I, DNA pol II, and DNA pol III. It is now known that DNA pol III is the enzyme required for DNA synthesis; DNA pol I and DNA pol II are primarily required for repair. DNA pol III adds deoxyribonucleotides each complementary to a nucleotide on the template strand, one by one to the 3’-OH group of the growing DNA chain. The addition of these nucleotides requires energy. This energy is present in the bonds of three phosphate groups attached to each nucleotide (a triphosphate nucleotide), similar to how energy is stored in the phosphate bonds of adenosine triphosphate (ATP) (Figure \(3\)). When the bond between the phosphates is broken and diphosphate is released, the energy released allows for the formation of a covalent phosphodiester bond by dehydration synthesis between the incoming nucleotide and the free 3’-OH group on the growing DNA strand.
Initiation
The initiation of replication occurs at specific nucleotide sequence called the origin of replication, where various proteins bind to begin the replication process. E. coli has a single origin of replication (as do most prokaryotes), called oriC, on its one chromosome. The origin of replication is approximately 245 base pairs long and is rich in adenine-thymine (AT) sequences.
Some of the proteins that bind to the origin of replication are important in making single-stranded regions of DNA accessible for replication. Chromosomal DNA is typically wrapped around histones (in eukaryotes and archaea) or histone-like proteins (in bacteria), and is supercoiled, or extensively wrapped and twisted on itself. This packaging makes the information in the DNA molecule inaccessible. However, enzymes called topoisomerases change the shape and supercoiling of the chromosome. For bacterial DNA replication to begin, the supercoiled chromosome is relaxed by topoisomerase II, also called DNA gyrase. An enzyme called helicase then separates the DNA strands by breaking the hydrogen bonds between the nitrogenous base pairs. Recall that AT sequences have fewer hydrogen bonds and, hence, have weaker interactions than guanine-cytosine (GC) sequences. These enzymes require ATP hydrolysis. As the DNA opens up, Y-shaped structures called replication forks are formed. Two replication forks are formed at the origin of replication, allowing for bidirectional replication and formation of a structure that looks like a bubble when viewed with a transmission electron microscope; as a result, this structure is called a replication bubble. The DNA near each replication fork is coated with single-stranded binding proteins to prevent the single-stranded DNA from rewinding into a double helix.
Once single-stranded DNA is accessible at the origin of replication, DNA replication can begin. However, DNA pol III is able to add nucleotides only in the 5’ to 3’ direction (a new DNA strand can be only extended in this direction). This is because DNA polymerase requires a free 3’-OH group to which it can add nucleotides by forming a covalent phosphodiester bond between the 3’-OH end and the 5’ phosphate of the next nucleotide. This also means that it cannot add nucleotides if a free 3’-OH group is not available, which is the case for a single strand of DNA. The problem is solved with the help of an RNA sequence that provides the free 3’-OH end. Because this sequence allows the start of DNA synthesis, it is appropriately called the primer. The primer is five to 10 nucleotides long and complementary to the parental or template DNA. It is synthesized by RNA primase, which is an RNA polymerase. Unlike DNA polymerases, RNA polymerases do not need a free 3’-OH group to synthesize an RNA molecule. Now that the primer provides the free 3’-OH group, DNA polymerase III can now extend this RNA primer, adding DNA nucleotides one by one that are complementary to the template strand (Figure \(1\)).
Elongation
During elongation in DNA replication, the addition of nucleotides occurs at its maximal rate of about 1000 nucleotides per second. DNA polymerase III can only extend in the 5’ to 3’ direction, which poses a problem at the replication fork. The DNA double helix is antiparallel; that is, one strand is oriented in the 5’ to 3’ direction and the other is oriented in the 3’ to 5’ direction (see Structure and Function of DNA). During replication, one strand, which is complementary to the 3’ to 5’ parental DNA strand, is synthesized continuously toward the replication fork because polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the leading strand. The other strand, complementary to the 5’ to 3’ parental DNA, grows away from the replication fork, so the polymerase must move back toward the replication fork to begin adding bases to a new primer, again in the direction away from the replication fork. It does so until it bumps into the previously synthesized strand and then it moves back again (Figure \(4\)). These steps produce small DNA sequence fragments known as Okazaki fragments, each separated by RNA primer. Okazaki fragments are named after the Japanese research team and married couple Reiji and Tsuneko Okazaki, who first discovered them in 1966. The strand with the Okazaki fragments is known as the lagging strand, and its synthesis is said to be discontinuous.
The leading strand can be extended from one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3’ to 5’, and that of the leading strand 5’ to 3’. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides. The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. Beyond its role in initiation, topoisomerase also prevents the overwinding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it. As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the exonuclease activity of DNA polymerase I, and the gaps are filled in. The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the enzyme DNA ligase that catalyzes the formation of covalent phosphodiester linkage between the 3’-OH end of one DNA fragment and the 5’ phosphate end of the other fragment, stabilizing the sugar-phosphate backbone of the DNA molecule.
Termination
Once the complete chromosome has been replicated, termination of DNA replication must occur. Although much is known about initiation of replication, less is known about the termination process. Following replication, the resulting complete circular genomes of prokaryotes are concatenated, meaning that the circular DNA chromosomes are interlocked and must be separated from each other. This is accomplished through the activity of bacterial topoisomerase IV, which introduces double-stranded breaks into DNA molecules, allowing them to separate from each other; the enzyme then reseals the circular chromosomes. The resolution of concatemers is an issue unique to prokaryotic DNA replication because of their circular chromosomes. Because both bacterial DNA gyrase and topoisomerase IV are distinct from their eukaryotic counterparts, these enzymes serve as targets for a class of antimicrobial drugs called quinolones.
Table \(1\): The Molecular Machinery Involved in Bacterial DNA Replication
Enzyme or Factor Function
DNA pol I Exonuclease activity removes RNA primer and replaces it with newly synthesized DNA
DNA pol III Main enzyme that adds nucleotides in the 5’ to 3’ direction
Helicase Opens the DNA helix by breaking hydrogen bonds between the nitrogenous bases
Ligase Seals the gaps between the Okazaki fragments on the lagging strand to create one continuous DNA strand
Primase Synthesizes RNA primers needed to start replication
Single-stranded binding proteins Bind to single-stranded DNA to prevent hydrogen bonding between DNA strands, reforming double-stranded DNA
Sliding clamp Helps hold DNA pol III in place when nucleotides are being added
Topoisomerase II (DNA gyrase) Relaxes supercoiled chromosome to make DNA more accessible for the initiation of replication; helps relieve the stress on DNA when unwinding, by causing breaks and then resealing the DNA
Topoisomerase IV Introduces single-stranded break into concatenated chromosomes to release them from each other, and then reseals the DNA
Exercise \(2\)
1. Which enzyme breaks the hydrogen bonds holding the two strands of DNA together so that replication can occur?
2. Is it the lagging strand or the leading strand that is synthesized in the direction toward the opening of the replication fork?
3. Which enzyme is responsible for removing the RNA primers in newly replicated bacterial DNA?
DNA Replication in Eukaryotes
Eukaryotic genomes are much more complex and larger than prokaryotic genomes and are typically composed of multiple linear chromosomes (Table \(2\)). The human genome, for example, has 3 billion base pairs per haploid set of chromosomes, and 6 billion base pairs are inserted during replication. There are multiple origins of replication on each eukaryotic chromosome (Figure \(5\)); the human genome has 30,000 to 50,000 origins of replication. The rate of replication is approximately 100 nucleotides per second—10 times slower than prokaryotic replication.
The essential steps of replication in eukaryotes are the same as in prokaryotes. Before replication can start, the DNA has to be made available as a template. Eukaryotic DNA is highly supercoiled and packaged, which is facilitated by many proteins, including histones (see Structure and Function of Cellular Genomes). At the origin of replication, a prereplication complex composed of several proteins, including helicase, forms and recruits other enzymes involved in the initiation of replication, including topoisomerase to relax supercoiling, single-stranded binding protein, RNA primase, and DNA polymerase. Following initiation of replication, in a process similar to that found in prokaryotes, elongation is facilitated by eukaryotic DNA polymerases. The leading strand is continuously synthesized by the eukaryotic polymerase enzyme pol δ, while the lagging strand is synthesized by pol ε. A sliding clamp protein holds the DNA polymerase in place so that it does not fall off the DNA. The enzyme ribonuclease H (RNase H), instead of a DNA polymerase as in bacteria, removes the RNA primer, which is then replaced with DNA nucleotides. The gaps that remain are sealed by DNA ligase.
Because eukaryotic chromosomes are linear, one might expect that their replication would be more straightforward. As in prokaryotes, the eukaryotic DNA polymerase can add nucleotides only in the 5’ to 3’ direction. In the leading strand, synthesis continues until it reaches either the end of the chromosome or another replication fork progressing in the opposite direction. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no place to make a primer for the DNA fragment to be copied at the end of the chromosome. These ends thus remain unpaired and, over time, they may get progressively shorter as cells continue to divide.
The ends of the linear chromosomes are known as telomeres and consist of noncoding repetitive sequences. The telomeres protect coding sequences from being lost as cells continue to divide. In humans, a six base-pair sequence, TTAGGG, is repeated 100 to 1000 times to form the telomere. The discovery of the enzyme telomerase (Figure \(6\)) clarified our understanding of how chromosome ends are maintained. Telomerase contains a catalytic part and a built-in RNA template. It attaches to the end of the chromosome, and complementary bases to the RNA template are added on the 3’ end of the DNA strand. Once the 3’ end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. In this way, the ends of the chromosomes are replicated. In humans, telomerase is typically active in germ cells and adult stem cells; it is not active in adult somatic cells and may be associated with the aging of these cells. Eukaryotic microbes including fungi and protozoans also produce telomerase to maintain chromosomal integrity. For her discovery of telomerase and its action, Elizabeth Blackburn (1948–) received the Nobel Prize for Medicine or Physiology in 2009.
Table \(2\): Comparison of Bacterial and Eukaryotic Replication
Property Bacteria Eukaryotes
Genome structure Single circular chromosome Multiple linear chromosomes
Number of origins per chromosome Single Multiple
Rate of replication 1000 nucleotides per second 100 nucleotides per second
Telomerase Not present Present
RNA primer removal DNA pol I RNase H
Strand elongation DNA pol III pol δ, pol ε
Exercise \(3\)
1. How does the origin of replication differ between eukaryotes and prokaryotes?
2. What polymerase enzymes are responsible for DNA synthesis during eukaryotic replication?
3. What is found at the ends of the chromosomes in eukaryotes and why?
DNA Replication of Extrachromosomal Elements: Plasmids and Viruses
To copy their nucleic acids, plasmids and viruses frequently use variations on the pattern of DNA replication described for prokaryote genomes. For more information on the wide range of viral replication strategies, see The Viral Life Cycle.
Rolling Circle Replication
Whereas many bacterial plasmids (see Unique Characteristics of Prokaryotic Cells) replicate by a process similar to that used to copy the bacterial chromosome, other plasmids, several bacteriophages, and some viruses of eukaryotes use rolling circle replication (Figure \(7\)). The circular nature of plasmids and the circularization of some viral genomes on infection make this possible. Rolling circle replication begins with the enzymatic nicking of one strand of the double-stranded circular molecule at the double-stranded origin (dso) site. In bacteria, DNA polymerase III binds to the 3’-OH group of the nicked strand and begins to unidirectionally replicate the DNA using the un-nicked strand as a template, displacing the nicked strand as it does so. Completion of DNA replication at the site of the original nick results in full displacement of the nicked strand, which may then recircularize into a single-stranded DNA molecule. RNA primase then synthesizes a primer to initiate DNA replication at the single-stranded origin (sso) site of the single-stranded DNA (ssDNA) molecule, resulting in a double-stranded DNA (dsDNA) molecule identical to the other circular DNA molecule.
Exercise \(4\)
Is there a lagging strand in rolling circle replication? Why or why not?
Key Concepts and Summary
• The DNA replication process is semiconservative, which results in two DNA molecules, each having one parental strand of DNA and one newly synthesized strand.
• In bacteria, the initiation of replication occurs at the origin of replication, where supercoiled DNA is unwound by DNA gyrase, made single-stranded by helicase, and bound by single-stranded binding protein to maintain its single-stranded state. Primase synthesizes a short RNA primer, providing a free 3’-OH group to which DNA polymerase III can add DNA nucleotides.
• During elongation, the leading strand of DNA is synthesized continuously from a single primer. The lagging strand is synthesized discontinuously in short Okazaki fragments, each requiring its own primer. The RNA primers are removed and replaced with DNA nucleotides by bacterial DNA polymerase I, and DNA ligase seals the gaps between these fragments.
• Termination of replication in bacteria involves the resolution of circular DNA concatemers by topoisomerase IV to release the two copies of the circular chromosome.
• Eukaryotes typically have multiple linear chromosomes, each with multiple origins of replication. Overall, replication in eukaryotes is similar to that in prokaryotes.
• The linear nature of eukaryotic chromosomes necessitates telomeres to protect genes near the end of the chromosomes. Telomerase extends telomeres, preventing their degradation, in some cell types.
• Rolling circle replication is a type of rapid unidirectional DNA synthesis of a circular DNA molecule used for the replication of some plasmids. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/11%3A_Mechanisms_of_Microbial_Genetics/11.02%3A_DNA_Replication.txt |
Learning Objectives
• Explain how RNA is synthesized using DNA as a template
• Distinguish between transcription in prokaryotes and eukaryotes
During the process of transcription, the information encoded within the DNA sequence of one or more genes is transcribed into a strand of RNA, also called an RNA transcript. The resulting single-stranded RNA molecule, composed of ribonucleotides containing the bases adenine (A), cytosine (C), guanine (G), and uracil (U), acts as a mobile molecular copy of the original DNA sequence. Transcription in prokaryotes and in eukaryotes requires the DNA double helix to partially unwind in the region of RNA synthesis. The unwound region is called a transcription bubble. Transcription of a particular gene always proceeds from one of the two DNA strands that acts as a template, the so-called antisense strand. The RNA product is complementary to the template strand of DNA and is almost identical to the nontemplate DNA strand, or the sense strand. The only difference is that in RNA, all of the T nucleotides are replaced with U nucleotides; during RNA synthesis, U is incorporated when there is an A in the complementary antisense strand.
Transcription in Bacteria
Bacteria use the same RNA polymerase to transcribe all of their genes. Like DNA polymerase, RNA polymerase adds nucleotides one by one to the 3’-OH group of the growing nucleotide chain. One critical difference in activity between DNA polymerase and RNA polymerase is the requirement for a 3’-OH onto which to add nucleotides: DNA polymerase requires such a 3’-OH group, thus necessitating a primer, whereas RNA polymerase does not. During transcription, a ribonucleotide complementary to the DNA template strand is added to the growing RNA strand and a covalent phosphodiester bond is formed by dehydration synthesis between the new nucleotide and the last one added. In E. coli, RNA polymerase comprises six polypeptide subunits, five of which compose the polymerase core enzyme responsible for adding RNA nucleotides to a growing strand. The sixth subunit is known as sigma (σ). The σ factor enables RNA polymerase to bind to a specific promoter, thus allowing for the transcription of various genes. There are various σ factors that allow for transcription of various genes.
Initiation
The initiation of transcription begins at a promoter, a DNA sequence onto which the transcription machinery binds and initiates transcription. The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5’ RNA nucleotide is transcribed is the initiation site. Nucleotides preceding the initiation site are designated “upstream,” whereas nucleotides following the initiation site are called “downstream” nucleotides. In most cases, promoters are located just upstream of the genes they regulate. Although promoter sequences vary among bacterial genomes, a few elements are conserved. At the –10 and –35 positions within the DNA prior to the initiation site (designated +1), there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species. The –10 consensus sequence, called the TATA box, is TATAAT. The –35 sequence is recognized and bound by σ.
Elongation
The elongation in transcription phase begins when the σ subunit dissociates from the polymerase, allowing the core enzyme to synthesize RNA complementary to the DNA template in a 5’ to 3’ direction at a rate of approximately 40 nucleotides per second. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it (Figure \(1\)).
Termination
Once a gene is transcribed, the bacterial polymerase must dissociate from the DNA template and liberate the newly made RNA. This is referred to as termination of transcription. The DNA template includes repeated nucleotide sequences that act as termination signals, causing RNA polymerase to stall and release from the DNA template, freeing the RNA transcript.
Exercise \(1\)
1. Where does σ factor of RNA polymerase bind DNA to start transcription?
2. What occurs to initiate the polymerization activity of RNA polymerase?
3. Where does the signal to end transcription come from?
Transcription in Eukaryotes
Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with a few significant differences (see Table \(1\)). Eukaryotes use three different polymerases, RNA polymerases I, II, and III, all structurally distinct from the bacterial RNA polymerase. Each transcribes a different subset of genes. Interestingly, archaea contain a single RNA polymerase that is more closely related to eukaryotic RNA polymerase II than to its bacterial counterpart. Eukaryotic mRNAs are also usually monocistronic, meaning that they each encode only a single polypeptide, whereas prokaryotic mRNAs of bacteria and archaea are commonly polycistronic, meaning that they encode multiple polypeptides.
The most important difference between prokaryotes and eukaryotes is the latter’s membrane-bound nucleus, which influences the ease of use of RNA molecules for protein synthesis. With the genes bound in a nucleus, the eukaryotic cell must transport protein-encoding RNA molecules to the cytoplasm to be translated. Protein-encoding primary transcripts, the RNA molecules directly synthesized by RNA polymerase, must undergo several processing steps to protect these RNA molecules from degradation during the time they are transferred from the nucleus to the cytoplasm and translated into a protein. For example, eukaryotic mRNAs may last for several hours, whereas the typical prokaryotic mRNA lasts no more than 5 seconds.
The primary transcript (also called pre-mRNA) is first coated with RNA-stabilizing proteins to protect it from degradation while it is processed and exported out of the nucleus. The first type of processing begins while the primary transcript is still being synthesized; a special 7-methylguanosine nucleotide, called the 5’ cap, is added to the 5’ end of the growing transcript. In addition to preventing degradation, factors involved in subsequent protein synthesis recognize the cap, which helps initiate translation by ribosomes. Once elongation is complete, another processing enzyme then adds a string of approximately 200 adenine nucleotides to the 3’ end, called the poly-A tail. This modification further protects the pre-mRNA from degradation and signals to cellular factors that the transcript needs to be exported to the cytoplasm.
Eukaryotic genes that encode polypeptides are composed of coding sequences called exons (ex-on signifies that they are expressed) and intervening sequences called introns (int-ron denotes their intervening role). Transcribed RNA sequences corresponding to introns do not encode regions of the functional polypeptide and are removed from the pre-mRNA during processing. It is essential that all of the intron-encoded RNA sequences are completely and precisely removed from a pre-mRNA before protein synthesis so that the exon-encoded RNA sequences are properly joined together to code for a functional polypeptide. If the process errs by even a single nucleotide, the sequences of the rejoined exons would be shifted, and the resulting polypeptide would be nonfunctional. The process of removing intron-encoded RNA sequences and reconnecting those encoded by exons is called RNA splicing and is facilitated by the action of a spliceosome containing small nuclear ribonucleo proteins (snRNPs). Intron-encoded RNA sequences are removed from the pre-mRNA while it is still in the nucleus. Although they are not translated, introns appear to have various functions, including gene regulation and mRNA transport. On completion of these modifications, the mature transcript, the mRNA that encodes a polypeptide, is transported out of the nucleus, destined for the cytoplasm for translation. Introns can be spliced out differently, resulting in various exons being included or excluded from the final mRNA product. This process is known as alternative splicing. The advantage of alternative splicing is that different types of mRNA transcripts can be generated, all derived from the same DNA sequence. In recent years, it has been shown that some archaea also have the ability to splice their pre-mRNA.
Table \(1\): Comparison of Transcription in Bacteria Versus Eukaryotes
Property Bacteria Eukaryotes
Number of polypeptides encoded per mRNA Monocistronic or polycistronic Exclusively monocistronic
Strand elongation core + σ = holoenzyme RNA polymerases I, II, or III
Addition of 5’ cap No Yes
Addition of 3’ poly-A tail No Yes
Splicing of pre-mRNA No Yes
Link to Learning
Visualize how mRNA splicing happens by watching the process in action in this video. See how introns are removed during RNA splicing here.
Exercise \(2\)
1. In eukaryotic cells, how is the RNA transcript from a gene for a protein modified after it is transcribed?
2. Do exons or introns contain information for protein sequences?
Clinical Focus: Part 2
In the emergency department, a nurse told Mark that he had made a good decision to come to the hospital because his symptoms indicated an infection that had gotten out of control. Mark’s symptoms had progressed, with the area of skin affected and the amount of swelling increasing. Within the affected area, a rash had begun, blistering and small gas pockets underneath the outermost layer of skin had formed, and some of the skin was becoming gray. Based on the putrid smell of the pus draining from one of the blisters, the rapid progression of the infection, and the visual appearance of the affected skin, the physician immediately began treatment for necrotizing fasciitis. Mark’s physician ordered a culture of the fluid draining from the blister and also ordered blood work, including a white blood cell count.
Mark was admitted to the intensive care unit and began intravenous administration of a broad-spectrum antibiotic to try to minimize further spread of the infection. Despite antibiotic therapy, Mark’s condition deteriorated quickly. Mark became confused and dizzy. Within a few hours of his hospital admission, his blood pressure dropped significantly and his breathing became shallower and more rapid. Additionally, blistering increased, with the blisters intensifying in color to purplish black, and the wound itself seemed to be progressing rapidly up Mark’s leg.
Exercise \(3\)
1. What are possible causative agents of Mark’s necrotizing fasciitis?
2. What are some possible explanations for why the antibiotic treatment does not seem to be working?
Key Concepts and Summary
• During transcription, the information encoded in DNA is used to make RNA.
• RNA polymerase synthesizes RNA, using the antisense strand of the DNA as template by adding complementary RNA nucleotides to the 3’ end of the growing strand.
• RNA polymerase binds to DNA at a sequence called a promoter during the initiation of transcription.
• Genes encoding proteins of related functions are frequently transcribed under the control of a single promoter in prokaryotes, resulting in the formation of a polycistronic mRNA molecule that encodes multiple polypeptides.
• Unlike DNA polymerase, RNA polymerase does not require a 3’-OH group to add nucleotides, so a primer is not needed during initiation.
• Termination of transcription in bacteria occurs when the RNA polymerase encounters specific DNA sequences that lead to stalling of the polymerase. This results in release of RNA polymerase from the DNA template strand, freeing the RNA transcript.
• Eukaryotes have three different RNA polymerases. Eukaryotes also have monocistronic mRNA, each encoding only a single polypeptide.
• Eukaryotic primary transcripts are processed in several ways, including the addition of a 5’ cap and a 3′-poly-A tail, as well as splicing, to generate a mature mRNA molecule that can be transported out of the nucleus and that is protected from degradation. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/11%3A_Mechanisms_of_Microbial_Genetics/11.03%3A_RNA_Transcription.txt |
Learning Objectives
• Describe the genetic code and explain why it is considered almost universal
• Explain the process of translation and the functions of the molecular machinery of translation
• Compare translation in eukaryotes and prokaryotes
The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other macromolecule of living organisms. They perform virtually every function of a cell, serving as both functional (e.g., enzymes) and structural elements. The process of translation, or protein synthesis, the second part of gene expression, involves the decoding by a ribosome of an mRNA message into a polypeptide product.
The Genetic Code
Translation of the mRNA template converts nucleotide-based genetic information into the “language” of amino acids to create a protein product. A protein sequence consists of 20 commonly occurring amino acids. Each amino acid is defined within the mRNA by a triplet of nucleotides called a codon. The relationship between an mRNA codon and its corresponding amino acid is called the genetic code.
The three-nucleotide code means that there is a total of 64 possible combinations (43, with four different nucleotides possible at each of the three different positions within the codon). This number is greater than the number of amino acids and a given amino acid is encoded by more than one codon (Figure \(1\)). This redundancy in the genetic code is called degeneracy. Typically, whereas the first two positions in a codon are important for determining which amino acid will be incorporated into a growing polypeptide, the third position, called the wobble position, is less critical. In some cases, if the nucleotide in the third position is changed, the same amino acid is still incorporated.
Whereas 61 of the 64 possible triplets code for amino acids, three of the 64 codons do not code for an amino acid; they terminate protein synthesis, releasing the polypeptide from the translation machinery. These are called stop codons or nonsense codons. Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also typically serves as the start codon to initiate translation. The reading frame, the way nucleotides in mRNA are grouped into codons, for translation is set by the AUG start codon near the 5’ end of the mRNA. Each set of three nucleotides following this start codon is a codon in the mRNA message.
The genetic code is nearly universal. With a few exceptions, virtually all species use the same genetic code for protein synthesis, which is powerful evidence that all extant life on earth shares a common origin. However, unusual amino acids such as selenocysteine and pyrrolysine have been observed in archaea and bacteria. In the case of selenocysteine, the codon used is UGA (normally a stop codon). However, UGA can encode for selenocysteine using a stem-loop structure (known as the selenocysteine insertion sequence, or SECIS element), which is found at the 3’ untranslated region of the mRNA. Pyrrolysine uses a different stop codon, UAG. The incorporation of pyrrolysine requires the pylS gene and a unique transfer RNA (tRNA) with a CUA anticodon.
Exercise \(1\)
1. How many bases are in each codon?
2. What amino acid is coded for by the codon AAU?
3. What happens when a stop codon is reached?
The Protein Synthesis Machinery
In addition to the mRNA template, many molecules and macromolecules contribute to the process of translation. The composition of each component varies across taxa; for instance, ribosomes may consist of different numbers of ribosomal RNAs (rRNAs) and polypeptides depending on the organism. However, the general structures and functions of the protein synthesis machinery are comparable from bacteria to human cells. Translation requires the input of an mRNA template, ribosomes, tRNAs, and various enzymatic factors.
Ribosomes
A ribosome is a complex macromolecule composed of catalytic rRNAs (called ribozymes) and structural rRNAs, as well as many distinct polypeptides. Mature rRNAs make up approximately 50% of each ribosome. Prokaryotes have 70S ribosomes, whereas eukaryotes have 80S ribosomes in the cytoplasm and rough endoplasmic reticulum, and 70S ribosomes in mitochondria and chloroplasts. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation. In E. coli, the small subunit is described as 30S (which contains the 16S rRNA subunit), and the large subunit is 50S (which contains the 5S and 23S rRNA subunits), for a total of 70S (Svedberg units are not additive). Eukaryote ribosomes have a small 40S subunit (which contains the 18S rRNA subunit) and a large 60S subunit (which contains the 5S, 5.8S and 28S rRNA subunits), for a total of 80S. The small subunit is responsible for binding the mRNA template, whereas the large subunit binds tRNAs (discussed in the next subsection).
Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5’ to 3’ and synthesizing the polypeptide from the N terminus to the C terminus. The complete structure containing an mRNA with multiple associated ribosomes is called a polyribosome (or polysome). In both bacteria and archaea, before transcriptional termination occurs, each protein-encoding transcript is already being used to begin synthesis of numerous copies of the encoded polypeptide(s) because the processes of transcription and translation can occur concurrently, forming polyribosomes (Figure \(2\)). The reason why transcription and translation can occur simultaneously is because both of these processes occur in the same 5’ to 3’ direction, they both occur in the cytoplasm of the cell, and because the RNA transcript is not processed once it is transcribed. This allows a prokaryotic cell to respond to an environmental signal requiring new proteins very quickly. In contrast, in eukaryotic cells, simultaneous transcription and translation is not possible. Although polyribosomes also form in eukaryotes, they cannot do so until RNA synthesis is complete and the RNA molecule has been modified and transported out of the nucleus.
Transfer RNAs
Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different types of tRNAs exist in the cytoplasm. Bacterial species typically have between 60 and 90 types. Serving as adaptors, each tRNA type binds to a specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually “translate” the language of RNA into the language of proteins. As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into such a small package. The tRNA molecule interacts with three factors: aminoacyl tRNA synthetases, ribosomes, and mRNA.
Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the single-stranded RNA molecule hydrogen bond with each other (Figure \(3\)). This shape positions the amino-acid binding site, called the CCA amino acid binding end, which is a cytosine-cytosine-adenine sequence at the 3’ end of the tRNA, and the anticodonat the other end. The anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing.
An amino acid is added to the end of a tRNA molecule through the process of tRNA “charging,” during which each tRNA molecule is linked to its correct or cognate amino acid by a group of enzymes called aminoacyl tRNA synthetases. At least one type of aminoacyl tRNA synthetase exists for each of the 20 amino acids. During this process, the amino acid is first activated by the addition of adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA, and AMP is released.
Exercise \(2\)
1. Describe the structure and composition of the prokaryotic ribosome.
2. In what direction is the mRNA template read?
3. Describe the structure and function of a tRNA.
The Mechanism of Protein Synthesis
Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli, a representative prokaryote, and specify any differences between bacterial and eukaryotic translation.
Initiation
The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli, this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine(fMet-tRNAfMet) (Figure \(4\)). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.
In eukaryotes, initiation complex formation is similar, with the following differences:
• The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
• Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5’ cap of the eukaryotic mRNA, then tracks along the mRNA in the 5’ to 3’ direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.
Elongation
In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.
Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3’ direction. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.
Termination
The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation init iation complex.
In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in Figure \(5\) and listed in Figure \(6\).
Protein Targeting, Folding, and Modification
During and after translation, polypeptides may need to be modified before they are biologically active. Post-translational modifications include:
1. removal of translated signal sequences—short tails of amino acids that aid in directing a protein to a specific cellular compartment
2. proper “folding” of the polypeptide and association of multiple polypeptide subunits, often facilitated by chaperone proteins, into a distinct three-dimensional structure
3. proteolytic processing of an inactive polypeptide to release an active protein component, and
4. various chemical modifications (e.g., phosphorylation, methylation, or glycosylation) of individual amino acids.
Exercise \(3\)
1. What are the components of the initiation complex for translation in prokaryotes?
2. What are two differences between initiation of prokaryotic and eukaryotic translation?
3. What occurs at each of the three active sites of the ribosome?
4. What causes termination of translation?
Key Concepts and Summary
• In translation, polypeptides are synthesized using mRNA sequences and cellular machinery, including tRNAs that match mRNA codons to specific amino acids and ribosomes composed of RNA and proteins that catalyze the reaction.
• The genetic code is degenerate in that several mRNA codons code for the same amino acids. The genetic code is almost universal among living organisms.
• Prokaryotic (70S) and cytoplasmic eukaryotic (80S) ribosomes are each composed of a large subunit and a small subunit of differing sizes between the two groups. Each subunit is composed of rRNA and protein. Organelle ribosomes in eukaryotic cells resemble prokaryotic ribosomes.
• Some 60 to 90 species of tRNA exist in bacteria. Each tRNA has a three-nucleotide anticodon as well as a binding site for a cognate amino acid. All tRNAs with a specific anticodon will carry the same amino acid.
• Initiation of translation occurs when the small ribosomal subunit binds with initiation factors and an initiator tRNA at the start codon of an mRNA, followed by the binding to the initiation complex of the large ribosomal subunit.
• In prokaryotic cells, the start codon codes for N-formyl-methionine carried by a special initiator tRNA. In eukaryotic cells, the start codon codes for methionine carried by a special initiator tRNA. In addition, whereas ribosomal binding of the mRNA in prokaryotes is facilitated by the Shine-Dalgarno sequence within the mRNA, eukaryotic ribosomes bind to the 5’ cap of the mRNA.
• During the elongation stage of translation, a charged tRNA binds to mRNA in the A site of the ribosome; a peptide bond is catalyzed between the two adjacent amino acids, breaking the bond between the first amino acid and its tRNA; the ribosome moves one codon along the mRNA; and the first tRNA is moved from the P site of the ribosome to the E site and leaves the ribosomal complex.
• Termination of translation occurs when the ribosome encounters a stop codon, which does not code for a tRNA. Release factors cause the polypeptide to be released, and the ribosomal complex dissociates.
• In prokaryotes, transcription and translation may be coupled, with translation of an mRNA molecule beginning as soon as transcription allows enough mRNA exposure for the binding of a ribosome, prior to transcription termination. Transcription and translation are not coupled in eukaryotes because transcription occurs in the nucleus, whereas translation occurs in the cytoplasm or in association with the rough endoplasmic reticulum.
• Polypeptides often require one or more post-translational modifications to become biologically active. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/11%3A_Mechanisms_of_Microbial_Genetics/11.04%3A_Protein_Synthesis_%28Translation%29.txt |
Learning Objectives
• Compare point mutations and frameshift mutations
• Describe the differences between missense, nonsense, and silent mutations
• Describe the differences between light and dark repair
• Explain how different mutagens act
• Explain why the Ames test can be used to detect carcinogens
• Analyze sequences of DNA and identify examples of types of mutations
A mutation is a heritable change in the DNA sequence of an organism. The resulting organism, called a mutant, may have a recognizable change in phenotype compared to the wild type, which is the phenotype most commonly observed in nature. A change in the DNA sequence is conferred to mRNA through transcription, and may lead to an altered amino acid sequence in a protein on translation. Because proteins carry out the vast majority of cellular functions, a change in amino acid sequence in a protein may lead to an altered phenotype for the cell and organism.
Effects of Mutations on DNA Sequence
There are several types of mutations that are classified according to how the DNA molecule is altered. One type, called a point mutation, affects a single base and most commonly occurs when one base is substituted or replaced by another. Mutations also result from the addition of one or more bases, known as an insertion, or the removal of one or more bases, known as a deletion.
Exercise \(1\)
What type of a mutation occurs when a gene has two fewer nucleotides in its sequence?
Effects of Mutations on Protein Structure and Function
Point mutations may have a wide range of effects on protein function (Figure \(1\)). As a consequence of the degeneracy of the genetic code, a point mutation will commonly result in the same amino acid being incorporated into the resulting polypeptide despite the sequence change. This change would have no effect on the protein’s structure, and is thus called a silent mutation. A missense mutation results in a different amino acid being incorporated into the resulting polypeptide. The effect of a missense mutation depends on how chemically different the new amino acid is from the wild-type amino acid. The location of the changed amino acid within the protein also is important. For example, if the changed amino acid is part of the enzyme’s active site, then the effect of the missense mutation may be significant. Many missense mutations result in proteins that are still functional, at least to some degree. Sometimes the effects of missense mutations may be only apparent under certain environmental conditions; such missense mutations are called conditional mutations. Rarely, a missense mutation may be beneficial. Under the right environmental conditions, this type of mutation may give the organism that harbors it a selective advantage. Yet another type of point mutation, called a nonsense mutation, converts a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon). Nonsense mutations result in the synthesis of proteins that are shorter than the wild type and typically not functional.
Deletions and insertions also cause various effects. Because codons are triplets of nucleotides, insertions or deletions in groups of three nucleotides may lead to the insertion or deletion of one or more amino acids and may not cause significant effects on the resulting protein’s functionality. However, frameshift mutations, caused by insertions or deletions of a number of nucleotides that are not a multiple of three are extremely problematic because a shift in the reading frame results (Figure \(1\)). Because ribosomes read the mRNA in triplet codons, frameshift mutations can change every amino acid after the point of the mutation. The new reading frame may also include a stop codon before the end of the coding sequence. Consequently, proteins made from genes containing frameshift mutations are nearly always nonfunctional.
Exercise \(2\)
1. What are the reasons a nucleotide change in a gene for a protein might not have any effect on the phenotype of that gene?
2. Is it possible for an insertion of three nucleotides together after the fifth nucleotide in a protein-coding gene to produce a protein that is shorter than normal? How or how not?
A Beneficial Mutation
Since the first case of infection with human immunodeficiency virus (HIV) was reported in 1981, nearly 40 million people have died from HIV infection,1 the virus that causes acquired immune deficiency syndrome (AIDS). The virus targets helper T cells that play a key role in bridging the innate and adaptive immune response, infecting and killing cells normally involved in the body’s response to infection. There is no cure for HIV infection, but many drugs have been developed to slow or block the progression of the virus. Although individuals around the world may be infected, the highest prevalence among people 15–49 years old is in sub-Saharan Africa, where nearly one person in 20 is infected, accounting for greater than 70% of the infections worldwide2 (Figure \(2\)). Unfortunately, this is also a part of the world where prevention strategies and drugs to treat the infection are the most lacking.
In recent years, scientific interest has been piqued by the discovery of a few individuals from northern Europe who are resistant to HIV infection. In 1998, American geneticist Stephen J. O’Brien at the National Institutes of Health (NIH) and colleagues published the results of their genetic analysis of more than 4,000 individuals. These indicated that many individuals of Eurasian descent (up to 14% in some ethnic groups) have a deletion mutation, called CCR5-delta 32, in the gene encoding CCR5. CCR5 is a coreceptor found on the surface of T cells that is necessary for many strains of the virus to enter the host cell. The mutation leads to the production of a receptor to which HIV cannot effectively bind and thus blocks viral entry. People homozygous for this mutation have greatly reduced susceptibility to HIV infection, and those who are heterozygous have some protection from infection as well.
It is not clear why people of northern European descent, specifically, carry this mutation, but its prevalence seems to be highest in northern Europe and steadily decreases in populations as one moves south. Research indicates that the mutation has been present since before HIV appeared and may have been selected for in European populations as a result of exposure to the plague or smallpox. This mutation may protect individuals from plague (caused by the bacterium Yersinia pestis) and smallpox (caused by the variola virus) because this receptor may also be involved in these diseases. The age of this mutation is a matter of debate, but estimates suggest it appeared between 1875 years to 225 years ago, and may have been spread from Northern Europe through Viking invasions.
This exciting finding has led to new avenues in HIV research, including looking for drugs to block CCR5 binding to HIV in individuals who lack the mutation. Although DNA testing to determine which individuals carry the CCR5-delta 32 mutation is possible, there are documented cases of individuals homozygous for the mutation contracting HIV. For this reason, DNA testing for the mutation is not widely recommended by public health officials so as not to encourage risky behavior in those who carry the mutation. Nevertheless, inhibiting the binding of HIV to CCR5 continues to be a valid strategy for the development of drug therapies for those infected with HIV.
Causes of Mutations
Mistakes in the process of DNA replication can cause spontaneous mutations to occur. The error rate of DNA polymerase is one incorrect base per billion base pairs replicated. Exposure to mutagens can cause induced mutations, which are various types of chemical agents or radiation (Table \(1\)). Exposure to a mutagen can increase the rate of mutation more than 1000-fold. Mutagens are often also carcinogens, agents that cause cancer. However, whereas nearly all carcinogens are mutagenic, not all mutagens are necessarily carcinogens.
Chemical Mutagens
Various types of chemical mutagens interact directly with DNA either by acting as nucleoside analogs or by modifying nucleotide bases. Chemicals called nucleoside analogs are structurally similar to normal nucleotide bases and can be incorporated into DNA during replication (Figure \(3\)). These base analogs induce mutations because they often have different base-pairing rules than the bases they replace. Other chemical mutagens can modify normal DNA bases, resulting in different base-pairing rules. For example, nitrous acid deaminates cytosine, converting it to uracil. Uracil then pairs with adenine in a subsequent round of replication, resulting in the conversion of a GC base pair to an AT base pair. Nitrous acid also deaminates adenine to hypoxanthine, which base pairs with cytosine instead of thymine, resulting in the conversion of a TA base pair to a CG base pair.
Chemical mutagens known as intercalating agents work differently. These molecules slide between the stacked nitrogenous bases of the DNA double helix, distorting the molecule and creating atypical spacing between nucleotide base pairs (Figure \(4\)). As a result, during DNA replication, DNA polymerase may either skip replicating several nucleotides (creating a deletion) or insert extra nucleotides (creating an insertion). Either outcome may lead to a frameshift mutation. Combustion products like polycyclic aromatic hydrocarbons are particularly dangerous intercalating agents that can lead to mutation-caused cancers. The intercalating agents ethidium bromide and acridine orange are commonly used in the laboratory to stain DNA for visualization and are potential mutagens.
Radiation
Exposure to either ionizing or nonionizing radiation can each induce mutations in DNA, although by different mechanisms. Strong ionizing radiation like X-rays and gamma rays can cause single- and double-stranded breaks in the DNA backbone through the formation of hydroxyl radicals on radiation exposure (Figure \(5\)). Ionizing radiation can also modify bases; for example, the deamination of cytosine to uracil, analogous to the action of nitrous acid.3 Ionizing radiation exposure is used to kill microbes to sterilize medical devices and foods, because of its dramatic nonspecific effect in damaging DNA, proteins, and other cellular components (see Using Physical Methods to Control Microorganisms).
Nonionizing radiation, like ultraviolet light, is not energetic enough to initiate these types of chemical changes. However, nonionizing radiation can induce dimer formation between two adjacent pyrimidine bases, commonly two thymines, within a nucleotide strand. During thymine dimer formation, the two adjacent thymines become covalently linked and, if left unrepaired, both DNA replication and transcription are stalled at this point. DNA polymerase may proceed and replicate the dimer incorrectly, potentially leading to frameshift or point mutations.
Table \(1\): A Summary of Mutagenic Agents
Mutagenic Agents Mode of Action Effect on DNA Resulting Type of Mutation
Nucleoside analogs
2-aminopurine Is inserted in place of A but base pairs with C Converts AT to GC base pair Point
5-bromouracil Is inserted in place of T but base pairs with G Converts AT to GC base pair Point
Nucleotide-modifying agent
Nitrous oxide Deaminates C to U Converts GC to AT base pair Point
Intercalating agents
Acridine orange, ethidium bromide, polycyclic aromatic hydrocarbons Distorts double helix, creates unusual spacing between nucleotides Introduces small deletions and insertions Frameshift
Ionizing radiation
X-rays, γ-rays Forms hydroxyl radicals Causes single- and double-strand DNA breaks Repair mechanisms may introduce mutations
X-rays, γ-rays Modifies bases (e.g., deaminating C to U) Converts GC to AT base pair Point
Nonionizing radiation
Ultraviolet Forms pyrimidine (usually thymine) dimers Causes DNA replication errors Frameshift or point
Exercise \(3\)
1. How does a base analog introduce a mutation?
2. How does an intercalating agent introduce a mutation?
3. What type of mutagen causes thymine dimers?
DNA Repair
The process of DNA replication is highly accurate, but mistakes can occur spontaneously or be induced by mutagens. Uncorrected mistakes can lead to serious consequences for the phenotype. Cells have developed several repair mechanisms to minimize the number of mutations that persist.
Proofreading
Most of the mistakes introduced during DNA replication are promptly corrected by most DNA polymerases through a function called proofreading. In proofreading, the DNA polymerase reads the newly added base, ensuring that it is complementary to the corresponding base in the template strand before adding the next one. If an incorrect base has been added, the enzyme makes a cut to release the wrong nucleotide and a new base is added.
Mismatch Repair
Some errors introduced during replication are corrected shortly after the replication machinery has moved. This mechanism is called mismatch repair. The enzymes involved in this mechanism recognize the incorrectly added nucleotide, excise it, and replace it with the correct base. One example is the methyl-directed mismatch repair in E. coli. The DNA is hemimethylated. This means that the parental strand is methylated while the newly synthesized daughter strand is not. It takes several minutes before the new strand is methylated. Proteins MutS, MutL, and MutH bind to the hemimethylated site where the incorrect nucleotide is found. MutH cuts the nonmethylated strand (the new strand). An exonuclease removes a portion of the strand (including the incorrect nucleotide). The gap formed is then filled in by DNA pol III and ligase.
Repair of Thymine Dimers
Because the production of thymine dimers is common (many organisms cannot avoid ultraviolet light), mechanisms have evolved to repair these lesions. In nucleotide excision repair (also called dark repair), enzymes remove the pyrimidine dimer and replace it with the correct nucleotides (Figure \(6\)). In E. coli, the DNA is scanned by an enzyme complex. If a distortion in the double helix is found that was introduced by the pyrimidine dimer, the enzyme complex cuts the sugar-phosphate backbone several bases upstream and downstream of the dimer, and the segment of DNA between these two cuts is then enzymatically removed. DNA pol I replaces the missing nucleotides with the correct ones and DNA ligase seals the gap in the sugar-phosphate backbone.
The direct repair (also called light repair) of thymine dimers occurs through the process of photoreactivation in the presence of visible light. An enzyme called photolyase recognizes the distortion in the DNA helix caused by the thymine dimer and binds to the dimer. Then, in the presence of visible light, the photolyase enzyme changes conformation and breaks apart the thymine dimer, allowing the thymines to again correctly base pair with the adenines on the complementary strand. Photoreactivation appears to be present in all organisms, with the exception of placental mammals, including humans. Photoreactivation is particularly important for organisms chronically exposed to ultraviolet radiation, like plants, photosynthetic bacteria, algae, and corals, to prevent the accumulation of mutations caused by thymine dimer formation.
Exercise \(4\)
1. During mismatch repair, how does the enzyme recognize which is the new and which is the old strand?
2. What type of mutation does photolyase repair?
Identifying Bacterial Mutants
One common technique used to identify bacterial mutants is called replica plating. This technique is used to detect nutritional mutants, called auxotrophs, which have a mutation in a gene encoding an enzyme in the biosynthesis pathway of a specific nutrient, such as an amino acid. As a result, whereas wild-type cells retain the ability to grow normally on a medium lacking the specific nutrient, auxotrophs are unable to grow on such a medium. During replica plating (Figure \(7\)), a population of bacterial cells is mutagenized and then plated as individual cells on a complex nutritionally complete plate and allowed to grow into colonies. Cells from these colonies are removed from this master plate, often using sterile velvet. This velvet, containing cells, is then pressed in the same orientation onto plates of various media. At least one plate should also be nutritionally complete to ensure that cells are being properly transferred between the plates. The other plates lack specific nutrients, allowing the researcher to discover various auxotrophic mutants unable to produce specific nutrients. Cells from the corresponding colony on the nutritionally complete plate can be used to recover the mutant for further study.
Exercise \(5\)
Why are cells plated on a nutritionally complete plate in addition to nutrient-deficient plates when looking for a mutant?
The Ames Test
The Ames test, developed by Bruce Ames (1928–) in the 1970s, is a method that uses bacteria for rapid, inexpensive screening of the carcinogenic potential of new chemical compounds. The test measures the mutation rate associated with exposure to the compound, which, if elevated, may indicate that exposure to this compound is associated with greater cancer risk. The Ames test uses as the test organism a strain of Salmonella typhimurium that is a histidine auxotroph, unable to synthesize its own histidine because of a mutation in an essential gene required for its synthesis. After exposure to a potential mutagen, these bacteria are plated onto a medium lacking histidine, and the number of mutants regaining the ability to synthesize histidine is recorded and compared with the number of such mutants that arise in the absence of the potential mutagen (Figure \(8\)). Chemicals that are more mutagenic will bring about more mutants with restored histidine synthesis in the Ames test. Because many chemicals are not directly mutagenic but are metabolized to mutagenic forms by liver enzymes, rat liver extract is commonly included at the start of this experiment to mimic liver metabolism. After the Ames test is conducted, compounds identified as mutagenic are further tested for their potential carcinogenic properties by using other models, including animal models like mice and rats.
Exercise \(6\)
1. What mutation is used as an indicator of mutation rate in the Ames test?
2. Why can the Ames test work as a test for carcinogenicity?
Key Concepts and Summary
• A mutation is a heritable change in DNA. A mutation may lead to a change in the amino-acid sequence of a protein, possibly affecting its function.
• A point mutation affects a single base pair. A point mutation may cause a silent mutation if the mRNA codon codes for the same amino acid, a missense mutation if the mRNA codon codes for a different amino acid, or a nonsense mutation if the mRNA codon becomes a stop codon.
• Missense mutations may retain function, depending on the chemistry of the new amino acid and its location in the protein. Nonsense mutations produce truncated and frequently nonfunctional proteins.
• A frameshift mutation results from an insertion or deletion of a number of nucleotides that is not a multiple of three. The change in reading frame alters every amino acid after the point of the mutation and results in a nonfunctional protein.
• Spontaneous mutations occur through DNA replication errors, whereas induced mutations occur through exposure to a mutagen.
• Mutagenic agents are frequently carcinogenic but not always. However, nearly all carcinogens are mutagenic.
• Chemical mutagens include base analogs and chemicals that modify existing bases. In both cases, mutations are introduced after several rounds of DNA replication.
• Ionizing radiation, such as X-rays and γ-rays, leads to breakage of the phosphodiester backbone of DNA and can also chemically modify bases to alter their base-pairing rules.
• Nonionizing radiation like ultraviolet light may introduce pyrimidine (thymine) dimers, which, during DNA replication and transcription, may introduce frameshift or point mutations.
• Cells have mechanisms to repair naturally occurring mutations. DNA polymerase has proofreading activity. Mismatch repair is a process to repair incorrectly incorporated bases after DNA replication has been completed.
• Pyrimidine dimers can also be repaired. In nucleotide excision repair (dark repair), enzymes recognize the distortion introduced by the pyrimidine dimer and replace the damaged strand with the correct bases, using the undamaged DNA strand as a template. Bacteria and other organisms may also use direct repair, in which the photolyase enzyme, in the presence of visible light, breaks apart the pyrimidines.
• Through comparison of growth on the complete plate and lack of growth on media lacking specific nutrients, specific loss-of-function mutants called auxotrophs can be identified.
• The Ames test is an inexpensive method that uses auxotrophic bacteria to measure mutagenicity of a chemical compound. Mutagenicity is an indicator of carcinogenic potential.
Footnotes
1. 1 World Health Organization. “ Global Health Observatory (GHO) Data, HIV/AIDS.” http://www.who.int/gho/hiv/en/. Accessed August 5, 2016.
2. 2 World Health Organization. “ Global Health Observatory (GHO) Data, HIV/AIDS.” http://www.who.int/gho/hiv/en/. Accessed August 5, 2016.
3. 3 K.R. Tindall et al. “Changes in DNA Base Sequence Induced by Gamma-Ray Mutagenesis of Lambda Phage and Prophage.” Genetics 118 no. 4 (1988):551–560. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/11%3A_Mechanisms_of_Microbial_Genetics/11.05%3A_Mutations.txt |
Learning Objectives
• Compare the processes of transformation, transduction, and conjugation
• Explain how asexual gene transfer results in prokaryotic genetic diversity
• Explain the structure and consequences for bacterial genetic diversity of transposons
Typically, when we consider genetic transfer, we think of vertical gene transfer, the transmission of genetic information from generation to generation. Vertical gene transfer is by far the main mode of transmission of genetic information in all cells. In sexually reproducing organisms, crossing-over events and independent assortment of individual chromosomes during meiosis contribute to genetic diversity in the population. Genetic diversity is also introduced during sexual reproduction, when the genetic information from two parents, each with different complements of genetic information, are combined, producing new combinations of parental genotypes in the diploid offspring. The occurrence of mutations also contributes to genetic diversity in a population. Genetic diversity of offspring is useful in changing or inconsistent environments and may be one reason for the evolutionary success of sexual reproduction.
When prokaryotes and eukaryotes reproduce asexually, they transfer a nearly identical copy of their genetic material to their offspring through vertical gene transfer. Although asexual reproduction produces more offspring more quickly, any benefits of diversity among those offspring are lost. How then do organisms whose dominant reproductive mode is asexual create genetic diversity? In prokaryotes, horizontal gene transfer (HGT), the introduction of genetic material from one organism to another organism within the same generation, is an important way to introduce genetic diversity. HGT allows even distantly related species to share genes, influencing their phenotypes. It is thought that HGT is more prevalent in prokaryotes but that only a small fraction of the prokaryotic genome may be transferred by this type of transfer at any one time. As the phenomenon is investigated more thoroughly, it may be revealed to be even more common. Many scientists believe that HGT and mutation are significant sources of genetic variation, the raw material for the process of natural selection, in prokaryotes. Although HGT is more common among evolutionarily related organisms, it may occur between any two species that live together in a natural community.
HGT in prokaryotes is known to occur by the three primary mechanisms that are illustrated in Figure \(1\):
1. Transformation: naked DNA is taken up from the environment
2. Transduction: genes are transferred between cells in a virus (see The Viral Life Cycle)
3. Conjugation: use of a hollow tube called a conjugation pilus to transfer genes between cells
Exercise \(1\)
1. What are three ways sexual reproduction introduces genetic variation into offspring?
2. What is a benefit of asexual reproduction?
3. What are the three mechanisms of horizontal gene transfer in prokaryotes?
Transformation
Frederick Griffith was the first to demonstrate the process of transformation. In 1928, he showed that live, nonpathogenic Streptococcus pneumoniae bacteria could be transformed into pathogenic bacteria through exposure to a heat-killed pathogenic strain. He concluded that some sort of agent, which he called the “transforming principle,” had been passed from the dead pathogenic bacteria to the live, nonpathogenic bacteria. In 1944, Oswald Avery (1877–1955), Colin MacLeod (1909–1972), and Maclyn McCarty (1911–2005) demonstrated that the transforming principle was DNA (see Using Microbiology to Discover the Secrets of Life).
In transformation, the prokaryote takes up naked DNA found in its environment and that is derived from other cells that have lysed on death and released their contents, including their genome, into the environment. Many bacteria are naturally competent, meaning that they actively bind to environmental DNA, transport it across their cell envelopes into their cytoplasm, and make it single stranded. Typically, double-stranded foreign DNA within cells is destroyed by nucleases as a defense against viral infection. However, these nucleases are usually ineffective against single-stranded DNA, so this single-stranded DNA within the cell has the opportunity to recombine into the bacterial genome. A molecule of DNA that contains fragments of DNA from different organisms is called recombinant DNA. (Recombinant DNA will be discussed in more detail in Microbes and the Tools of Genetic Engineering.) If the bacterium incorporates the new DNA into its own genome through recombination, the bacterial cell may gain new phenotypic properties. For example, if a nonpathogenic bacterium takes up DNA for a toxin gene from a pathogen and then incorporates it into its chromosome, it, too, may become pathogenic. Plasmid DNA may also be taken up by competent bacteria and confer new properties to the cell. Overall, transformation in nature is a relatively inefficient process because environmental DNA levels are low because of the activity of nucleases that are also released during cellular lysis. Additionally, genetic recombination is inefficient at incorporating new DNA sequences into the genome.
In nature, bacterial transformation is an important mechanism for the acquisition of genetic elements encoding virulence factors and antibiotic resistance. Genes encoding resistance to antimicrobial compounds have been shown to be widespread in nature, even in environments not influenced by humans. These genes, which allow microbes living in mixed communities to compete for limited resources, can be transferred within a population by transformation, as well as by the other processes of HGT. In the laboratory, we can exploit the natural process of bacterial transformation for genetic engineering to make a wide variety of medicinal products, as discussed in Microbes and the Tools of Genetic Engineering.
Exercise \(2\)
Why does a bacterial cell make environmental DNA brought into the cell into a single-stranded form?
Transduction
Viruses that infect bacteria (bacteriophages) may also move short pieces of chromosomal DNA from one bacterium to another in a process called transduction (see Figure 6.2.3). Recall that in generalized transduction, any piece of chromosomal DNA may be transferred to a new host cell by accidental packaging of chromosomal DNA into a phage head during phage assembly. By contrast, specialized transduction results from the imprecise excision of a lysogenic prophage from the bacterial chromosome such that it carries with it a piece of the bacterial chromosome from either side of the phage’s integration site to a new host cell. As a result, the host may acquire new properties. This process is called lysogenic conversion. Of medical significance, a lysogenic phage may carry with it a virulence gene to its new host. Once inserted into the new host’s chromosome, the new host may gain pathogenicity. Several pathogenic bacteria, including Corynebacterium diphtheriae (the causative agent of diphtheria) and Clostridium botulinum (the causative agent of botulism), are virulent because of the introduction of toxin-encoding genes by lysogenic bacteriophages, affirming the clinical relevance of transduction in the exchange of genes involved in infectious disease. Archaea have their own viruses that translocate genetic material from one individual to another.
Exercise \(3\)
1. What is the agent of transduction of prokaryotic cells?
2. In specialized transduction, where does the transducing piece of DNA come from?
The Clinical Consequences of Transduction
Paul, a 23-year-old relief worker from Atlanta, traveled to Haiti in 2011 to provide aid following the 2010 earthquake. After working there for several weeks, he suddenly began experiencing abdominal distress, including severe cramping, nausea, vomiting, and watery diarrhea. He also began to experience intense muscle cramping. At a local clinic, the physician suspected that Paul’s symptoms were caused by cholera because there had been a cholera outbreak after the earthquake. Because cholera is transmitted by the fecal-oral route, breaches in sanitation infrastructure, such as often occur following natural disasters, may precipitate outbreaks. The physician confirmed the presumptive diagnosis using a cholera dipstick test. He then prescribed Paul a single dose of doxycycline, as well as oral rehydration salts, instructing him to drink significant amounts of clean water.
Cholera is caused by the gram-negative curved rod Vibrio cholerae (Figure \(2\)). Its symptoms largely result from the production of the cholera toxin (CT), which ultimately activates a chloride transporter to pump chloride ions out of the epithelial cells into the gut lumen. Water then follows the chloride ions, causing the prolific watery diarrhea characteristic of cholera. The gene encoding the cholera toxin is incorporated into the bacterial chromosome of V. cholerae through infection of the bacterium with the lysogenic filamentous CTX phage, which carries the CT gene and introduces it into the chromosome on integration of the prophage. Thus, pathogenic strains of V. cholerae result from horizontal gene transfer by specialized transduction.
Exercise \(4\)
1. Why are outbreaks of cholera more common as a result of a natural disaster?
2. Why is muscle cramping a common symptom of cholera? Why is treatment with oral rehydration salts so important for the treatment of cholera?
3. In areas stricken by cholera, what are some strategies that people could use to prevent disease transmission?
Conjugation
In conjugation, DNA is directly transferred from one prokaryote to another by means of a conjugation pilus, which brings the organisms into contact with one another. In E. coli, the genes encoding the ability to conjugate are located on a bacterial plasmid called the F plasmid, also known as the fertility factor, and the conjugation pilus is called the F pilus. The F-plasmid genes encode both the proteins composing the F pilus and those involved in rolling circle replication of the plasmid. Cells containing the F plasmid, capable of forming an F pilus, are called F+ cells or donor cells, and those lacking an F plasmid are called F cells or recipient cells.
Conjugation of the F Plasmid
During typical conjugation in E. coli, the F pilus of an F+ cell comes into contact with an F cell and retracts, bringing the two cell envelopes into contact (Figure \(3\)). Then a cytoplasmic bridge forms between the two cells at the site of the conjugation pilus. As rolling circle replication of the F plasmid occurs in the F+ cell, a single-stranded copy of the F plasmid is transferred through the cytoplasmic bridge to the F cell, which then synthesizes the complementary strand, making it double stranded. The F cell now becomes an F+ cell capable of making its own conjugation pilus. Eventually, in a mixed bacterial population containing both F+ and F cells, all cells will become F+ cells. Genes on the E. coli F plasmid also encode proteins preventing conjugation between F+ cells.
Conjugation of F’ and Hfr Cells
Although typical conjugation in E. coli results in the transfer of the F-plasmid DNA only, conjugation may also transfer chromosomal DNA. This is because the F plasmid occasionally integrates into the bacterial chromosome through recombination between the plasmid and the chromosome, forming an Hfr cell (Figure \(4\)). “Hfr” refers to the high frequency of recombination seen when recipient F cells receive genetic information from Hfr cells through conjugation. Similar to the imprecise excision of a prophage during specialized transduction, the integrated F plasmid may also be imprecisely excised from the chromosome, producing an F’ plasmid that carries with it some chromosomal DNA adjacent to the integration site. On conjugation, this DNA is introduced to the recipient cell and may be either maintained as part of the F’ plasmid or be recombined into the recipient cell’s bacterial chromosome.
Hfr cells may also treat the bacterial chromosome like an enormous F plasmid and attempt to transfer a copy of it to a recipient F cell. Because the bacterial chromosome is so large, transfer of the entire chromosome takes a long time (Figure \(5\)). However, contact between bacterial cells during conjugation is transient, so it is unusual for the entire chromosome to be transferred. Host chromosomal DNA near the integration site of the F plasmid, displaced by the unidirectional process of rolling circle replication, is more likely to be transferred and recombined into a recipient cell’s chromosome than host genes farther away. Thus, the relative location of bacterial genes on the Hfr cell’s genome can be mapped based on when they are transferred through conjugation. As a result, prior to the age of widespread bacterial genome sequencing, distances on prokaryotic genome maps were often measured in minutes.
Consequences and Applications of Conjugation
Plasmids are an important type of extrachromosomal DNA element in bacteria and, in those cells that harbor them, are considered to be part of the bacterial genome. From a clinical perspective, plasmids often code for genes involved in virulence. For example, genes encoding proteins that make a bacterial cell resistant to a particular antibiotic are encoded on R plasmids. R plasmids, in addition to their genes for antimicrobial resistance, contain genes that control conjugation and transfer of the plasmid. R plasmids are able to transfer between cells of the same species and between cells of different species. Single R plasmids commonly contain multiple genes conferring resistance to multiple antibiotics.
Genes required for the production of various toxins and molecules important for colonization during infection may also be found encoded on plasmids. For example, verotoxin-producing strains of E. coli (VTEC) appear to have acquired the genes encoding the Shiga toxin from its gram-negative relative Shigella dysenteriae through the acquisition of a large plasmid encoding this toxin. VTEC causes severe diarrheal disease that may result in hemolytic uremic syndrome(HUS), which may be lead to kidney failure and death.
In nonclinical settings, bacterial genes that encode metabolic enzymes needed to degrade specialized atypical compounds like polycyclic aromatic hydrocarbons (PAHs) are also frequently encoded on plasmids. Additionally, certain plasmids have the ability to move from bacterial cells to other cell types, like those of plants and animals, through mechanisms distinct from conjugation. Such mechanisms and their use in genetic engineering are covered in Modern Applications of Microbial Genetics.
Exercise \(5\)
1. What type of replication occurs during conjugation?
2. What occurs to produce an Hfr E. coli cell?
3. What types of traits are encoded on plasmids?
Transposition
Genetic elements called transposons (transposable elements), or “jumping genes,” are molecules of DNA that include special inverted repeat sequences at their ends and a gene encoding the enzyme transposase (Figure \(6\)). Transposons allow the entire sequence to independently excise from one location in a DNA molecule and integrate into the DNA elsewhere through a process called transposition. Transposons were originally discovered in maize (corn) by American geneticist Barbara McClintock (1902–1992) in the 1940s. Transposons have since been found in all types of organisms, both prokaryotes and eukaryotes. Thus, unlike the three previous mechanisms discussed, transposition is not prokaryote-specific. Most transposons are nonreplicative, meaning they move in a “cut-and-paste” fashion. Some may be replicative, however, retaining their location in the DNA while making a copy to be inserted elsewhere (“copy and paste”). Because transposons can move within a DNA molecule, from one DNA molecule to another, or even from one cell to another, they have the ability to introduce genetic diversity. Movement within the same DNA molecule can alter phenotype by inactivating or activating a gene.
Transposons may carry with them additional genes, moving these genes from one location to another with them. For example, bacterial transposons can relocate antibiotic resistance genes, moving them from chromosomes to plasmids. This mechanism has been shown to be responsible for the colocalization of multiple antibiotic resistance genes on a single R plasmid in Shigella strains causing bacterial dysentery. Such an R plasmid can then be easily transferred among a bacterial population through the process of conjugation.
Exercise \(6\)
What are two ways a transposon can affect the phenotype of a cell it moves to?
Table \(1\) summarizes the processes discussed in this section. Table \(1\): Summary of Mechanisms of Genetic Diversity in Prokaryotes
Term Definition
Conjugation Transfer of DNA through direct contact using a conjugation pilus
Transduction Mechanism of horizontal gene transfer in bacteria in which genes are transferred through viral infection
Transformation Mechanism of horizontal gene transfer in which naked environmental DNA is taken up by a bacterial cell
Transposition Process whereby DNA independently excises from one location in a DNA molecule and integrates elsewhere
Clinical Focus: Part 3
Despite continued antibiotic treatment, Mark’s infection continued to progress rapidly. The infected region continued to expand, and he had to be put on a ventilator to help him breathe. Mark’s physician ordered surgical removal of the infected tissue. Following an initial surgery, Mark’s wound was monitored daily to ensure that the infection did not return, but it continued to spread.
After two additional rounds of surgery, the infection finally seemed to be contained. A few days later, Mark was removed from the ventilator and was able to breathe on his own. However, he had lost a great deal of skin and soft tissue on his lower leg.
Exercise \(7\)
1. Why does the removal of infected tissue stem the infection?
2. What are some likely complications of this method of treatment?
Key Concepts and Summary
• Horizontal gene transfer is an important way for asexually reproducing organisms like prokaryotes to acquire new traits.
• There are three mechanisms of horizontal gene transfer typically used by bacteria: transformation, transduction, and conjugation.
• Transformation allows for competent cells to take up naked DNA, released from other cells on their death, into their cytoplasm, where it may recombine with the host genome.
• In generalized transduction, any piece of chromosomal DNA may be transferred by accidental packaging of the degraded host chromosome into a phage head. In specialized transduction, only chromosomal DNA adjacent to the integration site of a lysogenic phage may be transferred as a result of imprecise excision of the prophage.
• Conjugation is mediated by the F plasmid, which encodes a conjugation pilus that brings an F plasmid-containing F+ cell into contact with an F- cell.
• The rare integration of the F plasmid into the bacterial chromosome, generating an Hfr cell, allows for transfer of chromosomal DNA from the donor to the recipient. Additionally, imprecise excision of the F plasmid from the chromosome may generate an F’ plasmid that may be transferred to a recipient by conjugation.
• Conjugation transfer of R plasmids is an important mechanism for the spread of antibiotic resistance in bacterial communities.
• Transposons are molecules of DNA with inverted repeats at their ends that also encode the enzyme transposase, allowing for their movement from one location in DNA to another. Although found in both prokaryotes and eukaryotes, transposons are clinically relevant in bacterial pathogens for the movement of virulence factors, including antibiotic resistance genes. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/11%3A_Mechanisms_of_Microbial_Genetics/11.06%3A_How_Asexual_Prokaryotes_Achieve_Genetic_Diversity.txt |
Learning Objectives
• Compare inducible operons and repressible operons
• Describe why regulation of operons is important
Each nucleated cell in a multicellular organism contains copies of the same DNA. Similarly, all cells in two pure bacterial cultures inoculated from the same starting colony contain the same DNA, with the exception of changes that arise from spontaneous mutations. If each cell in a multicellular organism has the same DNA, then how is it that cells in different parts of the organism’s body exhibit different characteristics? Similarly, how is it that the same bacterial cells within two pure cultures exposed to different environmental conditions can exhibit different phenotypes? In both cases, each genetically identical cell does not turn on, or express, the same set of genes. Only a subset of proteins in a cell at a given time is expressed.
Genomic DNA contains both structural genes, which encode products that serve as cellular structures or enzymes, and regulatory genes, which encode products that regulate gene expression. The expression of a gene is a highly regulated process. Whereas regulating gene expression in multicellular organisms allows for cellular differentiation, in single-celled organisms like prokaryotes, it primarily ensures that a cell’s resources are not wasted making proteins that the cell does not need at that time.
Elucidating the mechanisms controlling gene expression is important to the understanding of human health. Malfunctions in this process in humans lead to the development of cancer and other diseases. Understanding the interaction between the gene expression of a pathogen and that of its human host is important for the understanding of a particular infectious disease. Gene regulation involves a complex web of interactions within a given cell among signals from the cell’s environment, signaling molecules within the cell, and the cell’s DNA. These interactions lead to the expression of some genes and the suppression of others, depending on circumstances.
Prokaryotes and eukaryotes share some similarities in their mechanisms to regulate gene expression; however, gene expression in eukaryotes is more complicated because of the temporal and spatial separation between the processes of transcription and translation. Thus, although most regulation of gene expression occurs through transcriptional control in prokaryotes, regulation of gene expression in eukaryotes occurs at the transcriptional level and post-transcriptionally (after the primary transcript has been made).
Prokaryotic Gene Regulation
In bacteria and archaea, structural proteins with related functions are usually encoded together within the genome in a block called an operon and are transcribed together under the control of a single promoter, resulting in the formation of a polycistronic transcript (Figure \(1\)). In this way, regulation of the transcription of all of the structural genes encoding the enzymes that catalyze the many steps in a single biochemical pathway can be controlled simultaneously, because they will either all be needed at the same time, or none will be needed. For example, in E. coli, all of the structural genes that encode enzymes needed to use lactose as an energy source lie next to each other in the lactose (or lac) operon under the control of a single promoter, the lac promoter. French scientists François Jacob (1920–2013) and Jacques Monod at the Pasteur Institute were the first to show the organization of bacterial genes into operons, through their studies on the lac operon of E. coli. For this work, they won the Nobel Prize in Physiology or Medicine in 1965. Although eukaryotic genes are not organized into operons, prokaryotic operons are excellent models for learning about gene regulation generally. There are some gene clusters in eukaryotes that function similar to operons. Many of the principles can be applied to eukaryotic systems and contribute to our understanding of changes in gene expression in eukaryotes that can result pathological changes such as cancer.
Each operon includes DNA sequences that influence its own transcription; these are located in a region called the regulatory region. The regulatory region includes the promoter and the region surrounding the promoter, to which transcription factors, proteins encoded by regulatory genes, can bind. Transcription factors influence the binding of RNA polymerase to the promoter and allow its progression to transcribe structural genes. A repressor is a transcription factor that suppresses transcription of a gene in response to an external stimulus by binding to a DNA sequence within the regulatory region called the operator, which is located between the RNA polymerase binding site of the promoter and the transcriptional start site of the first structural gene. Repressor binding physically blocks RNA polymerase from transcribing structural genes. Conversely, an activator is a transcription factor that increases the transcription of a gene in response to an external stimulus by facilitating RNA polymerase binding to the promoter. An inducer, a third type of regulatory molecule, is a small molecule that either activates or represses transcription by interacting with a repressor or an activator.
In prokaryotes, there are examples of operons whose gene products are required rather consistently and whose expression, therefore, is unregulated. Such operons are constitutively expressed, meaning they are transcribed and translated continuously to provide the cell with constant intermediate levels of the protein products. Such genes encode enzymes involved in housekeeping functions required for cellular maintenance, including DNA replication, repair, and expression, as well as enzymes involved in core metabolism. In contrast, there are other prokaryotic operons that are expressed only when needed and are regulated by repressors, activators, and inducers.
Exercise \(1\)
1. What are the parts in the DNA sequence of an operon?
2. What types of regulatory molecules are there?
Regulation by Repression
Prokaryotic operons are commonly controlled by the binding of repressors to operator regions, thereby preventing the transcription of the structural genes. Such operons are classified as either repressible operons or inducible operons. Repressible operons, like the tryptophan (trp) operon, typically contain genes encoding enzymes required for a biosynthetic pathway. As long as the product of the pathway, like tryptophan, continues to be required by the cell, a repressible operon will continue to be expressed. However, when the product of the biosynthetic pathway begins to accumulate in the cell, removing the need for the cell to continue to make more, the expression of the operon is repressed. Conversely, inducible operons, like the lac operon of E. coli, often contain genes encoding enzymes in a pathway involved in the metabolism of a specific substrate like lactose. These enzymes are only required when that substrate is available, thus expression of the operons is typically induced only in the presence of the substrate.
The trp Operon: A Repressible Operon
E. coli can synthesize tryptophan using enzymes that are encoded by five structural genes located next to each other in the trp operon (Figure \(2\)). When environmental tryptophan is low, the operon is turned on. This means that transcription is initiated, the genes are expressed, and tryptophan is synthesized. However, if tryptophan is present in the environment, the trp operon is turned off. Transcription does not occur and tryptophan is not synthesized.
When tryptophan is not present in the cell, the repressor by itself does not bind to the operator; therefore, the operon is active and tryptophan is synthesized. However, when tryptophan accumulates in the cell, two tryptophan molecules bind to the trp repressor molecule, which changes its shape, allowing it to bind to the trp operator. This binding of the active form of the trp repressor to the operator blocks RNA polymerase from transcribing the structural genes, stopping expression of the operon. Thus, the actual product of the biosynthetic pathway controlled by the operon regulates the expression of the operon.
Link to Learning
Watch this video to learn more about the trp operon.
The lac Operon: An Inducible Operon
The lac operon is an example of an inducible operon that is also subject to activation in the absence of glucose (Figure \(3\)). The lac operon encodes three structural genes necessary to acquire and process the disaccharide lactose from the environment, breaking it down into the simple sugars glucose and galactose. For the lac operon to be expressed, lactose must be present. This makes sense for the cell because it would be energetically wasteful to create the enzymes to process lactose if lactose was not available.
In the absence of lactose, the lac repressor is bound to the operator region of the lac operon, physically preventing RNA polymerase from transcribing the structural genes. However, when lactose is present, the lactose inside the cell is converted to allolactose. Allolactose serves as an inducer molecule, binding to the repressor and changing its shape so that it is no longer able to bind to the operator DNA. Removal of the repressor in the presence of lactose allows RNA polymerase to move through the operator region and begin transcription of the lac structural genes.
The lac Operon: Activation by Catabolite Activator Protein
Bacteria typically have the ability to use a variety of substrates as carbon sources. However, because glucose is usually preferable to other substrates, bacteria have mechanisms to ensure that alternative substrates are only used when glucose has been depleted. Additionally, bacteria have mechanisms to ensure that the genes encoding enzymes for using alternative substrates are expressed only when the alternative substrate is available. In the 1940s, Jacques Monod was the first to demonstrate the preference for certain substrates over others through his studies of E. coli’s growth when cultured in the presence of two different substrates simultaneously. Such studies generated diauxic growth curves, like the one shown in Figure \(4\). Although the preferred substrate glucose is used first, E. coli grows quickly and the enzymes for lactose metabolism are absent. However, once glucose levels are depleted, growth rates slow, inducing the expression of the enzymes needed for the metabolism of the second substrate, lactose. Notice how the growth rate in lactose is slower, as indicated by the lower steepness of the growth curve.
The ability to switch from glucose use to another substrate like lactose is a consequence of the activity of an enzyme called Enzyme IIA (EIIA). When glucose levels drop, cells produce less ATP from catabolism (see Catabolism of Carbohydrates), and EIIA becomes phosphorylated. Phosphorylated EIIA activates adenylyl cyclase, an enzyme that converts some of the remaining ATP to cyclic AMP (cAMP), a cyclic derivative of AMP and important signaling molecule involved in glucose and energy metabolism in E. coli. As a result, cAMP levels begin to rise in the cell (Figure \(5\)).
The lac operon also plays a role in this switch from using glucose to using lactose. When glucose is scarce, the accumulating cAMP caused by increased adenylyl cyclase activity binds to catabolite activator protein (CAP), also known as cAMP receptor protein (CRP). The complex binds to the promoter region of the lac operon (Figure \(6\)). In the regulatory regions of these operons, a CAP binding site is located upstream of the RNA polymerase binding site in the promoter. Binding of the CAP-cAMP complex to this site increases the binding ability of RNA polymerase to the promoter region to initiate the transcription of the structural genes. Thus, in the case of the lac operon, for transcription to occur, lactose must be present (removing the lac repressor protein) and glucose levels must be depleted (allowing binding of an activating protein). When glucose levels are high, there is catabolite repression of operons encoding enzymes for the metabolism of alternative substrates. Because of low cAMP levels under these conditions, there is an insufficient amount of the CAP-cAMP complex to activate transcription of these operons. See Table \(1\) for a summary of the regulation of the lac operon.
Table \(1\): Conditions Affecting Transcription of the lac Operon
Glucose CAP binds Lactose Repressor binds Transcription
+ + No
+ + Some
+ + No
+ + Yes
Link to Learning
Watch an animated tutorial about the workings of lac operon here.
Exercise \(2\)
1. What affects the binding of the trp operon repressor to the operator?
2. How and when is the behavior of the lac repressor protein altered?
3. In addition to being repressible, how else is the lac operon regulated?
Global Responses of Prokaryotes
In prokaryotes, there are also several higher levels of gene regulation that have the ability to control the transcription of many related operons simultaneously in response to an environmental signal. A group of operons all controlled simultaneously is called a regulon.
Alarmones
When sensing impending stress, prokaryotes alter the expression of a wide variety of operons to respond in coordination. They do this through the production of alarmones, which are small intracellular nucleotide derivatives. Alarmones change which genes are expressed and stimulate the expression of specific stress-response genes. The use of alarmones to alter gene expression in response to stress appears to be important in pathogenic bacteria. On encountering host defense mechanisms and other harsh conditions during infection, many operons encoding virulence genes are upregulated in response to alarmone signaling. Knowledge of these responses is key to being able to fully understand the infection process of many pathogens and to the development of therapies to counter this process.
Alternate σ Factors
Since the σ subunit of bacterial RNA polymerase confers specificity as to which promoters should be transcribed, altering the σ factor used is another way for bacteria to quickly and globally change what regulons are transcribed at a given time. The σ factor recognizes sequences within a bacterial promoter, so different σ factors will each recognize slightly different promoter sequences. In this way, when the cell senses specific environmental conditions, it may respond by changing which σ factor it expresses, degrading the old one and producing a new one to transcribe the operons encoding genes whose products will be useful under the new environmental condition. For example, in sporulating bacteria of the genera Bacillus and Clostridium (which include many pathogens), a group of σ factors controls the expression of the many genes needed for sporulation in response to sporulation-stimulating signals.
Exercise \(3\)
1. What is the name given to a collection of operons that can be regulated as a group?
2. What type of stimulus would trigger the transcription of a different σ factor?
Additional Methods of Regulation in Bacteria: Attenuation and Riboswitches
Although most gene expression is regulated at the level of transcription initiation in prokaryotes, there are also mechanisms to control both the completion of transcription as well as translation concurrently. Since their discovery, these mechanisms have been shown to control the completion of transcription and translation of many prokaryotic operons. Because these mechanisms link the regulation of transcription and translation directly, they are specific to prokaryotes, because these processes are physically separated in eukaryotes.
One such regulatory system is attenuation, whereby secondary stem-loop structures formed within the 5’ end of an mRNA being transcribed determine if transcription to complete the synthesis of this mRNA will occur and if this mRNA will be used for translation. Beyond the transcriptional repression mechanism already discussed, attenuation also controls expression of the trp operon in E. coli (Figure \(7\)). The trp operon regulatory region contains a leader sequence called trpL between the operator and the first structural gene, which has four stretches of RNA that can base pair with each other in different combinations. When a terminator stem-loop forms, transcription terminates, releasing RNA polymerase from the mRNA. However, when an antiterminator stem-loop forms, this prevents the formation of the terminator stem-loop, so RNA polymerase can transcribe the structural genes.
A related mechanism of concurrent regulation of transcription and translation in prokaryotes is the use of a riboswitch, a small region of noncoding RNA found within the 5’ end of some prokaryotic mRNA molecules (Figure \(8\)). A riboswitch may bind to a small intracellular molecule to stabilize certain secondary structures of the mRNA molecule. The binding of the small molecule determines which stem-loop structure forms, thus influencing the completion of mRNA synthesis and protein synthesis.
Other Factors Affecting Gene Expression in Prokaryotes and Eukaryotes
Although the focus on our discussion of transcriptional control used prokaryotic operons as examples, eukaryotic transcriptional control is similar in many ways. As in prokaryotes, eukaryotic transcription can be controlled through the binding of transcription factors including repressors and activators. Interestingly, eukaryotic transcription can be influenced by the binding of proteins to regions of DNA, called enhancers, rather far away from the gene, through DNA looping facilitated between the enhancer and the promoter (Figure \(9\)). Overall, regulating transcription is a highly effective way to control gene expression in both prokaryotes and eukaryotes. However, the control of gene expression in eukaryotes in response to environmental and cellular stresses can be accomplished in additional ways without the binding of transcription factors to regulatory regions.
DNA-Level Control
In eukaryotes, the DNA molecules or associated histones can be chemically modified in such a way as to influence transcription; this is called epigenetic regulation. Methylation of certain cytosine nucleotides in DNA in response to environmental factors has been shown to influence use of such DNA for transcription, with DNA methylation commonly correlating to lowered levels of gene expression. Additionally, in response to environmental factors, histone proteins for packaging DNA can also be chemically modified in multiple ways, including acetylation and deacetylation, influencing the packaging state of DNA and thus affecting the availability of loosely wound DNA for transcription. These chemical modifications can sometimes be maintained through multiple rounds of cell division, making at least some of these epigenetic changes heritable.
Link to Learning
This video describes how epigenetic regulation controls gene expression.
Exercise \(\PageIndex{}\)
1. What stops or allows transcription to proceed when attenuation is operating?
2. What determines the state of a riboswitch?
3. Describe the function of an enhancer.
4. Describe two mechanisms of epigenetic regulation in eukaryotes.
Clinical Focus: Resolution
Although Mark survived his bout with necrotizing fasciitis, he would now have to undergo a skin-grafting surgery, followed by long-term physical therapy. Based on the amount of muscle mass he lost, it is unlikely that his leg will return to full strength, but his physical therapist is optimistic that he will regain some use of his leg.
Laboratory testing revealed the causative agent of Mark’s infection was a strain of group A streptococcus (Group A strep). As required by law, Mark’s case was reported to the state health department and ultimately to the Centers for Disease Control and Prevention (CDC). At the CDC, the strain of group A strep isolated from Mark was analyzed more thoroughly for methicillin resistance.
Methicillin resistance is genetically encoded and is becoming more common in group A strep through horizontal gene transfer. In necrotizing fasciitis, blood flow to the infected area is typically limited because of the action of various genetically encoded bacterial toxins. This is why there is typically little to no bleeding as a result of the incision test. Unfortunately, these bacterial toxins limit the effectiveness of intravenous antibiotics in clearing infection from the skin and underlying tissue, meaning that antibiotic resistance alone does not explain the ineffectiveness of Mark’s treatment. Nevertheless, intravenous antibiotic therapy was warranted to help minimize the possible outcome of sepsis, which is a common outcome of necrotizing fasciitis. Through genomic analysis by the CDC of the strain isolated from Mark, several of the important virulence genes were shown to be encoded on prophages, indicating that transduction is important in the horizontal gene transfer of these genes from one bacterial cell to another.
Key Concepts and Summary
• Gene expression is a tightly regulated process.
• Gene expression in prokaryotes is largely regulated at the point of transcription. Gene expression in eukaryotes is additionally regulated post-transcriptionally.
• Prokaryotic structural genes of related function are often organized into operons, all controlled by transcription from a single promoter. The regulatory region of an operon includes the promoter itself and the region surrounding the promoter to which transcription factors can bind to influence transcription.
• Although some operons are constitutively expressed, most are subject to regulation through the use of transcription factors (repressors and activators). A repressor binds to an operator, a DNA sequence within the regulatory region between the RNA polymerase binding site in the promoter and first structural gene, thereby physically blocking transcription of these operons. An activator binds within the regulatory region of an operon, helping RNA polymerase bind to the promoter, thereby enhancing the transcription of this operon. An inducerinfluences transcription through interacting with a repressor or activator.
• The trp operon is a classic example of a repressible operon. When tryptophan accumulates, tryptophan binds to a repressor, which then binds to the operator, preventing further transcription.
• The lac operon is a classic example an inducible operon. When lactose is present in the cell, it is converted to allolactose. Allolactose acts as an inducer, binding to the repressor and preventing the repressor from binding to the operator. This allows transcription of the structural genes.
• The lac operon is also subject to activation. When glucose levels are depleted, some cellular ATP is converted into cAMP, which binds to the catabolite activator protein (CAP). The cAMP-CAP complex activates transcription of the lac operon. When glucose levels are high, its presence prevents transcription of the lac operon and other operons by catabolite repression.
• Small intracellular molecules called alarmones are made in response to various environmental stresses, allowing bacteria to control the transcription of a group of operons, called a regulon.
• Bacteria have the ability to change which σ factor of RNA polymerase they use in response to environmental conditions to quickly and globally change which regulons are transcribed.
• Prokaryotes have regulatory mechanisms, including attenuation and the use of riboswitches, to simultaneously control the completion of transcription and translation from that transcript. These mechanisms work through the formation of stem loops in the 5’ end of an mRNA molecule currently being synthesized.
• There are additional points of regulation of gene expression in prokaryotes and eukaryotes. In eukaryotes, epigenetic regulation by chemical modification of DNA or histones, and regulation of RNA processing are two methods. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/11%3A_Mechanisms_of_Microbial_Genetics/11.07%3A_Gene_Regulation_-_Operon_Theory.txt |
11.1: What Are Genes?
A gene is composed of DNA that is “read” or transcribed to produce an RNA molecule during the process of transcription. One major type of RNA molecule, called messenger RNA (mRNA), provides the information for the ribosome to catalyze protein synthesis in a process called translation. The processes of transcription and translation are collectively referred to as gene expression.
Multiple Choice
DNA does all but which of the following?
1. serves as the genetic material passed from parent to offspring
2. remains constant despite changes in environmental conditions
3. provides the instructions for the synthesis of messenger RNA
4. is read by ribosomes during the process of translation
Answer
D
According to the central dogma, which of the following represents the flow of genetic information in cells?
1. protein to DNA to RNA
2. DNA to RNA to protein
3. RNA to DNA to protein
4. DNA to protein to RNA
Answer
B
True/False
Cells are always producing proteins from every gene they possess.
Answer
False
Fill in the Blank
The process of making an RNA copy of a gene is called ________.
Answer
transcription
A cell’s ________ remains constant whereas its phenotype changes in response to environmental influences.
Answer
genotype or genome
Short Answer
Can two observably different cells have the same genotype? Explain.
Critical Thinking
A pure culture of an unknown bacterium was streaked onto plates of a variety of media. You notice that the colony morphology is strikingly different on plates of minimal media with glucose compared to that seen on trypticase soy agar plates. How can you explain these differences in colony morphology?
11.2: DNA Replication
The DNA replication process is semiconservative, which results in two DNA molecules, each having one parental strand of DNA and one newly synthesized strand. In bacteria, the initiation of replication occurs at the origin of replication, where supercoiled DNA is unwound by DNA gyrase, made single-stranded by helicase, and bound by single-stranded binding protein to maintain its single-stranded state.
Multiple Choice
Which of the following is the enzyme that replaces the RNA nucleotides in a primer with DNA nucleotides?
1. DNA polymerase III
2. DNA polymerase I
3. primase
4. helicase
Answer
B
Which of the following is not involved in the initiation of replication?
1. ligase
2. DNA gyrase
3. single-stranded binding protein
4. primase
Answer
A
Which of the following enzymes involved in DNA replication is unique to eukaryotes?
1. helicase
2. DNA polymerase
3. ligase
4. telomerase
Answer
D
Which of the following would be synthesized using 5′-CAGTTCGGA-3′ as a template?
1. 3′-AGGCTTGAC-4′
2. 3′-TCCGAACTG-5′
3. 3′-GTCAAGCCT-5′
4. 3′-CAGTTCGGA-5′
Answer
C
Fill in the Blank
The enzyme responsible for relaxing supercoiled DNA to allow for the initiation of replication is called ________.
Answer
DNA gyrase or topoisomerase II
Unidirectional replication of a circular DNA molecule like a plasmid that involves nicking one DNA strand and displacing it while synthesizing a new strand is called ________.
Answer
rolling circle replication
True/False
More primers are used in lagging strand synthesis than in leading strand synthesis.
Answer
True
Short Answer
Why is primase required for DNA replication?
What is the role of single-stranded binding protein in DNA replication?
Below is a DNA sequence. Envision that this is a section of a DNA molecule that has separated in preparation for replication, so you are only seeing one DNA strand. Construct the complementary DNA sequence (indicating 5’ and 3’ ends).
DNA sequence: 3’-T A C T G A C T G A C G A T C-5’
Critical Thinking
Review Figure 11.2.1 and Figure 11.2.2. Why was it important that Meselson and Stahl continue their experiment to at least two rounds of replication after isotopic labeling of the starting DNA with 15N, instead of stopping the experiment after only one round of replication?
If deoxyribonucleotides that lack the 3’-OH groups are added during the replication process, what do you expect will occur?
11.3: RNA Transcription
During the process of transcription, the information encoded within the DNA sequence of one or more genes is transcribed into a strand of RNA, also called an RNA transcript. The resulting single-stranded RNA molecule, composed of ribonucleotides containing the bases adenine, cytosine, guanine, and uracil, acts as a mobile molecular copy of the original DNA sequence. Transcription in prokaryotes and in eukaryotes requires the DNA double helix to partially unwind in the region of RNA synthesis.
Multiple Choice
During which stage of bacterial transcription is the σ subunit of the RNA polymerase involved?
1. initiation
2. elongation
3. termination
4. splicing
Answer
A
Which of the following components is involved in the initiation of transcription?
1. primer
2. origin
3. promoter
4. start codon
Answer
C
Which of the following is not a function of the 5’ cap and 3’ poly-A tail of a mature eukaryotic mRNA molecule?
1. to facilitate splicing
2. to prevent mRNA degradation
3. to aid export of the mature transcript to the cytoplasm
4. to aid ribosome binding to the transcript
Answer
A
Mature mRNA from a eukaryote would contain each of these features except which of the following?
1. exon-encoded RNA
2. intron-encoded RNA
3. 5’ cap
4. 3’ poly-A tail
Answer
B
Fill in the Blank
A ________ mRNA is one that codes for multiple polypeptides.
Answer
polycistronic
The protein complex responsible for removing intron-encoded RNA sequences from primary transcripts in eukaryotes is called the ________.
Answer
Spliceosome
Short Answer
What is the purpose of RNA processing in eukaryotes? Why don’t prokaryotes require similar processing?
Below is a DNA sequence. Envision that this is a section of a DNA molecule that has separated in preparation for transcription, so you are only seeing the antisense strand. Construct the mRNA sequence transcribed from this template.
Antisense DNA strand: 3’-T A C T G A C T G A C G A T C-5’
Critical Thinking
Predict the effect of an alteration in the sequence of nucleotides in the –35 region of a bacterial promoter.
11.4: Protein Synthesis (Translation)
The synthesis of proteins consumes more of a cell’s energy than any other metabolic process. In turn, proteins account for more mass than any other macromolecule of living organisms. They perform virtually every function of a cell, serving as both functional (e.g., enzymes) and structural elements. The process of translation, or protein synthesis, the second part of gene expression, involves the decoding by a ribosome of an mRNA message into a polypeptide product.
Multiple Choice
Which of the following is the name of the three-base sequence in the mRNA that binds to a tRNA molecule?
1. P site
2. codon
3. anticodon
4. CCA binding site
Answer
B
Which component is the last to join the initiation complex during the initiation of translation?
1. the mRNA molecule
2. the small ribosomal subunit
3. the large ribosomal subunit
4. the initiator tRNA
Answer
C
During elongation in translation, to which ribosomal site does an incoming charged tRNA molecule bind?
1. A site
2. P site
3. E site
4. B site
Answer
A
Which of the following is the amino acid that appears at the N-terminus of all newly translated prokaryotic and eukaryotic polypeptides?
1. tryptophan
2. methionine
3. selenocysteine
4. glycine
Answer
B
When the ribosome reaches a nonsense codon, which of the following occurs?
1. a methionine is incorporated
2. the polypeptide is released
3. a peptide bond forms
4. the A site binds to a charged tRNA
Answer
B
Fill in the Blank
The third position within a codon, in which changes often result in the incorporation of the same amino acid into the growing polypeptide, is called the ________.
Answer
wobble position
The enzyme that adds an amino acid to a tRNA molecule is called ________.
Answer
aminoacyl-tRNA synthetase
True/False
Each codon within the genetic code encodes a different amino acid.
Answer
False
Short Answer
Why does translation terminate when the ribosome reaches a stop codon? What happens?
How does the process of translation differ between prokaryotes and eukaryotes?
What is meant by the genetic code being nearly universal?
Below is an antisense DNA sequence. Translate the mRNA molecule synthesized using the genetic code, recording the resulting amino acid sequence, indicating the N and C termini.
Antisense DNA strand: 3’-T A C T G A C T G A C G A T C-5’
Critical Thinking
Label the following in the figure: ribosomal E, P, and A sites; mRNA; codons; anticodons; growing polypeptide; incoming amino acid; direction of translocation; small ribosomal unit; large ribosomal unit.
Prior to the elucidation of the genetic code, prominent scientists, including Francis Crick, had predicted that each mRNA codon, coding for one of the 20 amino acids, needed to be at least three nucleotides long. Why is it not possible for codons to be any shorter?
11.5: Mutations
A mutation is a heritable change in the DNA sequence of an organism. The resulting organism, called a mutant, may have a recognizable change in phenotype compared to the wild type, which is the phenotype most commonly observed in nature. A change in the DNA sequence is conferred to mRNA through transcription, and may lead to an altered amino acid sequence in a protein on translation.
Multiple Choice
Which of the following is a change in the sequence that leads to formation of a stop codon?
1. missense mutation
2. nonsense mutation
3. silent mutation
4. deletion mutation
Answer
B
The formation of pyrimidine dimers results from which of the following?
1. spontaneous errors by DNA polymerase
2. exposure to gamma radiation
3. exposure to ultraviolet radiation
4. exposure to intercalating agents
Answer
C
Which of the following is an example of a frameshift mutation?
1. a deletion of a codon
2. missense mutation
3. silent mutation
4. deletion of one nucleotide
Answer
D
Which of the following is the type of DNA repair in which thymine dimers are directly broken down by the enzyme photolyase?
1. direct repair
2. nucleotide excision repair
3. mismatch repair
4. proofreading
Answer
A
Which of the following regarding the Ames test is true?
1. It is used to identify newly formed auxotrophic mutants.
2. It is used to identify mutants with restored biosynthetic activity.
3. It is used to identify spontaneous mutants.
4. It is used to identify mutants lacking photoreactivation activity.
Answer
B
Fill in the Blank
A chemical mutagen that is structurally similar to a nucleotide but has different base-pairing rules is called a ________.
Answer
nucleoside analog
The enzyme used in light repair to split thymine dimers is called ________.
Answer
photolyase
The phenotype of an organism that is most commonly observed in nature is called the ________.
Answer
wild type
True/False
Carcinogens are typically mutagenic.
Answer
True
Short Answer
Why is it more likely that insertions or deletions will be more detrimental to a cell than point mutations?
Critical Thinking
Below are several DNA sequences that are mutated compared with the wild-type sequence: 3’-T A C T G A C T G A C G A T C-5’. Envision that each is a section of a DNA molecule that has separated in preparation for transcription, so you are only seeing the template strand. Construct the complementary DNA sequences (indicating 5’ and 3’ ends) for each mutated DNA sequence, then transcribe (indicating 5’ and 3’ ends) the template strands, and translate the mRNA molecules using the genetic code, recording the resulting amino acid sequence (indicating the N and C termini). What type of mutation is each?
Mutated DNA Template Strand #1: 3’-T A C T G T C T G A C G A T C-5’
Complementary DNA sequence:
mRNA sequence transcribed from template:
Amino acid sequence of peptide:
Type of mutation:
Mutated DNA Template Strand #2: 3’-T A C G G A C T G A C G A T C-5’
Complementary DNA sequence:
mRNA sequence transcribed from template:
Amino acid sequence of peptide:
Type of mutation:
Mutated DNA Template Strand #3: 3’-T A C T G A C T G A C T A T C-5
Complementary DNA sequence:
mRNA sequence transcribed from template:
Amino acid sequence of peptide:
Type of mutation:
Mutated DNA Template Strand #4: 3’-T A C G A C T G A C T A T C-5’
Complementary DNA sequence:
mRNA sequence transcribed from template:
Amino acid sequence of peptide:
Type of mutation:
Why do you think the Ames test is preferable to the use of animal models to screen chemical compounds for mutagenicity?
11.6: How Asexual Prokaryotes Achieve Genetic Diversity
How do organisms whose dominant reproductive mode is asexual create genetic diversity? In prokaryotes, horizontal gene transfer (HGT), the introduction of genetic material from one organism to another organism within the same generation, is an important way to introduce genetic diversity. HGT allows even distantly related species to share genes, influencing their phenotypes.
Multiple Choice
Which is the mechanism by which improper excision of a prophage from a bacterial chromosome results in packaging of bacterial genes near the integration site into a phage head?
1. conjugation
2. generalized transduction
3. specialized transduction
4. transformation
Answer
C
Which of the following refers to the uptake of naked DNA from the surrounding environment?
1. conjugation
2. generalized transduction
3. specialized transduction
4. transformation
Answer
D
The F plasmid is involved in which of the following processes?
1. conjugation
2. transduction
3. transposition
4. transformation
Answer
A
Which of the following refers to the mechanism of horizontal gene transfer naturally responsible for the spread of antibiotic resistance genes within a bacterial population?
1. conjugation
2. generalized transduction
3. specialized transduction
4. transformation
Answer
A
Fill in the Blank
A small DNA molecule that has the ability to independently excise from one location in a larger DNA molecule and integrate into the DNA elsewhere is called a ________.
Answer
transposon or transposable element
________ is a group of mechanisms that allow for the introduction of genetic material from one organism to another organism within the same generation.
Answer
Horizontal gene transfer
True/False
Asexually reproducing organisms lack mechanisms for generating genetic diversity within a population.
Answer
False
Short Answer
Briefly describe two ways in which chromosomal DNA from a donor cell may be transferred to a recipient cell during the process of conjugation.
Describe what happens when a nonsense mutation is introduced into the gene encoding transposase within a transposon.
11.7: Gene Regulation - Operon Theory
Genomic DNA contains both structural genes, which encode products that serve as cellular structures or enzymes, and regulatory genes, which encode products that regulate gene expression. The expression of a gene is a highly regulated process. Whereas regulating gene expression in multicellular organisms allows for cellular differentiation, in single-celled organisms like prokaryotes, it ensures that a cell’s resources are not wasted making proteins that the cell does not need at that time.
Multiple Choice
An operon of genes encoding enzymes in a biosynthetic pathway is likely to be which of the following?
1. inducible
2. repressible
3. constitutive
4. monocistronic
Answer
B
An operon encoding genes that are transcribed and translated continuously to provide the cell with constant intermediate levels of the protein products is said to be which of the following?
1. repressible
2. inducible
3. constitutive
4. activated
Answer
C
Which of the following conditions leads to maximal expression of the lac operon?
1. lactose present, glucose absent
2. lactose present, glucose present
3. lactose absent, glucose absent
4. lactose absent, glucose present
Answer
A
Which of the following is a type of regulation of gene expression unique to eukaryotes?
1. attenuation
2. use of alternate σ factor
3. chemical modification of histones
4. alarmones
Answer
C
Fill in the Blank
The DNA sequence, to which repressors may bind, that lies between the promoter and the first structural gene is called the ________.
Answer
operator
The prevention of expression of operons encoding substrate use pathways for substrates other than glucose when glucose is present is called _______.
Answer
catabolite repression
Short Answer
What are two ways that bacteria can influence the transcription of multiple different operons simultaneously in response to a particular environmental condition?
Critical Thinking
The following figure is from Monod’s original work on diauxic growth showing the growth of E. coli in the simultaneous presence of xylose and glucose as the only carbon sources. Explain what is happening at points A–D with respect to the carbon source being used for growth, and explain whether the xylose-use operon is being expressed (and why). Note that expression of the enzymes required for xylose use is regulated in a manner similar to the expression of the enzymes required for lactose use. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/11%3A_Mechanisms_of_Microbial_Genetics/11.E%3A_Mechanisms_of_Microbial_Genetics_%28Exercises%29.txt |
Watson and Crick’s identification of the structure of DNA in 1953 was the seminal event in the field of genetic engineering. Since the 1970s, there has been a veritable explosion in scientists’ ability to manipulate DNA in ways that have revolutionized the fields of biology, medicine, diagnostics, forensics, and industrial manufacturing. Many of the molecular tools discovered in recent decades have been produced using prokaryotic microbes. In this chapter, we will explore some of those tools, especially as they relate to applications in medicine and health care.
As an example, the thermal cycler in Figure \(1\) is used to perform a diagnostic technique called the polymerase chain reaction (PCR), which relies on DNA polymerase enzymes from thermophilic bacteria. Other molecular tools, such as restriction enzymes and plasmids obtained from microorganisms, allow scientists to insert genes from humans or other organisms into microorganisms. The microorganisms are then grown on an industrial scale to synthesize products such as insulin, vaccines, and biodegradable polymers. These are just a few of the numerous applications of microbial genetics that we will explore in this chapter.
• 12.1: Microbes and the Tools of Genetic Engineering
The science of using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of plants and animals through farming and breeding practices is a type of biotechnology. However, in a contemporary sense, we associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering.
• 12.2: Visualizing and Characterizing DNA
Finding a gene of interest within a sample requires the use of a single-stranded DNA probe labeled with a molecular beacon (typically radioactivity or fluorescence) that can hybridize with a complementary single-stranded nucleic acid in the sample. Agarose gel electrophoresis allows for the separation of DNA molecules based on size. Restriction fragment length polymorphism (RFLP) analysis allows for the visualization by agarose gel electrophoresis of distinct variants of a DNA sequence.
• 12.3: Whole Genome Methods and Industrial Applications
Advances in molecular biology have led to the creation of entirely new fields of science. Among these are fields that study aspects of whole genomes, collectively referred to as whole-genome methods. In this section, we’ll provide a brief overview of the whole-genome fields of genomics, transcriptomics, and proteomics.
• 12.4: Genetic Engineering - Risks, Benefits, and Perceptions
Many types of genetic engineering have yielded clear benefits with few apparent risks. However, many emerging applications of genetic engineering are much more controversial, often because their potential benefits are pitted against significant risks, real or perceived. This is certainly the case for gene therapy, a clinical application of genetic engineering that may one day provide a cure for many diseases but is still largely an experimental approach to treatment.
• 12.E: Modern Applications of Microbial Genetics (Exercises)
Thumbnail: A group of Genetically modified GloFish fluorescent fish. (www.glofish.com).
12: Modern Applications of Microbial Genetics
Learning Objectives
• Identify tools of molecular genetics that are derived from microorganisms
• Describe the methods used to create recombinant DNA molecules
• Describe methods used to introduce DNA into prokaryotic cells
• List the types of genomic libraries and describe their uses
• Describe the methods used to introduce DNA into eukaryotic cells
Clinical Focus: Part 1
Kayla, a 24-year-old electrical engineer and running enthusiast, just moved from Arizona to New Hampshire to take a new job. On her weekends off, she loves to explore her new surroundings, going for long runs in the pine forests. In July she spent a week hiking through the mountains. In early August, Kayla developed a low fever, headache, and mild muscle aches, and she felt a bit fatigued. Not thinking much of it, she took some ibuprofen to combat her symptoms and vowed to get more rest.
Exercise \(1\)
What types of medical conditions might be responsible for Kayla’s symptoms?
The science of using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of plants and animals through farming and breeding practices is a type of biotechnology. However, in a contemporary sense, we associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering. Genetic engineering involves the use of recombinant DNA technology, the process by which a DNA sequence is manipulated in vitro, thus creating recombinant DNA molecules that have new combinations of genetic material. The recombinant DNA is then introduced into a host organism. If the DNA that is introduced comes from a different species, the host organism is now considered to be transgenic.
One example of a transgenic microorganism is the bacterial strain that produces human insulin (Figure \(1\)). The insulin gene from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these transgenic microbes are able to produce and secrete human insulin. Many prokaryotes are able to acquire foreign DNA and incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction), and taking up DNA from the environment (transformation). Recall that these mechanisms are examples of horizontal gene transfer—the transfer of genetic material between cells of the same generation.
Molecular Cloning
Herbert Boyer and Stanley Cohen first demonstrated the complete molecular cloning process in 1973 when they successfully cloned genes from the African clawed frog (Xenopus laevis) into a bacterial plasmid that was then introduced into the bacterial host Escherichia coli. Molecular cloning is a set of methods used to construct recombinant DNA and incorporate it into a host organism; it makes use of a number of molecular tools that are derived from microorganisms.
Restriction Enzymes and Ligases
In recombinant DNA technology, DNA molecules are manipulated using naturally occurring enzymes derived mainly from bacteria and viruses. The creation of recombinant DNA molecules is possible due to the use of naturally occurring restriction endonucleases (restriction enzymes), bacterial enzymes produced as a protection mechanism to cut and destroy foreign cytoplasmic DNA that is most commonly a result of bacteriophage infection. Stewart Linn and Werner Arber discovered restriction enzymes in their 1960s studies of how E. coli limits bacteriophage replication on infection. Today, we use restriction enzymes extensively for cutting DNA fragments that can then be spliced into another DNA molecule to form recombinant molecules. Each restriction enzyme cuts DNA at a characteristic recognition site, a specific, usually palindromic, DNA sequence typically between four to six base pairs in length. A palindrome is a sequence of letters that reads the same forward as backward. (The word “level” is an example of a palindrome.) Palindromic DNA sequences contain the same base sequences in the 5ʹ to 3ʹ direction on one strand as in the 5ʹ to 3ʹ direction on the complementary strand. A restriction enzyme recognizes the DNA palindrome and cuts each backbone at identical positions in the palindrome. Some restriction enzymes cut to produce molecules that have complementary overhangs (sticky ends) while others cut without generating such overhangs, instead producing blunt ends (Figure \(2\)).
Molecules with complementary sticky ends can easily anneal, or form hydrogen bonds between complementary bases, at their sticky ends. The annealing step allows hybridization of the single-stranded overhangs. Hybridization refers to the joining together of two complementary single strands of DNA. Blunt ends can also attach together, but less efficiently than sticky ends due to the lack of complementary overhangs facilitating the process. In either case, ligationby DNA ligase can then rejoin the two sugar-phosphate backbones of the DNA through covalent bonding, making the molecule a continuous double strand. In 1972, Paul Berg, a Stanford biochemist, was the first to produce a recombinant DNA molecule using this technique, combining the SV40 monkey virus with E. coli bacteriophage lambda to create a hybrid.
Plasmids
After restriction digestion, genes of interest are commonly inserted into plasmids, small pieces of typically circular, double-stranded DNA that replicate independently of the bacterial chromosome (see Unique Characteristics of Prokaryotic Cells). In recombinant DNA technology, plasmids are often used as vectors, DNA molecules that carry DNA fragments from one organism to another. Plasmids used as vectors can be genetically engineered by researchers and scientific supply companies to have specialized properties, as illustrated by the commonly used plasmid vector pUC19 (Figure \(3\)). Some plasmid vectors contain genes that confer antibiotic resistance; these resistance genes allow researchers to easily find plasmid-containing colonies by plating them on media containing the corresponding antibiotic. The antibiotic kills all host cells that do not harbor the desired plasmid vector, but those that contain the vector are able to survive and grow.
Plasmid vectors used for cloning typically have a polylinker site, or multiple cloning site (MCS). A polylinker site is a short sequence containing multiple unique restriction enzyme recognition sites that are used for inserting DNA into the plasmid after restriction digestion of both the DNA and the plasmid. Having these multiple restriction enzyme recognition sites within the polylinker site makes the plasmid vector versatile, so it can be used for many different cloning experiments involving different restriction enzymes.
This polylinker site is often found within a reporter gene, another gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion. The reporter gene allows a researcher to distinguish host cells that contain recombinant plasmids with cloned DNA fragments from host cells that only contain the non-recombinant plasmid vector. The most common reporter gene used in plasmid vectors is the bacterial lacZ gene encoding beta-galactosidase, an enzyme that naturally degrades lactose but can also degrade a colorless synthetic analog X-gal, thereby producing blue colonies on X-gal–containing media. The lacZ reporter gene is disabled when the recombinant DNA is spliced into the plasmid. Because the LacZ protein is not produced when the gene is disabled, X-gal is not degraded and white colonies are produced, which can then be isolated. This blue-white screening method is described later and shown in Figure \(4\). In addition to these features, some plasmids come pre-digested and with an enzyme linked to the linearized plasmid to aid in ligation after the insertion of foreign DNA fragments.
Molecular Cloning using Transformation
The most commonly used mechanism for introducing engineered plasmids into a bacterial cell is transformation, a process in which bacteria take up free DNA from their surroundings. In nature, free DNA typically comes from other lysed bacterial cells; in the laboratory, free DNA in the form of recombinant plasmids is introduced to the cell’s surroundings.
Some bacteria, such as Bacillus spp., are naturally competent, meaning they are able to take up foreign DNA. However, not all bacteria are naturally competent. In most cases, bacteria must be made artificially competent in the laboratory by increasing the permeability of the cell membrane. This can be achieved through chemical treatments that neutralize charges on the cell membrane or by exposing the bacteria to an electric field that creates microscopic pores in the cell membrane. These methods yield chemically competent or electrocompetent bacteria, respectively.
Following the transformation protocol, bacterial cells are plated onto an antibiotic-containing medium to inhibit the growth of the many host cells that were not transformed by the plasmid conferring antibiotic resistance. A technique called blue-white screening is then used for lacZ-encoding plasmid vectors such as pUC19. Blue colonies have a functional beta-galactosidase enzyme because the lacZ gene is uninterrupted, with no foreign DNA inserted into the polylinker site. These colonies typically result from the digested, linearized plasmid religating to itself. However, white colonies lack a functional beta-galactosidase enzyme, indicating the insertion of foreign DNA within the polylinker site of the plasmid vector, thus disrupting the lacZ gene. Thus, white colonies resulting from this blue-white screening contain plasmids with an insert and can be further screened to characterize the foreign DNA. To be sure the correct DNA was incorporated into the plasmid, the DNA insert can then be sequenced.
Link to Learning
View an animation of molecular cloning from the DNA Learning Center.
Exercise \(2\)
In blue-white screening, what does a blue colony mean and why is it blue?
Molecular Cloning Using Conjugation or Transduction
The bacterial process of conjugation (see How Asexual Prokaryotes Achieve Genetic Diversity) can also be manipulated for molecular cloning. F plasmids, or fertility plasmids, are transferred between bacterial cells through the process of conjugation. Recombinant DNA can be transferred by conjugation when bacterial cells containing a recombinant F plasmid are mixed with compatible bacterial cells lacking the plasmid. F plasmids encode a surface structure called an F pilus that facilitates contact between a cell containing an F plasmid and one without an F plasmid. On contact, a cytoplasmic bridge forms between the two cells and the F-plasmid-containing cell replicates its plasmid, transferring a copy of the recombinant F plasmid to the recipient cell. Once it has received the recombinant F plasmid, the recipient cell can produce its own F pilus and facilitate transfer of the recombinant F plasmid to an additional cell. The use of conjugation to transfer recombinant F plasmids to recipient cells is another effective way to introduce recombinant DNA molecules into host cells.
Alternatively, bacteriophages can be used to introduce recombinant DNA into host bacterial cells through a manipulation of the transduction process (see How Asexual Prokaryotes Achieve Genetic Diversity). In the laboratory, DNA fragments of interest can be engineered into phagemids, which are plasmids that have phage sequences that allow them to be packaged into bacteriophages. Bacterial cells can then be infected with these bacteriophages so that the recombinant phagemids can be introduced into the bacterial cells. Depending on the type of phage, the recombinant DNA may be integrated into the host bacterial genome (lysogeny), or it may exist as a plasmid in the host’s cytoplasm.
Exercise \(2\)
1. What is the original function of a restriction enzyme?
2. What two processes are exploited to get recombinant DNA into a bacterial host cell?
3. Distinguish the uses of an antibiotic resistance gene and a reporter gene in a plasmid vector.
Creating a Genomic Library
Molecular cloning may also be used to generate a genomic library. The library is a complete (or nearly complete) copy of an organism’s genome contained as recombinant DNA plasmids engineered into unique clones of bacteria. Having such a library allows a researcher to create large quantities of each fragment by growing the bacterial host for that fragment. These fragments can be used to determine the sequence of the DNA and the function of any genes present.
One method for generating a genomic library is to ligate individual restriction enzyme-digested genomic fragments into plasmid vectors cut with the same restriction enzyme (Figure \(5\)). After transformation into a bacterial host, each transformed bacterial cell takes up a single recombinant plasmid and grows into a colony of cells. All of the cells in this colony are identical clones and carry the same recombinant plasmid. The resulting library is a collection of colonies, each of which contains a fragment of the original organism’s genome, that are each separate and distinct and can each be used for further study. This makes it possible for researchers to screen these different clones to discover the one containing a gene of interest from the original organism’s genome.
To construct a genomic library using larger fragments of genomic DNA, an E. coli bacteriophage, such as lambda, can be used as a host (Figure \(6\)). Genomic DNA can be sheared or enzymatically digested and ligated into a pre-digested bacteriophage lambda DNA vector. Then, these recombinant phage DNA molecules can be packaged into phage particles and used to infect E. coli host cells on a plate. During infection within each cell, each recombinant phage will make many copies of itself and lyse the E. coli lawn, forming a plaque. Thus, each plaque from a phage library represents a unique recombinant phage containing a distinct genomic DNA fragment. Plaques can then be screened further to look for genes of interest. One advantage to producing a library using phages instead of plasmids is that a phage particle holds a much larger insert of foreign DNA compared with a plasmid vector, thus requiring a much smaller number of cultures to fully represent the entire genome of the original organism.
To focus on the expressed genes in an organism or even a tissue, researchers construct libraries using the organism’s messenger RNA (mRNA) rather than its genomic DNA. Whereas all cells in a single organism will have the same genomic DNA, different tissues express different genes, producing different complements of mRNA. For example, all human cells’ genomic DNA contains the gene for insulin, but only cells in the pancreas express mRNA directing the production of insulin. Because mRNA cannot be cloned directly, in the laboratory mRNA must be used as a template by the retroviral enzyme reverse transcriptase to make complementary DNA (cDNA). A cell’s full complement of mRNA can be reverse-transcribed into cDNA molecules, which can be used as a template for DNA polymerase to make double-stranded DNA copies; these fragments can subsequently be ligated into either plasmid vectors or bacteriophage to produce a cDNA library. The benefit of a cDNA library is that it contains DNA from only the expressed genes in the cell. This means that the introns, control sequences such as promoters, and DNA not destined to be translated into proteins are not represented in the library. The focus on translated sequences means that the library cannot be used to study the sequence and structure of the genome in its entirety. The construction of a cDNA genomic library is shown in Figure \(7\).
Exercise \(3\)
1. What are the hosts for the genomic libraries described?
2. What is cDNA?
Introducing Recombinant Molecules into Eukaryotic Hosts
The use of bacterial hosts for genetic engineering laid the foundation for recombinant DNA technology; however, researchers have also had great interest in genetically engineering eukaryotic cells, particularly those of plants and animals. The introduction of recombinant DNA molecules into eukaryotic hosts is called transfection. Genetically engineered plants, called transgenic plants, are of significant interest for agricultural and pharmaceutical purposes. The first transgenic plant sold commercially was the Flavr Savr delayed-ripening tomato, which came to market in 1994. Genetically engineered livestock have also been successfully produced, resulting, for example, in pigs with increased nutritional value1 and goats that secrete pharmaceutical products in their milk.2
Electroporation
Compared to bacterial cells, eukaryotic cells tend to be less amenable as hosts for recombinant DNA molecules. Because eukaryotes are typically neither competent to take up foreign DNA nor able to maintain plasmids, transfection of eukaryotic hosts is far more challenging and requires more intrusive techniques for success. One method used for transfecting cells in cell culture is called electroporation. A brief electric pulse induces the formation of transient pores in the phospholipid bilayers of cells through which the gene can be introduced. At the same time, the electric pulse generates a short-lived positive charge on one side of the cell’s interior and a negative charge on the opposite side; the charge difference draws negatively charged DNA molecules into the cell (Figure \(8\)).
Microinjection
An alternative method of transfection is called microinjection. Because eukaryotic cells are typically larger than those of prokaryotes, DNA fragments can sometimes be directly injected into the cytoplasm using a glass micropipette, as shown in Figure \(9\).
Gene Guns
Transfecting plant cells can be even more difficult than animal cells because of their thick cell walls. One approach involves treating plant cells with enzymes to remove their cell walls, producing protoplasts. Then, a gene gun is used to shoot gold or tungsten particles coated with recombinant DNA molecules into the plant protoplasts at high speeds. Recipient protoplast cells can then recover and be used to generate new transgenic plants (Figure \(10\)).
Shuttle Vectors
Another method of transfecting plants involves shuttle vectors, plasmids that can move between bacterial and eukaryotic cells. The tumor-inducing (Ti) plasmids originating from the bacterium Agrobacterium tumefaciens are commonly used as shuttle vectors for incorporating genes into plants (Figure \(11\)). In nature, the Ti plasmids of A. tumefaciens cause plants to develop tumors when they are transferred from bacterial cells to plant cells. Researchers have been able to manipulate these naturally occurring plasmids to remove their tumor-causing genes and insert desirable DNA fragments. The resulting recombinant Ti plasmids can be transferred into the plant genome through the natural transfer of Ti plasmids from the bacterium to the plant host. Once inside the plant host cell, the gene of interest recombines into the plant cell’s genome.
Viral Vectors
Viral vectors can also be used to transfect eukaryotic cells. In fact, this method is often used in gene therapy (see Gene Therapy) to introduce healthy genes into human patients suffering from diseases that result from genetic mutations. Viral genes can be deleted and replaced with the gene to be delivered to the patient;3 the virus then infects the host cell and delivers the foreign DNA into the genome of the targeted cell. Adenoviruses are often used for this purpose because they can be grown to high titer and can infect both nondividing and dividing host cells. However, use of viral vectors for gene therapy can pose some risks for patients, as discussed in Gene Therapy.
Exercise \(4\)
1. What are the methods used to introduce recombinant DNA vectors into animal cells?
2. Compare and contrast shuttle vectors and viral vectors.
Key Concepts and Summary
• Biotechology is the science of utilizing living systems to benefit humankind. In recent years, the ability to directly alter an organism’s genome through genetic engineering has been made possible due to advances in recombinant DNA technology, which allows researchers to create recombinant DNA molecules with new combinations of genetic material.
• Molecular cloning involves methods used to construct recombinant DNA and facilitate their replication in host organisms. These methods include the use of restriction enzymes (to cut both foreign DNA and plasmid vectors), ligation (to paste fragments of DNA together), and the introduction of recombinant DNA into a host organism (often bacteria).
• Blue-white screening allows selection of bacterial transformants that contain recombinant plasmids using the phenotype of a reporter gene that is disabled by insertion of the DNA fragment.
• Genomic libraries can be made by cloning genomic fragments from one organism into plasmid vectors or into bacteriophage.
• cDNA libraries can be generated to represent the mRNA molecules expressed in a cell at a given point.
• Transfection of eukaryotic hosts can be achieved through various methods using electroporation, gene guns, microinjection, shuttle vectors, and viral vectors.
Footnotes
1. 1 Liangxue Lai, Jing X. Kang, Rongfeng Li, Jingdong Wang, William T. Witt, Hwan Yul Yong, Yanhong Hao et al. “Generation of Cloned Transgenic Pigs Rich in Omega-3 Fatty Acids.” Nature Biotechnology 24 no. 4 (2006): 435–436.
2. 2 Raylene Ramos Moura, Luciana Magalhães Melo, and Vicente José de Figueirêdo Freitas. “Production of Recombinant Proteins in Milk of Transgenic and Non-Transgenic Goats.” Brazilian Archives of Biology and Technology 54 no. 5 (2011): 927–938.
3. 3 William S.M. Wold and Karoly Toth. “Adenovirus Vectors for Gene Therapy, Vaccination and Cancer Gene Therapy.” Current Gene Therapy 13 no. 6 (2013): 421. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/12%3A_Modern_Applications_of_Microbial_Genetics/12.01%3A_Microbes_and_the_Tools_of_Genetic_Engineering.txt |
Learning Objectives
• Explain the use of nucleic acid probes to visualize specific DNA sequences
• Explain the use of gel electrophoresis to separate DNA fragments
• Explain the principle of restriction fragment length polymorphism analysis and its uses
• Compare and contrast Southern and northern blots
• Explain the principles and uses of microarray analysis
• Describe the methods uses to separate and visualize protein variants
• Explain the method and uses of polymerase chain reaction and DNA sequencing
The sequence of a DNA molecule can help us identify an organism when compared to known sequences housed in a database. The sequence can also tell us something about the function of a particular part of the DNA, such as whether it encodes a particular protein. Comparing protein signatures—the expression levels of specific arrays of proteins—between samples is an important method for evaluating cellular responses to a multitude of environmental factors and stresses. Analysis of protein signatures can reveal the identity of an organism or how a cell is responding during disease.
The DNA and proteins of interest are microscopic and typically mixed in with many other molecules including DNA or proteins irrelevant to our interests. Many techniques have been developed to isolate and characterize molecules of interest. These methods were originally developed for research purposes, but in many cases they have been simplified to the point that routine clinical use is possible. For example, many pathogens, such as the bacterium Helicobacter pylori, which causes stomach ulcers, can be detected using protein-based tests. In addition, an increasing number of highly specific and accurate DNA amplification-based identification assays can now detect pathogens such as antibiotic-resistant enteric bacteria, herpes simplex virus, varicella-zoster virus, and many others.
Molecular Analysis of DNA
In this subsection, we will outline some of the basic methods used for separating and visualizing specific fragments of DNA that are of interest to a scientist. Some of these methods do not require knowledge of the complete sequence of the DNA molecule. Before the advent of rapid DNA sequencing, these methods were the only ones available to work with DNA, but they still form the basic arsenal of tools used by molecular geneticists to study the body’s responses to microbial and other diseases.
Nucleic Acid Probing
DNA molecules are small, and the information contained in their sequence is invisible. How does a researcher isolate a particular stretch of DNA, or having isolated it, determine what organism it is from, what its sequence is, or what its function is? One method to identify the presence of a certain DNA sequence uses artificially constructed pieces of DNA called probes. Probes can be used to identify different bacterial species in the environment and many DNA probes are now available to detect pathogens clinically. For example, DNA probes are used to detect the vaginal pathogens Candida albicans, Gardnerella vaginalis, and Trichomonas vaginalis.
To screen a genomic library for a particular gene or sequence of interest, researchers must know something about that gene. If researchers have a portion of the sequence of DNA for the gene of interest, they can design a DNA probe, a single-stranded DNA fragment that is complementary to part of the gene of interest and different from other DNA sequences in the sample. The DNA probe may be synthesized chemically by commercial laboratories, or it may be created by cloning, isolating, and denaturing a DNA fragment from a living organism. In either case, the DNA probe must be labeled with a molecular tag or beacon, such as a radioactive phosphorus atom (as is used for autoradiography) or a fluorescent dye (as is used in fluorescent in situ hybridization, or FISH), so that the probe and the DNA it binds to can be seen (Figure \(1\)). The DNA sample being probed must also be denatured to make it single-stranded so that the single-stranded DNA probe can anneal to the single-stranded DNA sample at locations where their sequences are complementary. While these techniques are valuable for diagnosis, their direct use on sputum and other bodily samples may be problematic due to the complex nature of these samples. DNA often must first be isolated from bodily samples through chemical extraction methods before a DNA probe can be used to identify pathogens.
Clinical Focus: Part 2
The mild, flu-like symptoms that Kayla is experiencing could be caused by any number of infectious agents. In addition, several non-infectious autoimmune conditions, such as multiple sclerosis, systemic lupus erythematosus (SLE), and amyotrophic lateral sclerosis (ALS), also have symptoms that are consistent with Kayla’s early symptoms. However, over the course of several weeks, Kayla’s symptoms worsened. She began to experience joint pain in her knees, heart palpitations, and a strange limpness in her facial muscles. In addition, she suffered from a stiff neck and painful headaches. Reluctantly, she decided it was time to seek medical attention.
Exercise \(1\)
1. Do Kayla’s new symptoms provide any clues as to what type of infection or other medical condition she may have?
2. What tests or tools might a health-care provider use to pinpoint the pathogen causing Kayla’s symptoms?
Agarose Gel Electrophoresis
There are a number of situations in which a researcher might want to physically separate a collection of DNA fragments of different sizes. A researcher may also digest a DNA sample with a restriction enzyme to form fragments. The resulting size and fragment distribution pattern can often yield useful information about the sequence of DNA bases that can be used, much like a bar-code scan, to identify the individual or species to which the DNA belongs.
Gel electrophoresis is a technique commonly used to separate biological molecules based on size and biochemical characteristics, such as charge and polarity. Agarose gel electrophoresis is widely used to separate DNA (or RNA) of varying sizes that may be generated by restriction enzyme digestion or by other means, such as the PCR (Figure \(2\)).
Due to its negatively charged backbone, DNA is strongly attracted to a positive electrode. In agarose gel electrophoresis, the gel is oriented horizontally in a buffer solution. Samples are loaded into sample wells on the side of the gel closest to the negative electrode, then drawn through the molecular sieve of the agarose matrix toward the positive electrode. The agarose matrix impedes the movement of larger molecules through the gel, whereas smaller molecules pass through more readily. Thus, the distance of migration is inversely correlated to the size of the DNA fragment, with smaller fragments traveling a longer distance through the gel. Sizes of DNA fragments within a sample can be estimated by comparison to fragments of known size in a DNA ladder also run on the same gel. To separate very large DNA fragments, such as chromosomes or viral genomes, agarose gel electrophoresis can be modified by periodically alternating the orientation of the electric field during pulsed-field gel electrophoresis (PFGE). In PFGE, smaller fragments can reorient themselves and migrate slightly faster than larger fragments and this technique can thus serve to separate very large fragments that would otherwise travel together during standard agarose gel electrophoresis. In any of these electrophoresis techniques, the locations of the DNA or RNA fragments in the gel can be detected by various methods. One common method is adding ethidium bromide, a stain that inserts into the nucleic acids at non-specific locations and can be visualized when exposed to ultraviolet light. Other stains that are safer than ethidium bromide, a potential carcinogen, are now available.
Restriction Fragment Length Polymorphism (RFLP) Analysis
Restriction enzyme recognition sites are short (only a few nucleotides long), sequence-specific palindromes, and may be found throughout the genome. Thus, differences in DNA sequences in the genomes of individuals will lead to differences in distribution of restriction-enzyme recognition sites that can be visualized as distinct banding patterns on a gel after agarose gel electrophoresis. Restriction fragment length polymorphism (RFLP) analysis compares DNA banding patterns of different DNA samples after restriction digestion (Figure \(3\)).
RFLP analysis has many practical applications in both medicine and forensic science. For example, epidemiologists use RFLP analysis to track and identify the source of specific microorganisms implicated in outbreaks of food poisoning or certain infectious diseases. RFLP analysis can also be used on human DNA to determine inheritance patterns of chromosomes with variant genes, including those associated with heritable diseases or to establish paternity.
Forensic scientists use RFLP analysis as a form of DNA fingerprinting, which is useful for analyzing DNA obtained from crime scenes, suspects, and victims. DNA samples are collected, the numbers of copies of the sample DNA molecules are increased using PCR, and then subjected to restriction enzyme digestion and agarose gel electrophoresis to generate specific banding patterns. By comparing the banding patterns of samples collected from the crime scene against those collected from suspects or victims, investigators can definitively determine whether DNA evidence collected at the scene was left behind by suspects or victims.
Southern Blots and Modifications
Several molecular techniques capitalize on sequence complementarity and hybridization between nucleic acids of a sample and DNA probes. Typically, probing nucleic-acid samples within a gel is unsuccessful because as the DNA probe soaks into a gel, the sample nucleic acids within the gel diffuse out. Thus, blotting techniques are commonly used to transfer nucleic acids to a thin, positively charged membrane made of nitrocellulose or nylon. In the Southern blottechnique, developed by Sir Edwin Southern in 1975, DNA fragments within a sample are first separated by agarose gel electrophoresis and then transferred to a membrane through capillary action (Figure \(4\)). The DNA fragments that bind to the surface of the membrane are then exposed to a specific single-stranded DNA probe labeled with a radioactive or fluorescent molecular beacon to aid in detection. Southern blots may be used to detect the presence of certain DNA sequences in a given DNA sample. Once the target DNA within the membrane is visualized, researchers can cut out the portion of the membrane containing the fragment to recover the DNA fragment of interest.
Variations of the Southern blot—the dot blot, slot blot, and the spot blot—do not involve electrophoresis, but instead concentrate DNA from a sample into a small location on a membrane. After hybridization with a DNA probe, the signal intensity detected is measured, allowing the researcher to estimate the amount of target DNA present within the sample.
A colony blot is another variation of the Southern blot in which colonies representing different clones in a genomic library are transferred to a membrane by pressing the membrane onto the culture plate. The cells on the membrane are lysed and the membrane can then be probed to determine which colonies within a genomic library harbor the target gene. Because the colonies on the plate are still growing, the cells of interest can be isolated from the plate.
In the northern blot, another variation of the Southern blot, RNA (not DNA) is immobilized on the membrane and probed. Northern blots are typically used to detect the amount of mRNA made through gene expression within a tissue or organism sample.
Microarray Analysis
Another technique that capitalizes on the hybridization between complementary nucleic acid sequences is called microarray analysis. Microarray analysis is useful for the comparison of gene-expression patterns between different cell types—for example, cells infected with a virus versus uninfected cells, or cancerous cells versus healthy cells (Figure \(5\)).
Typically, DNA or cDNA from an experimental sample is deposited on a glass slide alongside known DNA sequences. Each slide can hold more than 30,000 different DNA fragment types. Distinct DNA fragments (encompassing an organism’s entire genomic library) or cDNA fragments (corresponding to an organism’s full complement of expressed genes) can be individually spotted on a glass slide.
Once deposited on the slide, genomic DNA or mRNA can be isolated from the two samples for comparison. If mRNA is isolated, it is reverse-transcribed to cDNA using reverse transcriptase. Then the two samples of genomic DNA or cDNA are labeled with different fluorescent dyes (typically red and green). The labeled genomic DNA samples are then combined in equal amounts, added to the microarray chip, and allowed to hybridize to complementary spots on the microarray.
Hybridization of sample genomic DNA molecules can be monitored by measuring the intensity of fluorescence at particular spots on the microarray. Differences in the amount of hybridization between the samples can be readily observed. If only one sample’s nucleic acids hybridize to a particular spot on the microarray, then that spot will appear either green or red. However, if both samples’ nucleic acids hybridize, then the spot will appear yellow due to the combination of the red and green dyes.
Although microarray technology allows for a holistic comparison between two samples in a short time, it requires sophisticated (and expensive) detection equipment and analysis software. Because of the expense, this technology is typically limited to research settings. Researchers have used microarray analysis to study how gene expression is affected in organisms that are infected by bacteria or viruses or subjected to certain chemical treatments.
Link to Learning
Explore microchip technology at this interactive website.
Exercise \(2\)
1. What does a DNA probe consist of?
2. Why is a Southern blot used after gel electrophoresis of a DNA digest?
Molecular Analysis of Proteins
In many cases it may not be desirable or possible to study DNA or RNA directly. Proteins can provide species-specific information for identification as well as important information about how and whether a cell or tissue is responding to the presence of a pathogenic microorganism. Various proteins require different methods for isolation and characterization.
Polyacrylamide Gel Electrophoresis
A variation of gel electrophoresis, called polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins. In PAGE, the gel matrix is finer and composed of polyacrylamide instead of agarose. Additionally, PAGE is typically performed using a vertical gel apparatus (Figure \(6\)). Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins based on their net charges. Alternatively, proteins can be denatured and coated with a negatively charged detergent called sodium dodecyl sulfate (SDS), masking the native charges and allowing separation based on size only. PAGE can be further modified to separate proteins based on two characteristics, such as their charges at various pHs as well as their size, through the use of two-dimensional PAGE. In any of these cases, following electrophoresis, proteins are visualized through staining, commonly with either Coomassie blue or a silver stain.
Exercise \(3\)
On what basis are proteins separated in SDS-PAGE?
Clinical Focus: Part 3
When Kayla described her symptoms, her physician at first suspected bacterial meningitis, which is consistent with her headaches and stiff neck. However, she soon ruled this out as a possibility because meningitis typically progresses more quickly than what Kayla was experiencing. Many of her symptoms still paralleled those of amyotrophic lateral sclerosis (ALS) and systemic lupus erythematosus (SLE), and the physician also considered Lyme disease a possibility given how much time Kayla spends in the woods. Kayla did not recall any recent tick bites (the typical means by which Lyme disease is transmitted) and she did not have the typical bull’s-eye rash associated with Lyme disease (Figure \(7\)). However, 20–30% of patients with Lyme disease never develop this rash, so the physician did not want to rule it out.
Kayla’s doctor ordered an MRI of her brain, a complete blood count to test for anemia, blood tests assessing liver and kidney function, and additional tests to confirm or rule out SLE or Lyme disease. Her test results were inconsistent with both SLE and ALS, and the result of the test looking for Lyme disease antibodies was “equivocal,” meaning inconclusive. Having ruled out ALS and SLE, Kayla’s doctor decided to run additional tests for Lyme disease.
Exercise \(4\)
1. Why would Kayla’s doctor still suspect Lyme disease even if the test results did not detect Lyme antibodies in the blood?
2. What type of molecular test might be used for the detection of blood antibodies to Lyme disease?
Amplification-Based DNA Analysis Methods
Various methods can be used for obtaining sequences of DNA, which are useful for studying disease-causing organisms. With the advent of rapid sequencing technology, our knowledge base of the entire genomes of pathogenic organisms has grown phenomenally. We start with a description of the polymerase chain reaction, which is not a sequencing method but has allowed researchers and clinicians to obtain the large quantities of DNA needed for sequencing and other studies. The polymerase chain reaction eliminates the dependence we once had on cells to make multiple copies of DNA, achieving the same result through relatively simple reactions outside the cell.
Polymerase Chain Reaction (PCR)
Most methods of DNA analysis, such as restriction enzyme digestion and agarose gel electrophoresis, or DNA sequencing require large amounts of a specific DNA fragment. In the past, large amounts of DNA were produced by growing the host cells of a genomic library. However, libraries take time and effort to prepare and DNA samples of interest often come in minute quantities. The polymerase chain reaction (PCR) permits rapid amplification in the number of copies of specific DNA sequences for further analysis (Figure \(8\)). One of the most powerful techniques in molecular biology, PCR was developed in 1983 by Kary Mullis while at Cetus Corporation. PCR has specific applications in research, forensic, and clinical laboratories, including:
• determining the sequence of nucleotides in a specific region of DNA
• amplifying a target region of DNA for cloning into a plasmid vector
• identifying the source of a DNA sample left at a crime scene
• analyzing samples to determine paternity
• comparing samples of ancient DNA with modern organisms
• determining the presence of difficult to culture, or unculturable, microorganisms in humans or environmental samples
PCR is an in vitro laboratory technique that takes advantage of the natural process of DNA replication. The heat-stable DNA polymerase enzymes used in PCR are derived from hyperthermophilic prokaryotes. Taq DNA polymerase, commonly used in PCR, is derived from the Thermus aquaticus bacterium isolated from a hot spring in Yellowstone National Park. DNA replication requires the use of primers for the initiation of replication to have free 3ʹ-hydroxyl groups available for the addition of nucleotides by DNA polymerase. However, while primers composed of RNA are normally used in cells, DNA primers are used for PCR. DNA primers are preferable due to their stability, and DNA primers with known sequences targeting a specific DNA region can be chemically synthesized commercially. These DNA primers are functionally similar to the DNA probes used for the various hybridization techniques described earlier, binding to specific targets due to complementarity between the target DNA sequence and the primer.
PCR occurs over multiple cycles, each containing three steps: denaturation, annealing, and extension. Machines called thermal cyclers are used for PCR; these machines can be programmed to automatically cycle through the temperatures required at each step (Figure 12.1). First, double-stranded template DNA containing the target sequence is denatured at approximately 95 °C. The high temperature required to physically (rather than enzymatically) separate the DNA strands is the reason the heat-stable DNA polymerase is required. Next, the temperature is lowered to approximately 50 °C. This allows the DNA primers complementary to the ends of the target sequence to anneal (stick) to the template strands, with one primer annealing to each strand. Finally, the temperature is raised to 72 °C, the optimal temperature for the activity of the heat-stable DNA polymerase, allowing for the addition of nucleotides to the primer using the single-stranded target as a template. Each cycle doubles the number of double-stranded target DNA copies. Typically, PCR protocols include 25–40 cycles, allowing for the amplification of a single target sequence by tens of millions to over a trillion.
Natural DNA replication is designed to copy the entire genome, and initiates at one or more origin sites. Primers are constructed during replication, not before, and do not consist of a few specific sequences. PCR targets specific regions of a DNA sample using sequence-specific primers. In recent years, a variety of isothermal PCR amplification methods that circumvent the need for thermal cycling have been developed, taking advantage of accessory proteins that aid in the DNA replication process. As the development of these methods continues and their use becomes more widespread in research, forensic, and clinical labs, thermal cyclers may become obsolete.
Link to Learning
Deepen your understanding of the polymerase chain reaction by viewing this animation and working through an interactive exercise.
PCR Variations
Several later modifications to PCR further increase the utility of this technique. Reverse transcriptase PCR (RT-PCR) is used for obtaining DNA copies of a specific mRNA molecule. RT-PCR begins with the use of the reverse transcriptase enzyme to convert mRNA molecules into cDNA. That cDNA is then used as a template for traditional PCR amplification. RT-PCR can detect whether a specific gene has been expressed in a sample. Another recent application of PCR is real-time PCR, also known as quantitative PCR (qPCR). Standard PCR and RT-PCR protocols are not quantitative because any one of the reagents may become limiting before all of the cycles within the protocol are complete, and samples are only analyzed at the end. Because it is not possible to determine when in the PCR or RT-PCR protocol a given reagent has become limiting, it is not possible to know how many cycles were completed prior to this point, and thus it is not possible to determine how many original template molecules were present in the sample at the start of PCR. In qPCR, however, the use of fluorescence allows one to monitor the increase in a double-stranded template during a PCR reaction as it occurs. These kinetics data can then be used to quantify the amount of the original target sequence. The use of qPCR in recent years has further expanded the capabilities of PCR, allowing researchers to determine the number of DNA copies, and sometimes organisms, present in a sample. In clinical settings, qRT-PCR is used to determine viral load in HIV-positive patients to evaluate the effectiveness of their therapy.
DNA Sequencing
A basic sequencing technique is the chain termination method, also known as the dideoxy method or the Sanger DNA sequencing method, developed by Frederick Sanger in 1972. The chain termination method involves DNA replication of a single-stranded template with the use of a DNA primer to initiate synthesis of a complementary strand, DNA polymerase, a mix of the four regular deoxynucleotide (dNTP) monomers, and a small proportion of dideoxynucleotides (ddNTPs), each labeled with a molecular beacon. The ddNTPs are monomers missing a hydroxyl group (–OH) at the site at which another nucleotide usually attaches to form a chain (Figure \(9\)). Every time a ddNTP is randomly incorporated into the growing complementary strand, it terminates the process of DNA replication for that particular strand. This results in multiple short strands of replicated DNA that are each terminated at a different point during replication. When the reaction mixture is subjected to gel electrophoresis, the multiple newly replicated DNA strands form a ladder of differing sizes. Because the ddNTPs are labeled, each band on the gel reflects the size of the DNA strand when the ddNTP terminated the reaction.
In Sanger’s day, four reactions were set up for each DNA molecule being sequenced, each reaction containing only one of the four possible ddNTPs. Each ddNTP was labeled with a radioactive phosphorus molecule. The products of the four reactions were then run in separate lanes side by side on long, narrow PAGE gels, and the bands of varying lengths were detected by autoradiography. Today, this process has been simplified with the use of ddNTPs, each labeled with a different colored fluorescent dye or fluorochrome (Figure \(10\)), in one sequencing reaction containing all four possible ddNTPs for each DNA molecule being sequenced (Figure \(11\)). These fluorochromes are detected by fluorescence spectroscopy. Determining the fluorescence color of each band as it passes by the detector produces the nucleotide sequence of the template strand.
Since 2005, automated sequencing techniques used by laboratories fall under the umbrella of next generation sequencing, which is a group of automated techniques used for rapid DNA sequencing. These methods have revolutionized the field of molecular genetics because the low-cost sequencers can generate sequences of hundreds of thousands or millions of short fragments (25 to 600 base pairs) just in one day. Although several variants of next generation sequencing technologies are made by different companies (for example, 454 Life Sciences’ pyrosequencing and Illumina’s Solexa technology), they all allow millions of bases to be sequenced quickly, making the sequencing of entire genomes relatively easy, inexpensive, and commonplace. In 454 sequencing (pyrosequencing), for example, a DNA sample is fragmented into 400–600-bp single-strand fragments, modified with the addition of DNA adapters to both ends of each fragment. Each DNA fragment is then immobilized on a bead and amplified by PCR, using primers designed to anneal to the adapters, creating a bead containing many copies of that DNA fragment. Each bead is then put into a separate well containing sequencing enzymes. To the well, each of the four nucleotides is added one after the other; when each one is incorporated, pyrophosphate is released as a byproduct of polymerization, emitting a small flash of light that is recorded by a detector. This provides the order of nucleotides incorporated as a new strand of DNA is made and is an example of synthesis sequencing. Next generation sequencers use sophisticated software to get through the cumbersome process of putting all the fragments in order. Overall, these technologies continue to advance rapidly, decreasing the cost of sequencing and increasing the availability of sequence data from a wide variety of organisms quickly.
The National Center for Biotechnology Information houses a widely used genetic sequence database called GenBankwhere researchers deposit genetic information for public use. Upon publication of sequence data, researchers upload it to GenBank, giving other researchers access to the information. The collaboration allows researchers to compare newly discovered or unknown sample sequence information with the vast array of sequence data that already exists.
Link to Learning
View an animation about 454 sequencing to deepen your understanding of this method.
Using a NAAT to Diagnose a C. difficile Infection
Javier, an 80-year-old patient with a history of heart disease, recently returned home from the hospital after undergoing an angioplasty procedure to insert a stent into a cardiac artery. To minimize the possibility of infection, Javier was administered intravenous broad-spectrum antibiotics during and shortly after his procedure. He was released four days after the procedure, but a week later, he began to experience mild abdominal cramping and watery diarrhea several times a day. He lost his appetite, became severely dehydrated, and developed a fever. He also noticed blood in his stool. Javier’s wife called the physician, who instructed her to take him to the emergency room immediately.
The hospital staff ran several tests and found that Javier’s kidney creatinine levels were elevated compared with the levels in his blood, indicating that his kidneys were not functioning well. Javier’s symptoms suggested a possible infection with Clostridium difficile, a bacterium that is resistant to many antibiotics. The hospital collected and cultured a stool sample to look for the production of toxins A and B by C. difficile, but the results came back negative. However, the negative results were not enough to rule out a C. difficile infection because culturing of C. difficile and detection of its characteristic toxins can be difficult, particularly in some types of samples. To be safe, they proceeded with a diagnostic nucleic acid amplification test (NAAT). Currently NAATs are the clinical diagnostician’s gold standard for detecting the genetic material of a pathogen. In Javier’s case, qPCR was used to look for the gene encoding C. difficile toxin B (tcdB). When the qPCR analysis came back positive, the attending physician concluded that Javier was indeed suffering from a C. difficile infection and immediately prescribed the antibiotic vancomycin, to be administered intravenously. The antibiotic cleared the infection and Javier made a full recovery.
Because infections with C. difficile were becoming widespread in Javier’s community, his sample was further analyzed to see whether the specific strain of C. difficile could be identified. Javier’s stool sample was subjected to ribotyping and repetitive sequence-based PCR (rep-PCR) analysis. In ribotyping, a short sequence of DNA between the 16S rRNA and 23S rRNA genes is amplified and subjected to restriction digestion (Figure \(12\)). This sequence varies between strains of C. difficile, so restriction enzymes will cut in different places. In rep-PCR, DNA primers designed to bind to short sequences commonly found repeated within the C. difficile genome were used for PCR. Following restriction digestion, agarose gel electrophoresis was performed in both types of analysis to examine the banding patterns that resulted from each procedure (Figure \(13\)). Rep-PCR can be used to further subtype various ribotypes, increasing resolution for detecting differences between strains. The ribotype of the strain infecting Javier was found to be ribotype 27, a strain known for its increased virulence, resistance to antibiotics, and increased prevalence in the United States, Canada, Japan, and Europe.1
Exercise \(5\)
1. How do banding patterns differ between strains of C. difficile?
2. Why do you think laboratory tests were unable to detect toxin production directly?
Exercise \(6\)
1. How is PCR similar to the natural DNA replication process in cells? How is it different?
2. Compare RT-PCR and qPCR in terms of their respective purposes.
3. In chain-termination sequencing, how is the identity of each nucleotide in a sequence determined?
Key Concepts and Summary
• Finding a gene of interest within a sample requires the use of a single-stranded DNA probe labeled with a molecular beacon (typically radioactivity or fluorescence) that can hybridize with a complementary single-stranded nucleic acid in the sample.
• Agarose gel electrophoresis allows for the separation of DNA molecules based on size.
• Restriction fragment length polymorphism (RFLP) analysis allows for the visualization by agarose gel electrophoresis of distinct variants of a DNA sequence caused by differences in restriction sites.
• Southern blot analysis allows researchers to find a particular DNA sequence within a sample whereas northern blot analysis allows researchers to detect a particular mRNA sequence expressed in a sample.
• Microarray technology is a nucleic acid hybridization technique that allows for the examination of many thousands of genes at once to find differences in genes or gene expression patterns between two samples of genomic DNA or cDNA,
• Polyacrylamide gel electrophoresis (PAGE) allows for the separation of proteins by size, especially if native protein charges are masked through pretreatment with SDS.
• Polymerase chain reaction allows for the rapid amplification of a specific DNA sequence. Variations of PCR can be used to detect mRNA expression (reverse transcriptase PCR) or to quantify a particular sequence in the original sample (real-time PCR).
• Although the development of Sanger DNA sequencing was revolutionary, advances in next generation sequencing allow for the rapid and inexpensive sequencing of the genomes of many organisms, accelerating the volume of new sequence data.
Footnotes
1. 1 Patrizia Spigaglia, Fabrizio Barbanti, Anna Maria Dionisi, and Paola Mastrantonio. “Clostridium difficile Isolates Resistant to Fluoroquinolones in Italy: Emergence of PCR Ribotype 018.” Journal of Clinical Microbiology 48 no. 8 (2010): 2892–2896. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/12%3A_Modern_Applications_of_Microbial_Genetics/12.02%3A_Visualizing_and_Characterizing_DNA.txt |
Learning Objectives
• Explain the uses of genome-wide comparative analyses
• Summarize the advantages of genetically engineered pharmaceutical products
Advances in molecular biology have led to the creation of entirely new fields of science. Among these are fields that study aspects of whole genomes, collectively referred to as whole-genome methods. In this section, we’ll provide a brief overview of the whole-genome fields of genomics, transcriptomics, and proteomics.
Genomics, Transcriptomics, and Proteomics
The study and comparison of entire genomes, including the complete set of genes and their nucleotide sequence and organization, is called genomics. This field has great potential for future medical advances through the study of the human genome as well as the genomes of infectious organisms. Analysis of microbial genomes has contributed to the development of new antibiotics, diagnostic tools, vaccines, medical treatments, and environmental cleanup techniques.
The field of transcriptomics is the science of the entire collection of mRNA molecules produced by cells. Scientists compare gene expression patterns between infected and uninfected host cells, gaining important information about the cellular responses to infectious disease. Additionally, transcriptomics can be used to monitor the gene expression of virulence factors in microorganisms, aiding scientists in better understanding pathogenic processes from this viewpoint.
When genomics and transcriptomics are applied to entire microbial communities, we use the terms metagenomics and metatranscriptomics, respectively. Metagenomics and metatranscriptomics allow researchers to study genes and gene expression from a collection of multiple species, many of which may not be easily cultured or cultured at all in the laboratory. A DNA microarray (discussed in the previous section) can be used in metagenomics studies.
Another up-and-coming clinical application of genomics and transcriptomics is pharmacogenomics, also called toxicogenomics, which involves evaluating the effectiveness and safety of drugs on the basis of information from an individual’s genomic sequence. Genomic responses to drugs can be studied using experimental animals (such as laboratory rats or mice) or live cells in the laboratory before embarking on studies with humans. Changes in gene expression in the presence of a drug can sometimes be an early indicator of the potential for toxic effects. Personal genome sequence information may someday be used to prescribe medications that will be most effective and least toxic on the basis of the individual patient’s genotype.
The study of proteomics is an extension of genomics that allows scientists to study the entire complement of proteins in an organism, called the proteome. Even though all cells of a multicellular organism have the same set of genes, cells in various tissues produce different sets of proteins. Thus, the genome is constant, but the proteome varies and is dynamic within an organism. Proteomics may be used to study which proteins are expressed under various conditions within a single cell type or to compare protein expression patterns between different organisms.
The most prominent disease being studied with proteomic approaches is cancer, but this area of study is also being applied to infectious diseases. Research is currently underway to examine the feasibility of using proteomic approaches to diagnose various types of hepatitis, tuberculosis, and HIV infection, which are rather difficult to diagnose using currently available techniques.1
A recent and developing proteomic analysis relies on identifying proteins called biomarkers, whose expression is affected by the disease process. Biomarkers are currently being used to detect various forms of cancer as well as infections caused by pathogens such as Yersinia pestis and Vaccinia virus.2
Other “-omic” sciences related to genomics and proteomics include metabolomics, glycomics, and lipidomics, which focus on the complete set of small-molecule metabolites, sugars, and lipids, respectively, found within a cell. Through these various global approaches, scientists continue to collect, compile, and analyze large amounts of genetic information. This emerging field of bioinformatics can be used, among many other applications, for clues to treating diseases and understanding the workings of cells.
Additionally, researchers can use reverse genetics, a technique related to classic mutational analysis, to determine the function of specific genes. Classic methods of studying gene function involved searching for the genes responsible for a given phenotype. Reverse genetics uses the opposite approach, starting with a specific DNA sequence and attempting to determine what phenotype it produces. Alternatively, scientists can attach known genes (called reporter genes) that encode easily observable characteristics to genes of interest, and the location of expression of such genes of interest can be easily monitored. This gives the researcher important information about what the gene product might be doing or where it is located in the organism. Common reporter genes include bacterial lacZ, which encodes beta-galactosidase and whose activity can be monitored by changes in colony color in the presence of X-gal as previously described, and the gene encoding the jellyfish protein green fluorescent protein (GFP) whose activity can be visualized in colonies under ultraviolet light exposure (Figure \(1\)).
Exercise \(1\)
1. How is genomics different from traditional genetics?
2. If you wanted to study how two different cells in the body respond to an infection, what –omics field would you apply?
3. What are the biomarkers uncovered in proteomics used for?
Clinical Focus: Resolution
Because Kayla’s symptoms were persistent and serious enough to interfere with daily activities, Kayla’s physician decided to order some laboratory tests. The physician collected samples of Kayla’s blood, cerebrospinal fluid (CSF), and synovial fluid (from one of her swollen knees) and requested PCR analysis on all three samples. The PCR tests on the CSF and synovial fluid came back positive for the presence of Borrelia burgdorferi, the bacterium that causes Lyme disease.
Kayla’s physician immediately prescribed a full course of the antibiotic doxycycline. Fortunately, Kayla recovered fully within a few weeks and did not suffer from the long-term symptoms of post-treatment Lyme disease syndrome (PTLDS), which affects 10–20% of Lyme disease patients. To prevent future infections, Kayla’s physician advised her to use insect repellant and wear protective clothing during her outdoor adventures. These measures can limit exposure to Lyme-bearing ticks, which are common in many regions of the United States during the warmer months of the year. Kayla was also advised to make a habit of examining herself for ticks after returning from outdoor activities, as prompt removal of a tick greatly reduces the chances of infection.
Lyme disease is often difficult to diagnose. B. burgdorferi is not easily cultured in the laboratory, and the initial symptoms can be very mild and resemble those of many other diseases. But left untreated, the symptoms can become quite severe and debilitating. In addition to two antibody tests, which were inconclusive in Kayla’s case, and the PCR test, a Southern blot could be used with B. burgdorferi-specific DNA probes to identify DNA from the pathogen. Sequencing of surface protein genes of Borrelia species is also being used to identify strains within the species that may be more readily transmitted to humans or cause more severe disease.
Recombinant DNA Technology and Pharmaceutical Production
Genetic engineering has provided a way to create new pharmaceutical products called recombinant DNA pharmaceuticals. Such products include antibiotic drugs, vaccines, and hormones used to treat various diseases. Table \(1\) lists examples of recombinant DNA products and their uses.
For example, the naturally occurring antibiotic synthesis pathways of various Streptomyces spp., long known for their antibiotic production capabilities, can be modified to improve yields or to create new antibiotics through the introduction of genes encoding additional enzymes. More than 200 new antibiotics have been generated through the targeted inactivation of genes and the novel combination of antibiotic synthesis genes in antibiotic-producing Streptomyces hosts.3
Genetic engineering is also used to manufacture subunit vaccines, which are safer than other vaccines because they contain only a single antigenic molecule and lack any part of the genome of the pathogen (see Vaccines). For example, a vaccine for hepatitis B is created by inserting a gene encoding a hepatitis B surface protein into a yeast; the yeast then produces this protein, which the human immune system recognizes as an antigen. The hepatitis B antigen is purified from yeast cultures and administered to patients as a vaccine. Even though the vaccine does not contain the hepatitis B virus, the presence of the antigenic protein stimulates the immune system to produce antibodies that will protect the patient against the virus in the event of exposure.4 5
Genetic engineering has also been important in the production of other therapeutic proteins, such as insulin, interferons, and human growth hormone, to treat a variety of human medical conditions. For example, at one time, it was possible to treat diabetes only by giving patients pig insulin, which caused allergic reactions due to small differences between the proteins expressed in human and pig insulin. However, since 1978, recombinant DNA technology has been used to produce large-scale quantities of human insulin using E. coli in a relatively inexpensive process that yields a more consistently effective pharmaceutical product. Scientists have also genetically engineered E. coli capable of producing human growth hormone (HGH), which is used to treat growth disorders in children and certain other disorders in adults. The HGH gene was cloned from a cDNA library and inserted into E. coli cells by cloning it into a bacterial vector. Eventually, genetic engineering will be used to produce DNA vaccines and various gene therapies, as well as customized medicines for fighting cancer and other diseases.
Table \(1\): Some Genetically Engineered Pharmaceutical Products and Applications
Recombinant DNA Product Application
Atrial natriuretic peptide Treatment of heart disease (e.g., congestive heart failure), kidney disease, high blood pressure
DNase Treatment of viscous lung secretions in cystic fibrosis
Erythropoietin Treatment of severe anemia with kidney damage
Factor VIII Treatment of hemophilia
Hepatitis B vaccine Prevention of hepatitis B infection
Human growth hormone Treatment of growth hormone deficiency, Turner’s syndrome, burns
Human insulin Treatment of diabetes
Interferons Treatment of multiple sclerosis, various cancers (e.g., melanoma), viral infections (e.g., Hepatitis B and C)
Tetracenomycins Used as antibiotics
Tissue plasminogen activator Treatment of pulmonary embolism in ischemic stroke, myocardial infarction
Exercise \(2\)
1. What bacterium has been genetically engineered to produce human insulin for the treatment of diabetes?
2. Explain how microorganisms can be engineered to produce vaccines.
RNA Interference Technology
In Structure and Function of RNA, we described the function of mRNA, rRNA, and tRNA. In addition to these types of RNA, cells also produce several types of small noncoding RNA molecules that are involved in the regulation of gene expression. These include antisense RNA molecules, which are complementary to regions of specific mRNA molecules found in both prokaryotes and eukaryotic cells. Non-coding RNA molecules play a major role in RNA interference (RNAi), a natural regulatory mechanism by which mRNA molecules are prevented from guiding the synthesis of proteins. RNA interference of specific genes results from the base pairing of short, single-stranded antisense RNA molecules to regions within complementary mRNA molecules, preventing protein synthesis. Cells use RNA interference to protect themselves from viral invasion, which may introduce double-stranded RNA molecules as part of the viral replication process (Figure \(2\)).
Researchers are currently developing techniques to mimic the natural process of RNA interference as a way to treat viral infections in eukaryotic cells. RNA interference technology involves using small interfering RNAs (siRNAs) or microRNAs (miRNAs) (Figure \(3\)). siRNAs are completely complementary to the mRNA transcript of a specific gene of interest while miRNAs are mostly complementary. These double-stranded RNAs are bound to DICER, an endonuclease that cleaves the RNA into short molecules (approximately 20 nucleotides long). The RNAs are then bound to RNA-induced silencing complex (RISC), a ribonucleoprotein. The siRNA-RISC complex binds to mRNA and cleaves it. For miRNA, only one of the two strands binds to RISC. The miRNA-RISC complex then binds to mRNA, inhibiting translation. If the miRNA is completely complementary to the target gene, then the mRNA can be cleaved. Taken together, these mechanisms are known as gene silencing.
Key Concepts and Summary
• The science of genomics allows researchers to study organisms on a holistic level and has many applications of medical relevance.
• Transcriptomics and proteomics allow researchers to compare gene expression patterns between different cells and shows great promise in better understanding global responses to various conditions.
• The various –omics technologies complement each other and together provide a more complete picture of an organism’s or microbial community’s (metagenomics) state.
• The analysis required for large data sets produced through genomics, transcriptomics, and proteomics has led to the emergence of bioinformatics.
• Reporter genes encoding easily observable characteristics are commonly used to track gene expression patterns of genes of unknown function.
• The use of recombinant DNA technology has revolutionized the pharmaceutical industry, allowing for the rapid production of high-quality recombinant DNA pharmaceuticals used to treat a wide variety of human conditions.
• RNA interference technology has great promise as a method of treating viral infections by silencing the expression of specific genes.
Footnotes
1. 1 E.O. List, D.E. Berryman, B. Bower, L. Sackmann-Sala, E. Gosney, J. Ding, S. Okada, and J.J. Kopchick. “The Use of Proteomics to Study Infectious Diseases.” Infectious Disorders-Drug Targets (Formerly Current Drug Targets-Infectious Disorders) 8 no. 1 (2008): 31–45.
2. 2 Mohan Natesan, and Robert G. Ulrich. “Protein Microarrays and Biomarkers of Infectious Disease.” International Journal of Molecular Sciences 11 no. 12 (2010): 5165–5183.
3. 3 Jose-Luis Adrio and Arnold L. Demain. “Recombinant Organisms for Production of Industrial Products.” Bioengineered Bugs 1 no. 2 (2010): 116–131.
4. 4 U.S. Department of Health and Human Services. “Types of Vaccines.” 2013. www.vaccines.gov/more_info/types/#subunit. Accessed May 27, 2016.
5. 5 The Internet Drug List. Recombivax. 2015. http://www.rxlist.com/recombivax-drug.htm. Accessed May 27, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/12%3A_Modern_Applications_of_Microbial_Genetics/12.03%3A_Whole_Genome_Methods_and_Industrial_Applications.txt |
Learning Objectives
• Summarize the mechanisms, risks, and potential benefits of gene therapy
• Identify ethical issues involving gene therapy and the regulatory agencies that provide oversight for clinical trials
• Compare somatic-cell and germ-line gene therapy
Many types of genetic engineering have yielded clear benefits with few apparent risks. Few would question, for example, the value of our now abundant supply of human insulin produced by genetically engineered bacteria. However, many emerging applications of genetic engineering are much more controversial, often because their potential benefits are pitted against significant risks, real or perceived. This is certainly the case for gene therapy, a clinical application of genetic engineering that may one day provide a cure for many diseases but is still largely an experimental approach to treatment.
Mechanisms and Risks of Gene Therapy
Human diseases that result from genetic mutations are often difficult to treat with drugs or other traditional forms of therapy because the signs and symptoms of disease result from abnormalities in a patient’s genome. For example, a patient may have a genetic mutation that prevents the expression of a specific protein required for the normal function of a particular cell type. This is the case in patients with Severe Combined Immunodeficiency (SCID), a genetic disease that impairs the function of certain white blood cells essential to the immune system.
Gene therapy attempts to correct genetic abnormalities by introducing a nonmutated, functional gene into the patient’s genome. The nonmutated gene encodes a functional protein that the patient would otherwise be unable to produce. Viral vectors such as adenovirus are sometimes used to introduce the functional gene; part of the viral genome is removed and replaced with the desired gene (Figure \(1\)). More advanced forms of gene therapy attempt to correct the mutation at the original site in the genome, such as is the case with treatment of SCID.
So far, gene therapies have proven relatively ineffective, with the possible exceptions of treatments for cystic fibrosisand adenosine deaminase deficiency, a type of SCID. Other trials have shown the clear hazards of attempting genetic manipulation in complex multicellular organisms like humans. In some patients, the use of an adenovirus vector can trigger an unanticipated inflammatory response from the immune system, which may lead to organ failure. Moreover, because viruses can often target multiple cell types, the virus vector may infect cells not targeted for the therapy, damaging these other cells and possibly leading to illnesses such as cancer. Another potential risk is that the modified virus could revert to being infectious and cause disease in the patient. Lastly, there is a risk that the inserted gene could unintentionally inactivate another important gene in the patient’s genome, disrupting normal cell cycling and possibly leading to tumor formation and cancer. Because gene therapy involves so many risks, candidates for gene therapy need to be fully informed of these risks before providing informed consent to undergo the therapy.
Gene Therapy Gone Wrong
The risks of gene therapy were realized in the 1999 case of Jesse Gelsinger, an 18-year-old patient who received gene therapy as part of a clinical trial at the University of Pennsylvania. Jesse received gene therapy for a condition called ornithine transcarbamylase (OTC) deficiency, which leads to ammonia accumulation in the blood due to deficient ammonia processing. Four days after the treatment, Jesse died after a massive immune response to the adenovirus vector.1
Until that point, researchers had not really considered an immune response to the vector to be a legitimate risk, but on investigation, it appears that the researchers had some evidence suggesting that this was a possible outcome. Prior to Jesse’s treatment, several other human patients had suffered side effects of the treatment, and three monkeys used in a trial had died as a result of inflammation and clotting disorders. Despite this information, it appears that neither Jesse nor his family were made aware of these outcomes when they consented to the therapy. Jesse’s death was the first patient death due to a gene therapy treatment and resulted in the immediate halting of the clinical trial in which he was involved, the subsequent halting of all other gene therapy trials at the University of Pennsylvania, and the investigation of all other gene therapy trials in the United States. As a result, the regulation and oversight of gene therapy overall was reexamined, resulting in new regulatory protocols that are still in place today.
Exercise \(1\)
1. Explain how gene therapy works in theory.
2. Identify some risks of gene therapy.
Oversight of Gene Therapy
Presently, there is significant oversight of gene therapy clinical trials. At the federal level, three agencies regulate gene therapy in parallel: the Food and Drug Administration (FDA), the Office of Human Research Protection (OHRP), and the Recombinant DNA Advisory Committee (RAC) at the National Institutes of Health (NIH). Along with several local agencies, these federal agencies interact with the institutional review board to ensure that protocols are in place to protect patient safety during clinical trials. Compliance with these protocols is enforced mostly on the local level in cooperation with the federal agencies. Gene therapies are currently under the most extensive federal and local review compared to other types of therapies, which are more typically only under the review of the FDA. Some researchers believe that these extensive regulations actually inhibit progress in gene therapy research. In 2013, the Institute of Medicine (now the National Academy of Medicine) called upon the NIH to relax its review of gene therapy trials in most cases.2 However, ensuring patient safety continues to be of utmost concern.
Ethical Concerns
Beyond the health risks of gene therapy, the ability to genetically modify humans poses a number of ethical issues related to the limits of such “therapy.” While current research is focused on gene therapy for genetic diseases, scientists might one day apply these methods to manipulate other genetic traits not perceived as desirable. This raises questions such as:
Exercise \(2\)
1. Which genetic traits are worthy of being “corrected”?
2. Should gene therapy be used for cosmetic reasons or to enhance human abilities?
3. Should genetic manipulation be used to impart desirable traits to the unborn?
4. Is everyone entitled to gene therapy, or could the cost of gene therapy create new forms of social inequality?
5. Who should be responsible for regulating and policing inappropriate use of gene therapies?
The ability to alter reproductive cells using gene therapy could also generate new ethical dilemmas. To date, the various types of gene therapies have been targeted to somatic cells, the non-reproductive cells within the body. Because somatic cell traits are not inherited, any genetic changes accomplished by somatic-cell gene therapy would not be passed on to offspring. However, should scientists successfully introduce new genes to germ cells (eggs or sperm), the resulting traits could be passed on to offspring. This approach, called germ-line gene therapy, could potentially be used to combat heritable diseases, but it could also lead to unintended consequences for future generations. Moreover, there is the question of informed consent, because those impacted by germ-line gene therapy are unborn and therefore unable to choose whether they receive the therapy. For these reasons, the U.S. government does not currently fund research projects investigating germ-line gene therapies in humans.
Risky Gene Therapies
While there are currently no gene therapies on the market in the United States, many are in the pipeline and it is likely that some will eventually be approved. With recent advances in gene therapies targeting p53, a gene whose somatic cell mutations have been implicated in over 50% of human cancers,3 cancer treatments through gene therapies could become much more widespread once they reach the commercial market.
Bringing any new therapy to market poses ethical questions that pit the expected benefits against the risks. How quickly should new therapies be brought to the market? How can we ensure that new therapies have been sufficiently tested for safety and effectiveness before they are marketed to the public? The process by which new therapies are developed and approved complicates such questions, as those involved in the approval process are often under significant pressure to get a new therapy approved even in the face of significant risks.
To receive FDA approval for a new therapy, researchers must collect significant laboratory data from animal trials and submit an Investigational New Drug (IND) application to the FDA’s Center for Drug Evaluation and Research (CDER). Following a 30-day waiting period during which the FDA reviews the IND, clinical trials involving human subjects may begin. If the FDA perceives a problem prior to or during the clinical trial, the FDA can order a “clinical hold” until any problems are addressed. During clinical trials, researchers collect and analyze data on the therapy’s effectiveness and safety, including any side effects observed. Once the therapy meets FDA standards for effectiveness and safety, the developers can submit a New Drug Application (NDA) that details how the therapy will be manufactured, packaged, monitored, and administered.
Because new gene therapies are frequently the result of many years (even decades) of laboratory and clinical research, they require a significant financial investment. By the time a therapy has reached the clinical trials stage, the financial stakes are high for pharmaceutical companies and their shareholders. This creates potential conflicts of interest that can sometimes affect the objective judgment of researchers, their funders, and even trial participants. The Jesse Gelsinger case (see Case in Point: Gene Therapy Gone Wrong) is a classic example. Faced with a life-threatening disease and no reasonable treatments available, it is easy to see why a patient might be eager to participate in a clinical trial no matter the risks. It is also easy to see how a researcher might view the short-term risks for a small group of study participants as a small price to pay for the potential benefits of a game-changing new treatment.
Gelsinger’s death led to increased scrutiny of gene therapy, and subsequent negative outcomes of gene therapy have resulted in the temporary halting of clinical trials pending further investigation. For example, when children in France treated with gene therapy for SCID began to develop leukemia several years after treatment, the FDA temporarily stopped clinical trials of similar types of gene therapy occurring in the United States.4 Cases like these highlight the need for researchers and health professionals not only to value human well-being and patients’ rights over profitability, but also to maintain scientific objectivity when evaluating the risks and benefits of new therapies.
Exercise \(3\)
1. Why is gene therapy research so tightly regulated?
2. What is the main ethical concern associated with germ-line gene therapy?
Key Concepts and Summary
• While gene therapy shows great promise for the treatment of genetic diseases, there are also significant risks involved.
• There is considerable federal and local regulation of the development of gene therapies by pharmaceutical companies for use in humans.
• Before gene therapy use can increase dramatically, there are many ethical issues that need to be addressed by the medical and research communities, politicians, and society at large.
Footnotes
1. 1 Barbara Sibbald. “Death but One Unintended Consequence of Gene-Therapy Trial.” Canadian Medical Association Journal 164 no. 11 (2001): 1612–1612.
2. 2 Kerry Grens. “Report: Ease Gene Therapy Reviews.” The Scientist, December 9, 2013. http://www.the-scientist.com/?articl...erapy-Reviews/. Accessed May 27, 2016.
3. 3 Zhen Wang and Yi Sun. “Targeting p53 for Novel Anticancer Therapy.” Translational Oncology 3, no. 1 (2010): 1–12.
4. 4 Erika Check. “Gene Therapy: A Tragic Setback.” Nature 420 no. 6912 (2002): 116–118. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/12%3A_Modern_Applications_of_Microbial_Genetics/12.04%3A_Genetic_Engineering_-_Risks_Benefits_and_Perceptions.txt |
12.1: Microbes and the Tools of Genetic Engineering
The science of using living systems to benefit humankind is called biotechnology. Technically speaking, the domestication of plants and animals through farming and breeding practices is a type of biotechnology. However, in a contemporary sense, we associate biotechnology with the direct alteration of an organism’s genetics to achieve desirable traits through the process of genetic engineering.
Multiple Choice
Which of the following is required for repairing the phosphodiester backbone of DNA during molecular cloning?
1. cDNA
2. reverse transcriptase
3. restriction enzymes
4. DNA ligase
Answer
D
All of the following are processes used to introduce DNA molecules into bacterial cells except:
1. transformation
2. transduction
3. transcription
4. conjugation
Answer
C
The enzyme that uses RNA as a template to produce a DNA copy is called:
1. a restriction enzyme
2. DNA ligase
3. reverse transcriptase
4. DNA polymerase
Answer
C
In blue-white screening, what do blue colonies represent?
1. cells that have not taken up the plasmid vector
2. cells with recombinant plasmids containing a new insert
3. cells containing empty plasmid vectors
4. cells with a non-functional lacZ gene
Answer
C
The Ti plasmid is used for introducing genes into:
1. animal cells
2. plant cells
3. bacteriophages
4. E. coli cells
Answer
B
True/False
Recombination is a process not usually observed in nature.
Answer
false
It is generally easier to introduce recombinant DNA into prokaryotic cells than into eukaryotic cells.
Answer
true
Fill in the Blank
The process of introducing DNA molecules into eukaryotic cells is called ________.
Answer
transfection
Short answer
Name three elements incorporated into a plasmid vector for efficient cloning.
When would a scientist want to generate a cDNA library instead of a genomic library?
What is one advantage of generating a genomic library using phages instead of plasmids?
Critical Thinking
Is biotechnology always associated with genetic engineering? Explain your answer.
Which is more efficient: blunt-end cloning or sticky-end cloning? Why?
12.2: Visualizing and Characterizing DNA
Finding a gene of interest within a sample requires the use of a single-stranded DNA probe labeled with a molecular beacon (typically radioactivity or fluorescence) that can hybridize with a complementary single-stranded nucleic acid in the sample. Agarose gel electrophoresis allows for the separation of DNA molecules based on size. Restriction fragment length polymorphism (RFLP) analysis allows for the visualization by agarose gel electrophoresis of distinct variants of a DNA sequence.
Multiple Choice
Which technique is used to separate protein fragments based on size?
1. polyacrylamide gel electrophoresis
2. Southern blot
3. agarose gel electrophoresis
4. polymerase chain reaction
Answer
A
Which technique uses restriction enzyme digestion followed by agarose gel electrophoresis to generate a banding pattern for comparison to another sample processed in the same way?
1. qPCR
2. RT-PCR
3. RFLP
4. 454 sequencing
Answer
C
All of the following techniques involve hybridization between single-stranded nucleic acid molecules except:
1. Southern blot analysis
2. RFLP analysis
3. northern blot analysis
4. microarray analysis
Answer
B
Fill in the Blank
The __________ blot technique is used to find an RNA fragment within a sample that is complementary to a DNA probe.
Answer
northern
The PCR step during which the double-stranded template molecule becomes single-stranded is called _____________.
Answer
denaturation
The sequencing method involving the incorporation of ddNTPs is called __________.
Answer
Sanger sequencing, dideoxy method, or chain termination method
True/False
In agarose gel electrophoresis, DNA will be attracted to the negative electrode.
Answer
false
Short answer
Why is it important that a DNA probe be labeled with a molecular beacon?
When separating proteins strictly by size, why is exposure to SDS first required?
Why must the DNA polymerase used during PCR be heat-stable?
Critical Thinking
Suppose you are working in a molecular biology laboratory and are having difficulty performing the PCR successfully. You decide to double-check the PCR protocol programmed into the thermal cycler and discover that the annealing temperature was programmed to be 65 °C instead of 50 °C, as you had intended. What effects would this mistake have on the PCR reaction? Refer to Figure 12.2.8.
What is the advantage of microarray analysis over northern blot analysis in monitoring changes in gene expression?
What is the difference between reverse transcriptase PCR (RT-PCR) and real-time quantitative PCR (qPCR)?
12.3: Whole Genome Methods and Industrial Applications
Advances in molecular biology have led to the creation of entirely new fields of science. Among these are fields that study aspects of whole genomes, collectively referred to as whole-genome methods. In this section, we provide a brief overview of the whole-genome fields of genomics, transcriptomics, and proteomics.
Multiple Choice
The science of studying the entire collection of mRNA molecules produced by cells, allowing scientists to monitor differences in gene expression patterns between cells, is called:
1. genomics
2. transcriptomics
3. proteomics
4. pharmacogenomics
Answer
B
The science of studying genomic fragments from microbial communities, allowing researchers to study genes from a collection of multiple species, is called:
1. pharmacogenomics
2. transcriptomics
3. metagenomics
4. proteomics
Answer
C
The insulin produced by recombinant DNA technology is
1. a combination of E. coli and human insulin.
2. identical to human insulin produced in the pancreas.
3. cheaper but less effective than pig insulin for treating diabetes.
4. engineered to be more effective than human insulin.
Answer
B
Fill in the Blank
The application of genomics to evaluate the effectiveness and safety of drugs on the basis of information from an individual’s genomic sequence is called ____________.
Answer
pharmacogenomics or toxicogenomics
A gene whose expression can be easily visualized and monitored is called a ________.
Answer
reporter gene
True/False
RNA interference does not influence the sequence of genomic DNA.
Answer
true
Short answer
If all cellular proteins are encoded by the cell’s genes, what information does proteomics provide that genomics cannot?
Critical Thinking
What are some advantages of cloning human genes into bacteria to treat human diseases caused by specific protein deficiencies?
12.4: Genetic Engineering - Risks, Benefits, and Perceptions
Many types of genetic engineering have yielded clear benefits with few apparent risks. However, many emerging applications of genetic engineering are much more controversial, often because their potential benefits are pitted against significant risks, real or perceived. This is certainly the case for gene therapy, a clinical application of genetic engineering that may one day provide a cure for many diseases but is still largely an experimental approach to treatment.
Multiple Choice
At what point can the FDA halt the development or use of gene therapy?
1. on submission of an IND application
2. during clinical trials
3. after manufacturing and marketing of the approved therapy
4. all of the answers are correct
Answer
D
Fill in the Blank
_____________ is a common viral vector used in gene therapy for introducing a new gene into a specifically targeted cell type.
Answer
Adenovirus
Short Answer
Briefly describe the risks associated with somatic cell gene therapy.
Critical Thinking
Compare the ethical issues involved in the use of somatic cell gene therapy and germ-line gene therapy. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/12%3A_Modern_Applications_of_Microbial_Genetics/12.E%3A_Modern_Applications_of_Microbial_Genetics_%28Exercises%29.txt |
How clean is clean? People wash their cars and vacuum the carpets, but most would not want to eat from these surfaces. Similarly, we might eat with silverware cleaned in a dishwasher, but we could not use the same dishwasher to clean surgical instruments. As these examples illustrate, “clean” is a relative term. Car washing, vacuuming, and dishwashing all reduce the microbial load on the items treated, thus making them “cleaner.” But whether they are “clean enough” depends on their intended use. Because people do not normally eat from cars or carpets, these items do not require the same level of cleanliness that silverware does. Likewise, because silverware is not used for invasive surgery, these utensils do not require the same level of cleanliness as surgical equipment, which requires sterilization to prevent infection.
Why not play it safe and sterilize everything? Sterilizing everything we come in contact with is impractical, as well as potentially dangerous. As this chapter will demonstrate, sterilization protocols often require time- and labor-intensive treatments that may degrade the quality of the item being treated or have toxic effects on users. Therefore, the user must consider the item’s intended application when choosing a cleaning method to ensure that it is “clean enough.”
• 13.1: Controlling Microbial Growth
Inanimate items, such as doorknobs, toys, or towels, which may harbor microbes and aid in disease transmission, are called fomites. Two factors heavily influence the level of cleanliness required for a particular fomite and, hence, the protocol chosen to achieve this level. The first factor is the application for which the item will be used and the second factor is the level of resistance to antimicrobial treatment by potential pathogens.
• 13.2: Using Physical Methods to Control Microorganisms
For thousands of years, humans have used various physical methods of microbial control for food preservation. Common control methods include the application of high temperatures, radiation, filtration, and desiccation (drying), among others. Many of these methods nonspecifically kill cells by disrupting membranes, changing membrane permeability, or damaging proteins and nucleic acids by denaturation, degradation, or chemical modification.
• 13.3: Using Chemicals to Control Microorganisms
In addition to physical methods of microbial control, chemicals are also used to control microbial growth. A wide variety of chemicals can be used as disinfectants or antiseptics. This section describes the variety of chemicals used as disinfectants and antiseptics, including their mechanisms of action and common uses.
• 13.4: Testing the Effectiveness of Antiseptics and Disinfectants
Several environmental conditions influence the potency of an antimicrobial agent and its effectiveness. For example, length of exposure is particularly important, with longer exposure increasing efficacy. Similarly, the concentration of the chemical agent is also important, with higher concentrations being more effective than lower ones. Temperature, pH, and other factors can also affect the potency of a disinfecting agent.
• 13.E: Control of Microbial Growth (Exercises)
Footnotes
1. 1 R.E. Stephenson et al. “Elucidation of Bacteria Found in Car Interiors and Strategies to Reduce the Presence of Potential Pathogens.” Biofouling 30 no. 3 (2014):337–346.
Thumbnail: Scanning electron microscope image of Vibrio cholerae bacteria, which infect the digestive system. (Public Domain; T.J. Kirn, M.J. Lafferty, C.M.P Sandoe and R.K. Taylor).
13: Control of Microbial Growth
Learning Objectives
• Compare disinfectants, antiseptics, and sterilants
• Describe the principles of controlling the presence of microorganisms through sterilization and disinfection
• Differentiate between microorganisms of various biological safety levels and explain methods used for handling microbes at each level
Clinical Focus: Part 1
Roberta is a 46-year-old real estate agent who recently underwent a cholecystectomy (surgery to remove painful gallstones). The surgery was performed laparoscopically with the aid of a duodenoscope, a specialized endoscope that allows surgeons to see inside the body with the aid of a tiny camera. On returning home from the hospital, Roberta developed abdominal pain and a high fever. She also experienced a burning sensation during urination and noticed blood in her urine. She notified her surgeon of these symptoms, per her postoperative instructions.
Exercise \(1\)
What are some possible causes of Roberta’s symptoms?
To prevent the spread of human disease, it is necessary to control the growth and abundance of microbes in or on various items frequently used by humans. Inanimate items, such as doorknobs, toys, or towels, which may harbor microbes and aid in disease transmission, are called fomites. Two factors heavily influence the level of cleanliness required for a particular fomite and, hence, the protocol chosen to achieve this level. The first factor is the application for which the item will be used. For example, invasive applications that require insertion into the human body require a much higher level of cleanliness than applications that do not. The second factor is the level of resistance to antimicrobial treatment by potential pathogens. For example, foods preserved by canning often become contaminated with the bacterium Clostridium botulinum, which produces the neurotoxin that causes botulism. Because C. botulinum can produce endospores that can survive harsh conditions, extreme temperatures and pressures must be used to eliminate the endospores. Other organisms may not require such extreme measures and can be controlled by a procedure such as washing clothes in a laundry machine.
Laboratory Biological Safety Levels
For researchers or laboratory personnel working with pathogens, the risks associated with specific pathogens determine the levels of cleanliness and control required. The Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH) have established four classification levels, called “biological safety levels” (BSLs). Various organizations around the world, including the World Health Organization (WHO) and the European Union (EU), use a similar classification scheme. According to the CDC, the BSL is determined by the agent’s infectivity, ease of transmission, and potential disease severity, as well as the type of work being done with the agent.1
Each BSL requires a different level of biocontainment to prevent contamination and spread of infectious agents to laboratory personnel and, ultimately, the community. For example, the lowest BSL, BSL-1, requires the fewest precautions because it applies to situations with the lowest risk for microbial infection.
BSL-1 agents are those that generally do not cause infection in healthy human adults. These include noninfectious bacteria, such as nonpathogenic strains of Escherichia coli and Bacillus subtilis, and viruses known to infect animals other than humans, such as baculoviruses (insect viruses). Because working with BSL-1 agents poses very little risk, few precautions are necessary. Laboratory workers use standard aseptic technique and may work with these agents at an open laboratory bench or table, wearing personal protective equipment (PPE) such as a laboratory coat, goggles, and gloves, as needed. Other than a sink for handwashing and doors to separate the laboratory from the rest of the building, no additional modifications are needed.
Agents classified as BSL-2 include those that pose moderate risk to laboratory workers and the community, and are typically “indigenous,” meaning that they are commonly found in that geographical area. These include bacteria such as Staphylococcus aureus and Salmonella spp., and viruses like hepatitis, mumps, and measles viruses. BSL-2 laboratories require additional precautions beyond those of BSL-1, including restricted access; required PPE, including a face shield in some circumstances; and the use of biological safety cabinets for procedures that may disperse agents through the air (called “aerosolization”). BSL-2 laboratories are equipped with self-closing doors, an eyewash station, and an autoclave, which is a specialized device for sterilizing materials with pressurized steam before use or disposal. BSL-1 laboratories may also have an autoclave.
BSL-3 agents have the potential to cause lethal infections by inhalation. These may be either indigenous or “exotic,” meaning that they are derived from a foreign location, and include pathogens such as Mycobacterium tuberculosis, Bacillus anthracis, West Nile virus, and human immunodeficiency virus (HIV). Because of the serious nature of the infections caused by BSL-3 agents, laboratories working with them require restricted access. Laboratory workers are under medical surveillance, possibly receiving vaccinations for the microbes with which they work. In addition to the standard PPE already mentioned, laboratory personnel in BSL-3 laboratories must also wear a respirator and work with microbes and infectious agents in a biological safety cabinet at all times. BSL-3 laboratories require a hands-free sink, an eyewash station near the exit, and two sets of self-closing and locking doors at the entrance. These laboratories are equipped with directional airflow, meaning that clean air is pulled through the laboratory from clean areas to potentially contaminated areas. This air cannot be recirculated, so a constant supply of clean air is required.
BSL-4 agents are the most dangerous and often fatal. These microbes are typically exotic, are easily transmitted by inhalation, and cause infections for which there are no treatments or vaccinations. Examples include Ebola virus and Marburg virus, both of which cause hemorrhagic fevers, and smallpox virus. There are only a small number of laboratories in the United States and around the world appropriately equipped to work with these agents. In addition to BSL-3 precautions, laboratory workers in BSL-4 facilities must also change their clothing on entering the laboratory, shower on exiting, and decontaminate all material on exiting. While working in the laboratory, they must either wear a full-body protective suit with a designated air supply or conduct all work within a biological safety cabinet with a high-efficiency particulate air (HEPA)-filtered air supply and a doubly HEPA-filtered exhaust. If wearing a suit, the air pressure within the suit must be higher than that outside the suit, so that if a leak in the suit occurs, laboratory air that may be contaminated cannot be drawn into the suit (Figure \(1\)). The laboratory itself must be located either in a separate building or in an isolated portion of a building and have its own air supply and exhaust system, as well as its own decontamination system. The BSLs are summarized in Figure \(2\).
Link to Learning
To learn more about the four BSLs, visit the CDC’s website.
Exercise \(2\)
What are some factors used to determine the BSL necessary for working with a specific pathogen?
Sterilization
The most extreme protocols for microbial control aim to achieve sterilization: the complete removal or killing of all vegetative cells, endospores, and viruses from the targeted item or environment. Sterilization protocols are generally reserved for laboratory, medical, manufacturing, and food industry settings, where it may be imperative for certain items to be completely free of potentially infectious agents. Sterilization can be accomplished through either physical means, such as exposure to high heat, pressure, or filtration through an appropriate filter, or by chemical means. Chemicals that can be used to achieve sterilization are called sterilants. Sterilants effectively kill all microbes and viruses, and, with appropriate exposure time, can also kill endospores.
For many clinical purposes, aseptic technique is necessary to prevent contamination of sterile surfaces. Aseptic technique involves a combination of protocols that collectively maintain sterility, or asepsis, thus preventing contamination of the patient with microbes and infectious agents. Failure to practice aseptic technique during many types of clinical procedures may introduce microbes to the patient’s body and put the patient at risk for sepsis, a systemic inflammatory response to an infection that results in high fever, increased heart and respiratory rates, shock, and, possibly, death. Medical procedures that carry risk of contamination must be performed in a sterile field, a designated area that is kept free of all vegetative microbes, endospores, and viruses. Sterile fields are created according to protocols requiring the use of sterilized materials, such as packaging and drapings, and strict procedures for washing and application of sterilants. Other protocols are followed to maintain the sterile field while the medical procedure is being performed.
One food sterilization protocol, commercial sterilization, uses heat at a temperature low enough to preserve food quality but high enough to destroy common pathogens responsible for food poisoning, such as C. botulinum. Because C. botulinum and its endospores are commonly found in soil, they may easily contaminate crops during harvesting, and these endospores can later germinate within the anaerobic environment once foods are canned. Metal cans of food contaminated with C. botulinum will bulge due to the microbe’s production of gases; contaminated jars of food typically bulge at the metal lid. To eliminate the risk for C. botulinum contamination, commercial food-canning protocols are designed with a large margin of error. They assume an impossibly large population of endospores (1012 per can) and aim to reduce this population to 1 endospore per can to ensure the safety of canned foods. For example, low- and medium-acid foods are heated to 121 °C for a minimum of 2.52 minutes, which is the time it would take to reduce a population of 1012 endospores per can down to 1 endospore at this temperature. Even so, commercial sterilization does not eliminate the presence of all microbes; rather, it targets those pathogens that cause spoilage and foodborne diseases, while allowing many nonpathogenic organisms to survive. Therefore, “sterilization” is somewhat of a misnomer in this context, and commercial sterilization may be more accurately described as “quasi-sterilization.”
Exercise \(3\)
What is the difference between sterilization and aseptic technique?
Link to Learning
The Association of Surgical Technologists publishes standards for aseptic technique, including creating and maintaining a sterile field.
Other Methods of Control
Sterilization protocols require procedures that are not practical, or necessary, in many settings. Various other methods are used in clinical and nonclinical settings to reduce the microbial load on items. Although the terms for these methods are often used interchangeably, there are important distinctions (Figure \(3\)).
The process of disinfection inactivates most microbes on the surface of a fomite by using antimicrobial chemicals or heat. Because some microbes remain, the disinfected item is not considered sterile. Ideally, disinfectants should be fast acting, stable, easy to prepare, inexpensive, and easy to use. An example of a natural disinfectant is vinegar; its acidity kills most microbes. Chemical disinfectants, such as chlorine bleach or products containing chlorine, are used to clean nonliving surfaces such as laboratory benches, clinical surfaces, and bathroom sinks. Typical disinfection does not lead to sterilization because endospores tend to survive even when all vegetative cells have been killed.
Unlike disinfectants, antiseptics are antimicrobial chemicals safe for use on living skin or tissues. Examples of antiseptics include hydrogen peroxide and isopropyl alcohol. The process of applying an antiseptic is called antisepsis. In addition to the characteristics of a good disinfectant, antiseptics must also be selectively effective against microorganisms and able to penetrate tissue deeply without causing tissue damage.
The type of protocol required to achieve the desired level of cleanliness depends on the particular item to be cleaned. For example, those used clinically are categorized as critical, semicritical, and noncritical. Critical items must be sterile because they will be used inside the body, often penetrating sterile tissues or the bloodstream; examples of critical items include surgical instruments, catheters, and intravenous fluids. Gastrointestinal endoscopes and various types of equipment for respiratory therapies are examples of semicritical items; they may contact mucous membranes or nonintact skin but do not penetrate tissues. Semicritical items do not typically need to be sterilized but do require a high level of disinfection. Items that may contact but not penetrate intact skin are noncritical items; examples are bed linens, furniture, crutches, stethoscopes, and blood pressure cuffs. These articles need to be clean but not highly disinfected.
The act of handwashing is an example of degerming, in which microbial numbers are significantly reduced by gently scrubbing living tissue, most commonly skin, with a mild chemical (e.g., soap) to avoid the transmission of pathogenic microbes. Wiping the skin with an alcohol swab at an injection site is another example of degerming. These degerming methods remove most (but not all) microbes from the skin’s surface.
The term sanitization refers to the cleansing of fomites to remove enough microbes to achieve levels deemed safe for public health. For example, commercial dishwashers used in the food service industry typically use very hot water and air for washing and drying; the high temperatures kill most microbes, sanitizing the dishes. Surfaces in hospital rooms are commonly sanitized using a chemical disinfectant to prevent disease transmission between patients. Figure \(3\) summarizes common protocols, definitions, applications, and agents used to control microbial growth.
Exercise \(4\)
1. What is the difference between a disinfectant and an antiseptic?
2. Which is most effective at removing microbes from a product: sanitization, degerming, or sterilization? Explain.
Clinical Focus: Part 2
Roberta’s physician suspected that a bacterial infection was responsible for her sudden-onset high fever, abdominal pain, and bloody urine. Based on these symptoms, the physician diagnosed a urinary tract infection (UTI). A wide variety of bacteria may cause UTIs, which typically occur when bacteria from the lower gastrointestinal tract are introduced to the urinary tract. However, Roberta’s recent gallstone surgery caused the physician to suspect that she had contracted a nosocomial (hospital-acquired) infection during her surgery. The physician took a urine sample and ordered a urine culture to check for the presence of white blood cells, red blood cells, and bacteria. The results of this test would help determine the cause of the infection. The physician also prescribed a course of the antibiotic ciprofloxacin, confident that it would clear Roberta’s infection.
Exercise \(5\)
What are some possible ways that bacteria could have been introduced to Roberta’s urinary tract during her surgery?
Measuring Microbial Control
Physical and chemical methods of microbial control that kill the targeted microorganism are identified by the suffix -cide (or -cidal). The prefix indicates the type of microbe or infectious agent killed by the treatment method: bactericides kill bacteria, viricides kill or inactivate viruses, and fungicides kill fungi. Other methods do not kill organisms but, instead, stop their growth, making their population static; such methods are identified by the suffix -stat (or -static). For example, bacteriostatic treatments inhibit the growth of bacteria, whereas fungistatic treatments inhibit the growth of fungi. Factors that determine whether a particular treatment is -cidal or -static include the types of microorganisms targeted, the concentration of the chemical used, and the nature of the treatment applied.
Although -static treatments do not actually kill infectious agents, they are often less toxic to humans and other animals, and may also better preserve the integrity of the item treated. Such treatments are typically sufficient to keep the microbial population of an item in check. The reduced toxicity of some of these -static chemicals also allows them to be impregnated safely into plastics to prevent the growth of microbes on these surfaces. Such plastics are used in products such as toys for children and cutting boards for food preparation. When used to treat an infection, -static treatments are typically sufficient in an otherwise healthy individual, preventing the pathogen from multiplying, thus allowing the individual’s immune system to clear the infection.
The degree of microbial control can be evaluated using a microbial death curve to describe the progress and effectiveness of a particular protocol. When exposed to a particular microbial control protocol, a fixed percentage of the microbes within the population will die. Because the rate of killing remains constant even when the population size varies, the percentage killed is more useful information than the absolute number of microbes killed. Death curves are often plotted as semilog plots just like microbial growth curves because the reduction in microorganisms is typically logarithmic (Figure \(4\)). The amount of time it takes for a specific protocol to produce a one order-of-magnitude decrease in the number of organisms, or the death of 90% of the population, is called the decimal reduction time (DRT) or D-value.
Several factors contribute to the effectiveness of a disinfecting agent or microbial control protocol. First, as demonstrated in Figure \(4\), the length of time of exposure is important. Longer exposure times kill more microbes. Because microbial death of a population exposed to a specific protocol is logarithmic, it takes longer to kill a high-population load than a low-population load exposed to the same protocol. A shorter treatment time (measured in multiples of the D-value) is needed when starting with a smaller number of organisms. Effectiveness also depends on the susceptibility of the agent to that disinfecting agent or protocol. The concentration of disinfecting agent or intensity of exposure is also important. For example, higher temperatures and higher concentrations of disinfectants kill microbes more quickly and effectively. Conditions that limit contact between the agent and the targeted cells cells—for example, the presence of bodily fluids, tissue, organic debris (e.g., mud or feces), or biofilms on surfaces—increase the cleaning time or intensity of the microbial control protocol required to reach the desired level of cleanliness. All these factors must be considered when choosing the appropriate protocol to control microbial growth in a given situation.
Exercise \(6\)
1. What are two possible reasons for choosing a bacteriostatic treatment over a bactericidal one?
2. Name at least two factors that can compromise the effectiveness of a disinfecting agent.
Key Concepts and Summary
• Inanimate items that may harbor microbes and aid in their transmission are called fomites. The level of cleanliness required for a fomite depends both on the item’s use and the infectious agent with which the item may be contaminated.
• The CDC and the NIH have established four biological safety levels (BSLs) for laboratories performing research on infectious agents. Each level is designed to protect laboratory personnel and the community. These BSLs are determined by the agent’s infectivity, ease of transmission, and potential disease severity, as well as the type of work being performed with the agent.
• Disinfection removes potential pathogens from a fomite, whereas antisepsis uses antimicrobial chemicals safe enough for tissues; in both cases, microbial load is reduced, but microbes may remain unless the chemical used is strong enough to be a sterilant.
• The amount of cleanliness (sterilization versus high-level disinfection versus general cleanliness) required for items used clinically depends on whether the item will come into contact with sterile tissues (critical item), mucous membranes (semicritical item), or intact skin (noncritical item).
• Medical procedures with a risk for contamination should be carried out in a sterile field maintained by proper aseptic technique to prevent sepsis.
• Sterilization is necessary for some medical applications as well as in the food industry, where endospores of Clostridium botulinum are killed through commercial sterilization protocols.
• Physical or chemical methods to control microbial growth that result in death of the microbe are indicated by the suffixes -cide or -cidal (e.g., as with bactericides, viricides, and fungicides), whereas those that inhibit microbial growth are indicated by the suffixes -stat or-static (e.g., bacteriostatic, fungistatic).
• Microbial death curves display the logarithmic decline of living microbes exposed to a method of microbial control. The time it takes for a protocol to yield a 1-log (90%) reduction in the microbial population is the decimal reduction time, or D-value.
• When choosing a microbial control protocol, factors to consider include the length of exposure time, the type of microbe targeted, its susceptibility to the protocol, the intensity of the treatment, the presence of organics that may interfere with the protocol, and the environmental conditions that may alter the effectiveness of the protocol.
Footnotes
1. 1 US Centers for Disease Control and Prevention. “Recognizing the Biosafety Levels.” http://www.cdc.gov/training/quicklearns/biosafety/. Accessed June 7, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/13%3A_Control_of_Microbial_Growth/13.01%3A_Controlling_Microbial_Growth.txt |
Learning Objectives
• Understand and compare various physical methods of controlling microbial growth, including heating, refrigeration, freezing, high-pressure treatment, desiccation, lyophilization, irradiation, and filtration
For thousands of years, humans have used various physical methods of microbial control for food preservation. Common control methods include the application of high temperatures, radiation, filtration, and desiccation (drying), among others. Many of these methods nonspecifically kill cells by disrupting membranes, changing membrane permeability, or damaging proteins and nucleic acids by denaturation, degradation, or chemical modification. Various physical methods used for microbial control are described in this section.
Heat
Heating is one of the most common—and oldest—forms of microbial control. It is used in simple techniques like cooking and canning. Heat can kill microbes by altering their membranes and denaturing proteins. The thermal death point (TDP) of a microorganism is the lowest temperature at which all microbes are killed in a 10-minute exposure. Different microorganisms will respond differently to high temperatures, with some (e.g., endospore-formers such as C. botulinum) being more heat tolerant. A similar parameter, the thermal death time (TDT), is the length of time needed to kill all microorganisms in a sample at a given temperature. These parameters are often used to describe sterilization procedures that use high heat, such as autoclaving. Boiling is one of the oldest methods of moist-heat control of microbes, and it is typically quite effective at killing vegetative cells and some viruses. However, boiling is less effective at killing endospores; some endospores are able to survive up to 20 hours of boiling. Additionally, boiling may be less effective at higher altitudes, where the boiling point of water is lower and the boiling time needed to kill microbes is therefore longer. For these reasons, boiling is not considered a useful sterilization technique in the laboratory or clinical setting.
Many different heating protocols can be used for sterilization in the laboratory or clinic, and these protocols can be broken down into two main categories: dry-heat sterilization and moist-heat sterilization. Aseptic technique in the laboratory typically involves some dry-heat sterilization protocols using direct application of high heat, such as sterilizing inoculating loops (Figure \(1\)). Incineration at very high temperatures destroys all microorganisms. Dry heat can also be applied for relatively long periods of time (at least 2 hours) at temperatures up to 170 °C by using a dry-heat sterilizer, such as an oven. However, moist-heat sterilization is typically the more effective protocol because it penetrates cells better than dry heat does.
Autoclaves
Autoclaves rely on moist-heat sterilization. They are used to raise temperatures above the boiling point of water to sterilize items such as surgical equipment from vegetative cells, viruses, and especially endospores, which are known to survive boiling temperatures, without damaging the items. Charles Chamberland (1851–1908) designed the modern autoclave in 1879 while working in the laboratory of Louis Pasteur. The autoclave is still considered the most effective method of sterilization (Figure \(2\)). Outside laboratory and clinical settings, large industrial autoclaves called retorts allow for moist-heat sterilization on a large scale.
In general, the air in the chamber of an autoclave is removed and replaced with increasing amounts of steam trapped within the enclosed chamber, resulting in increased interior pressure and temperatures above the boiling point of water. The two main types of autoclaves differ in the way that air is removed from the chamber. In gravity displacement autoclaves, steam is introduced into the chamber from the top or sides. Air, which is heavier than steam, sinks to the bottom of the chamber, where it is forced out through a vent. Complete displacement of air is difficult, especially in larger loads, so longer cycles may be required for such loads. In prevacuum sterilizers, air is removed completely using a high-speed vacuum before introducing steam into the chamber. Because air is more completely eliminated, the steam can more easily penetrate wrapped items. Many autoclaves are capable of both gravity and prevacuum cycles, using the former for the decontamination of waste and sterilization of media and unwrapped glassware, and the latter for sterilization of packaged instruments.
Standard operating temperatures for autoclaves are 121 °C or, in some cases, 132 °C, typically at a pressure of 15 to 20 pounds per square inch (psi). The length of exposure depends on the volume and nature of material being sterilized, but it is typically 20 minutes or more, with larger volumes requiring longer exposure times to ensure sufficient heat transfer to the materials being sterilized. The steam must directly contact the liquids or dry materials being sterilized, so containers are left loosely closed and instruments are loosely wrapped in paper or foil. The key to autoclaving is that the temperature must be high enough to kill endospores to achieve complete sterilization.
Because sterilization is so important to safe medical and laboratory protocols, quality control is essential. Autoclaves may be equipped with recorders to document the pressures and temperatures achieved during each run. Additionally, internal indicators of various types should be autoclaved along with the materials to be sterilized to ensure that the proper sterilization temperature has been reached (Figure \(3\)). One common type of indicator is the use of heat-sensitive autoclave tape, which has white stripes that turn black when the appropriate temperature is achieved during a successful autoclave run. This type of indicator is relatively inexpensive and can be used during every run. However, autoclave tape provides no indication of length of exposure, so it cannot be used as an indicator of sterility. Another type of indicator, a biological indicator spore test, uses either a strip of paper or a liquid suspension of the endospores of Geobacillus stearothermophilus to determine whether the endospores are killed by the process. The endospores of the obligate thermophilic bacterium G. stearothermophilus are the gold standard used for this purpose because of their extreme heat resistance. Biological spore indicators can also be used to test the effectiveness of other sterilization protocols, including ethylene oxide, dry heat, formaldehyde, gamma radiation, and hydrogen peroxide plasma sterilization using either G. stearothermophilus, Bacillus atrophaeus, B. subtilis, or B. pumilus spores. In the case of validating autoclave function, the endospores are incubated after autoclaving to ensure no viable endospores remain. Bacterial growth subsequent to endospore germination can be monitored by biological indicator spore tests that detect acid metabolites or fluorescence produced by enzymes derived from viable G. stearothermophilus. A third type of autoclave indicator is the Diack tube, a glass ampule containing a temperature-sensitive pellet that melts at the proper sterilization temperature. Spore strips or Diack tubes are used periodically to ensure the autoclave is functioning properly.
Pasteurization
Although complete sterilization is ideal for many medical applications, it is not always practical for other applications and may also alter the quality of the product. Boiling and autoclaving are not ideal ways to control microbial growth in many foods because these methods may ruin the consistency and other organoleptic (sensory) qualities of the food. Pasteurization is a form of microbial control for food that uses heat but does not render the food sterile. Traditional pasteurization kills pathogens and reduces the number of spoilage-causing microbes while maintaining food quality. The process of pasteurization was first developed by Louis Pasteur in the 1860s as a method for preventing the spoilage of beer and wine. Today, pasteurization is most commonly used to kill heat-sensitive pathogens in milk and other food products (e.g., apple juice and honey) (Figure \(4\)). However, because pasteurized food products are not sterile, they will eventually spoil.
The methods used for milk pasteurization balance the temperature and the length of time of treatment. One method, high-temperature short-time (HTST) pasteurization, exposes milk to a temperature of 72 °C for 15 seconds, which lowers bacterial numbers while preserving the quality of the milk. An alternative is ultra-high-temperature (UHT) pasteurization, in which the milk is exposed to a temperature of 138 °C for 2 or more seconds. UHT pasteurized milk can be stored for a long time in sealed containers without being refrigerated; however, the very high temperatures alter the proteins in the milk, causing slight changes in the taste and smell. Still, this method of pasteurization is advantageous in regions where access to refrigeration is limited.
Exercise \(1\)
1. In an autoclave, how are temperatures above boiling achieved?
2. How would the onset of spoilage compare between HTST-pasteurized and UHT-pasteurized milk?
3. Why is boiling not used as a sterilization method in a clinical setting?
Refrigeration and Freezing
Just as high temperatures are effective for controlling microbial growth, exposing microbes to low temperatures can also be an easy and effective method of microbial control, with the exception of psychrophiles, which prefer cold temperatures (see Temperature and Microbial Growth). Refrigerators used in home kitchens or in the laboratory maintain temperatures between 0 °C and 7 °C. This temperature range inhibits microbial metabolism, slowing the growth of microorganisms significantly and helping preserve refrigerated products such as foods or medical supplies. Certain types of laboratory cultures can be preserved by refrigeration for later use.
Freezing below −2 °C may stop microbial growth and even kill susceptible organisms. According to the US Department of Agriculture (USDA), the only safe ways that frozen foods can be thawed are in the refrigerator, immersed in cold water changed every 30 minutes, or in the microwave, keeping the food at temperatures not conducive for bacterial growth.1In addition, halted bacterial growth can restart in thawed foods, so thawed foods should be treated like fresh perishables.
Bacterial cultures and medical specimens requiring long-term storage or transport are often frozen at ultra-low temperatures of −70 °C or lower. These ultra-low temperatures can be achieved by storing specimens on dry ice in an ultra-low freezer or in special liquid nitrogen tanks, which maintain temperatures lower than −196 °C (Figure \(5\)).
Exercise \(2\)
Does placing food in a refrigerator kill bacteria on the food?
Pressure
Exposure to high pressure kills many microbes. In the food industry, high-pressure processing (also called pascalization) is used to kill bacteria, yeast, molds, parasites, and viruses in foods while maintaining food quality and extending shelf life. The application of high pressure between 100 and 800 MPa (sea level atmospheric pressure is about 0.1 MPa) is sufficient to kill vegetative cells by protein denaturation, but endospores may survive these pressures.23
In clinical settings, hyperbaric oxygen therapy is sometimes used to treat infections. In this form of therapy, a patient breathes pure oxygen at a pressure higher than normal atmospheric pressure, typically between 1 and 3 atmospheres (atm). This is achieved by placing the patient in a hyperbaric chamber or by supplying the pressurized oxygen through a breathing tube. Hyperbaric oxygen therapy helps increase oxygen saturation in tissues that become hypoxic due to infection and inflammation. This increased oxygen concentration enhances the body’s immune response by increasing the activities of neutrophils and macrophages, white blood cells that fight infections. Increased oxygen levels also contribute to the formation of toxic free radicals that inhibit the growth of oxygen-sensitive or anaerobic bacteria like as Clostridium perfringens, a common cause of gas gangrene. In C. perfringens infections, hyperbaric oxygen therapy can also reduce secretion of a bacterial toxin that causes tissue destruction. Hyperbaric oxygen therapy also seems to enhance the effectiveness of antibiotic treatments. Unfortunately, some rare risks include oxygen toxicity and effects on delicate tissues, such as the eyes, middle ear, and lungs, which may be damaged by the increased air pressure.
High pressure processing is not commonly used for disinfection or sterilization of fomites. Although the application of pressure and steam in an autoclave is effective for killing endospores, it is the high temperature achieved, and not the pressure directly, that results in endospore death.
A Streak of Bad Potluck
One Monday in spring 2015, an Ohio woman began to experience blurred, double vision; difficulty swallowing; and drooping eyelids. She was rushed to the emergency department of her local hospital. During the examination, she began to experience abdominal cramping, nausea, paralysis, dry mouth, weakness of facial muscles, and difficulty speaking and breathing. Based on these symptoms, the hospital’s incident command center was activated, and Ohio public health officials were notified of a possible case of botulism. Meanwhile, other patients with similar symptoms began showing up at other local hospitals. Because of the suspicion of botulism, antitoxin was shipped overnight from the CDC to these medical facilities, to be administered to the affected patients. The first patient died of respiratory failure as a result of paralysis, and about half of the remaining victims required additional hospitalization following antitoxin administration, with at least two requiring ventilators for breathing.
Public health officials investigated each of the cases and determined that all of the patients had attended the same church potluck the day before. Moreover, they traced the source of the outbreak to a potato salad made with home-canned potatoes. More than likely, the potatoes were canned using boiling water, a method that allows endospores of Clostridium botulinum to survive. C. botulinum produces botulinum toxin, a neurotoxin that is often deadly once ingested. According to the CDC, the Ohio case was the largest botulism outbreak in the United States in nearly 40 years.4
Killing C. botulinum endospores requires a minimum temperature of 116 °C (240 °F), well above the boiling point of water. This temperature can only be reached in a pressure canner, which is recommended for home canning of low-acid foods such as meat, fish, poultry, and vegetables (Figure \(6\)). Additionally, the CDC recommends boiling home-canned foods for about 10 minutes before consumption. Since the botulinum toxin is heat labile (meaning that it is denatured by heat), 10 minutes of boiling will render nonfunctional any botulinum toxin that the food may contain.
Link to Learning
To learn more about proper home-canning techniques, visit the CDC’s website.
Desiccation
Drying, also known as desiccation or dehydration, is a method that has been used for millennia to preserve foods such as raisins, prunes, and jerky. It works because all cells, including microbes, require water for their metabolism and survival. Although drying controls microbial growth, it might not kill all microbes or their endospores, which may start to regrow when conditions are more favorable and water content is restored.
In some cases, foods are dried in the sun, relying on evaporation to achieve desiccation. Freeze-drying, or lyophilization, is another method of dessication in which an item is rapidly frozen (“snap-frozen”) and placed under vacuum so that water is lost by sublimation. Lyophilization combines both exposure to cold temperatures and desiccation, making it quite effective for controlling microbial growth. In addition, lyophilization causes less damage to an item than conventional desiccation and better preserves the item’s original qualities. Lyophilized items may be stored at room temperature if packaged appropriately to prevent moisture acquisition. Lyophilization is used for preservation in the food industry and is also used in the laboratory for the long-term storage and transportation of microbial cultures.
The water content of foods and materials, called the water activity, can be lowered without physical drying by the addition of solutes such as salts or sugars. At very high concentrations of salts or sugars, the amount of available water in microbial cells is reduced dramatically because water will be drawn from an area of low solute concentration (inside the cell) to an area of high solute concentration (outside the cell) (Figure \(7\)). Many microorganisms do not survive these conditions of high osmotic pressure. Honey, for example, is 80% sucrose, an environment in which very few microorganisms are capable of growing, thereby eliminating the need for refrigeration. Salted meats and fish, like ham and cod, respectively, were critically important foods before the age of refrigeration. Fruits were preserved by adding sugar, making jams and jellies. However, certain microbes, such as molds and yeasts, tend to be more tolerant of desiccation and high osmotic pressures, and, thus, may still contaminate these types of foods.
Exercise \(3\)
How does the addition of salt or sugar to food affect its water activity?
Radiation
Radiation in various forms, from high-energy radiation to sunlight, can be used to kill microbes or inhibit their growth. Ionizing radiation includes X-rays, gamma rays, and high-energy electron beams. Ionizing radiation is strong enough to pass into the cell, where it alters molecular structures and damages cell components. For example, ionizing radiation introduces double-strand breaks in DNA molecules. This may directly cause DNA mutations to occur, or mutations may be introduced when the cell attempts to repair the DNA damage. As these mutations accumulate, they eventually lead to cell death.
Both X-rays and gamma rays easily penetrate paper and plastic and can therefore be used to sterilize many packaged materials. In the laboratory, ionizing radiation is commonly used to sterilize materials that cannot be autoclaved, such as plastic Petri dishes and disposable plastic inoculating loops. For clinical use, ionizing radiation is used to sterilize gloves, intravenous tubing, and other latex and plastic items used for patient care. Ionizing radiation is also used for the sterilization of other types of delicate, heat-sensitive materials used clinically, including tissues for transplantation, pharmaceutical drugs, and medical equipment.
In Europe, gamma irradiation for food preservation is widely used, although it has been slow to catch on in the United States (see the Micro Connections box on this topic). Packaged dried spices are also often gamma-irradiated. Because of their ability to penetrate paper, plastic, thin sheets of wood and metal, and tissue, great care must be taken when using X-rays and gamma irradiation. These types of ionizing irradiation cannot penetrate thick layers of iron or lead, so these metals are commonly used to protect humans who may be potentially exposed.
Another type of radiation, nonionizing radiation, is commonly used for sterilization and uses less energy than ionizing radiation. It does not penetrate cells or packaging. Ultraviolet (UV) light is one example; it causes thymine dimers to form between adjacent thymines within a single strand of DNA (Figure \(8\)). When DNA polymerase encounters the thymine dimer, it does not always incorporate the appropriate complementary nucleotides (two adenines), and this leads to formation of mutations that can ultimately kill microorganisms.
UV light can be used effectively by both consumers and laboratory personnel to control microbial growth. UV lamps are now commonly incorporated into water purification systems for use in homes. In addition, small portable UV lights are commonly used by campers to purify water from natural environments before drinking. Germicidal lamps are also used in surgical suites, biological safety cabinets, and transfer hoods, typically emitting UV light at a wavelength of 260 nm. Because UV light does not penetrate surfaces and will not pass through plastics or glass, cells must be exposed directly to the light source.
Sunlight has a very broad spectrum that includes UV and visible light. In some cases, sunlight can be effective against certain bacteria because of both the formation of thymine dimers by UV light and by the production of reactive oxygen products induced in low amounts by exposure to visible light.
Exercise \(4\)
1. What are two advantages of ionizing radiation as a sterilization method?
2. How does the effectiveness of ionizing radiation compare with that of nonionizing radiation?
Irradiated Food: Would You Eat That?
Of all the ways to prevent food spoilage and foodborne illness, gamma irradiation may be the most unappetizing. Although gamma irradiation is a proven method of eliminating potentially harmful microbes from food, the public has yet to buy in. Most of their concerns, however, stem from misinformation and a poor understanding of the basic principles of radiation.
The most common method of irradiation is to expose food to cobalt-60 or cesium-137 by passing it through a radiation chamber on a conveyor belt. The food does not directly contact the radioactive material and does not become radioactive itself. Thus, there is no risk for exposure to radioactive material through eating gamma-irradiated foods. Additionally, irradiated foods are not significantly altered in terms of nutritional quality, aside from the loss of certain vitamins, which is also exacerbated by extended storage. Alterations in taste or smell may occur in irradiated foods with high fat content, such as fatty meats and dairy products, but this effect can be minimized by using lower doses of radiation at colder temperatures.
In the United States, the CDC, Environmental Protection Agency (EPA), and the Food and Drug Administration (FDA) have deemed irradiation safe and effective for various types of meats, poultry, shellfish, fresh fruits and vegetables, eggs with shells, and spices and seasonings. Gamma irradiation of foods has also been approved for use in many other countries, including France, the Netherlands, Portugal, Israel, Russia, China, Thailand, Belgium, Australia, and South Africa. To help ameliorate consumer concern and assist with education efforts, irradiated foods are now clearly labeled and marked with the international irradiation symbol, called the “radura” (Figure \(9\)). Consumer acceptance seems to be rising, as indicated by several recent studies.
Sonication
The use of high-frequency ultrasound waves to disrupt cell structures is called sonication. Application of ultrasound waves causes rapid changes in pressure within the intracellular liquid; this leads to cavitation, the formation of bubbles inside the cell, which can disrupt cell structures and eventually cause the cell to lyse or collapse. Sonication is useful in the laboratory for efficiently lysing cells to release their contents for further research; outside the laboratory, sonication is used for cleaning surgical instruments, lenses, and a variety of other objects such as coins, tools, and musical instruments.
Filtration
Filtration is a method of physically separating microbes from samples. Air is commonly filtered through high-efficiency particulate air (HEPA) filters (Figure \(10\)). HEPA filters have effective pore sizes of 0.3 µm, small enough to capture bacterial cells, endospores, and many viruses, as air passes through these filters, nearly sterilizing the air on the other side of the filter. HEPA filters have a variety of applications and are used widely in clinical settings, in cars and airplanes, and even in the home. For example, they may be found in vacuum cleaners, heating and air-conditioning systems, and air purifiers.
Biological Safety Cabinets
Biological safety cabinets are a good example of the use of HEPA filters. HEPA filters in biological safety cabinets (BSCs) are used to remove particulates in the air either entering the cabinet (air intake), leaving the cabinet (air exhaust), or treating both the intake and exhaust. Use of an air-intake HEPA filter prevents environmental contaminants from entering the BSC, creating a clean area for handling biological materials. Use of an air-exhaust HEPA filter prevents laboratory pathogens from contaminating the laboratory, thus maintaining a safe work area for laboratory personnel.
There are three classes of BSCs: I, II, and III. Each class is designed to provide a different level of protection for laboratory personnel and the environment; BSC II and III are also designed to protect the materials or devices in the cabinet. Table \(1\) summarizes the level of safety provided by each class of BSC for each BSL.
Table \(1\): Biological risks and BSCs
Biological Risk Assessed BSC Class Protection of Personnel Protection of Environment Protection of Product
BSL-1, BSL-2, BSL-3 I Yes Yes No
BSL-1, BSL-2, BSL-3 II Yes Yes Yes
BSL-4 III; II when used in suit room with suit Yes Yes Yes
Class I BSCs protect laboratory workers and the environment from a low to moderate risk for exposure to biological agents used in the laboratory. Air is drawn into the cabinet and then filtered before exiting through the building’s exhaust system. Class II BSCs use directional air flow and partial barrier systems to contain infectious agents. Class III BSCs are designed for working with highly infectious agents like those used in BSL-4 laboratories. They are gas tight, and materials entering or exiting the cabinet must be passed through a double-door system, allowing the intervening space to be decontaminated between uses. All air is passed through one or two HEPA filters and an air incineration system before being exhausted directly to the outdoors (not through the building’s exhaust system). Personnel can manipulate materials inside the Class III cabinet by using long rubber gloves sealed to the cabinet.
Link to Learning
This video shows how BSCs are designed and explains how they protect personnel, the environment, and the product.
Filtration in Hospitals
HEPA filters are also commonly used in hospitals and surgical suites to prevent contamination and the spread of airborne microbes through ventilation systems. HEPA filtration systems may be designed for entire buildings or for individual rooms. For example, burn units, operating rooms, or isolation units may require special HEPA-filtration systems to remove opportunistic pathogens from the environment because patients in these rooms are particularly vulnerable to infection.
Membrane Filters
Filtration can also be used to remove microbes from liquid samples using membrane filtration. Membrane filters for liquids function similarly to HEPA filters for air. Typically, membrane filters that are used to remove bacteria have an effective pore size of 0.2 µm, smaller than the average size of a bacterium (1 µm), but filters with smaller pore sizes are available for more specific needs. Membrane filtration is useful for removing bacteria from various types of heat-sensitive solutions used in the laboratory, such as antibiotic solutions and vitamin solutions. Large volumes of culture media may also be filter sterilized rather than autoclaved to protect heat-sensitive components. Often when filtering small volumes, syringe filters are used, but vacuum filters are typically used for filtering larger volumes (Figure \(11\)).
Exercise \(5\)
1. Would membrane filtration with a 0.2-µm filter likely remove viruses from a solution? Explain.
2. Name at least two common uses of HEPA filtration in clinical or laboratory settings.
Figure \(12\) and Figure \(13\) summarize the physical methods of control discussed in this section.
Key Concepts and Summary
• Heat is a widely used and highly effective method for controlling microbial growth.
• Dry-heat sterilization protocols are used commonly in aseptic techniques in the laboratory. However, moist-heat sterilization is typically the more effective protocol because it penetrates cells better than dry heat does.
• Pasteurization is used to kill pathogens and reduce the number of microbes that cause food spoilage. High-temperature, short-time pasteurization is commonly used to pasteurize milk that will be refrigerated; ultra-high temperature pasteurization can be used to pasteurize milk for long-term storage without refrigeration.
• Refrigeration slows microbial growth; freezing stops growth, killing some organisms. Laboratory and medical specimens may be frozen on dry ice or at ultra-low temperatures for storage and transport.
• High-pressure processing can be used to kill microbes in food. Hyperbaric oxygen therapy to increase oxygen saturation has also been used to treat certain infections.
• Desiccation has long been used to preserve foods and is accelerated through the addition of salt or sugar, which decrease water activity in foods.
• Lyophilization combines cold exposure and desiccation for the long-term storage of foods and laboratory materials, but microbes remain and can be rehydrated.
• Ionizing radiation, including gamma irradiation, is an effective way to sterilize heat-sensitive and packaged materials. Nonionizing radiation, like ultraviolet light, is unable to penetrate surfaces but is useful for surface sterilization.
• HEPA filtration is commonly used in hospital ventilation systems and biological safety cabinets in laboratories to prevent transmission of airborne microbes. Membrane filtration is commonly used to remove bacteria from heat-sensitive solutions.
Footnotes
1. 1 US Department of Agriculture. “Freezing and Food Safety.” 2013. http://www.fsis.usda.gov/wps/portal/...afety/CT_Index. Accessed June 8, 2016.
2. 2 C. Ferstl. “High Pressure Processing: Insights on Technology and Regulatory Requirements.” Food for Thought/White Paper. Series Volume 10. Livermore, CA: The National Food Lab; July 2013.
3. 3 US Food and Drug Administration. “Kinetics of Microbial Inactivation for Alternative Food Processing Technologies: High Pressure Processing.” 2000. www.fda.gov/Food/FoodScienceR.../ucm101456.htm. Accessed July 19, 2106.
4. 4 CL McCarty et al. “Large Outbreak of Botulism Associated with a Church Potluck Meal-Ohio, 2015.” Morbidity and Mortality Weekly Report 64, no. 29 (2015):802–803.
5. 5 AM Johnson et al. “Consumer Acceptance of Electron-Beam Irradiated Ready-to-Eat Poultry Meats.” Food Processing Preservation, 28 no. 4 (2004):302–319. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/13%3A_Control_of_Microbial_Growth/13.02%3A_Using_Physical_Methods_to_Control_Microorganisms.txt |
Learning Objectives
• Understand and compare various chemicals used to control microbial growth, including their uses, advantages and disadvantages, chemical structure, and mode of action
In addition to physical methods of microbial control, chemicals are also used to control microbial growth. A wide variety of chemicals can be used as disinfectants or antiseptics. When choosing which to use, it is important to consider the type of microbe targeted; how clean the item needs to be; the disinfectant’s effect on the item’s integrity; its safety to animals, humans, and the environment; its expense; and its ease of use. This section describes the variety of chemicals used as disinfectants and antiseptics, including their mechanisms of action and common uses.
Phenolics
In the 1800s, scientists began experimenting with a variety of chemicals for disinfection. In the 1860s, British surgeon Joseph Lister (1827–1912) began using carbolic acid, known as phenol, as a disinfectant for the treatment of surgical wounds (see Foundations of Modern Cell Theory). In 1879, Lister’s work inspired the American chemist Joseph Lawrence (1836–1909) to develop Listerine, an alcohol-based mixture of several related compounds that is still used today as an oral antiseptic. Today, carbolic acid is no longer used as a surgical disinfectant because it is a skin irritant, but the chemical compounds found in antiseptic mouthwashes and throat lozenges are called phenolics.
Chemically, phenol consists of a benzene ring with an –OH group, and phenolics are compounds that have this group as part of their chemical structure (Figure \(1\)). Phenolics such as thymol and eucalyptol occur naturally in plants. Other phenolics can be derived from creosote, a component of coal tar. Phenolics tend to be stable, persistent on surfaces, and less toxic than phenol. They inhibit microbial growth by denaturing proteins and disrupting membranes.
Since Lister’s time, several phenolic compounds have been used to control microbial growth. Phenolics like cresols(methylated phenols) and o-phenylphenol were active ingredients in various formulations of Lysol since its invention in 1889. o-Phenylphenol was also commonly used in agriculture to control bacterial and fungal growth on harvested crops, especially citrus fruits, but its use in the United States is now far more limited. The bisphenol hexachlorophene, a disinfectant, is the active ingredient in pHisoHex, a topical cleansing detergent widely used for handwashing in hospital settings. pHisoHex is particularly effective against gram-positive bacteria, including those causing staphylococcal and streptococcal skin infections. pHisoHex was formerly used for bathing infants, but this practice has been discontinued because it has been shown that exposure to hexachlorophene can lead to neurological problems.
Triclosan is another bisphenol compound that has seen widespread application in antibacterial products over the last several decades. Initially used in toothpastes, triclosan is now commonly used in hand soaps and is frequently impregnated into a wide variety of other products, including cutting boards, knives, shower curtains, clothing, and concrete, to make them antimicrobial. It is particularly effective against gram-positive bacteria on the skin, as well as certain gram-negative bacteria and yeasts.1
Triclosan: Antibacterial Overkill?
Hand soaps and other cleaning products are often marketed as “antibacterial,” suggesting that they provide a level of cleanliness superior to that of conventional soaps and cleansers. But are the antibacterial ingredients in these products really safe and effective?
About 75% of antibacterial liquid hand soaps and 30% of bar soaps contain the chemical triclosan, a phenolic, (Figure \(2\)).2 Triclosan blocks an enzyme in the bacterial fatty acid-biosynthesis pathway that is not found in the comparable human pathway. Although the use of triclosan in the home increased dramatically during the 1990s, more than 40 years of research by the FDA have turned up no conclusive evidence that washing with triclosan-containing products provides increased health benefits compared with washing with traditional soap. Although some studies indicate that fewer bacteria may remain on a person’s hands after washing with triclosan-based soap, compared with traditional soap, no evidence points to any reduction in the transmission of bacteria that cause respiratory and gastrointestinal illness. In short, soaps with triclosan may remove or kill a few more germs but not enough to reduce the spread of disease.
Perhaps more disturbing, some clear risks associated with triclosan-based soaps have come to light. The widespread use of triclosan has led to an increase in triclosan-resistant bacterial strains, including those of clinical importance, such as Salmonella enterica; this resistance may render triclosan useless as an antibacterial in the long run.34 Bacteria can easily gain resistance to triclosan through a change to a single gene encoding the targeted enzyme in the bacterial fatty acid-synthesis pathway. Other disinfectants with a less specific mode of action are much less prone to engendering resistance because it would take much more than a single genetic change.
Use of triclosan over the last several decades has also led to a buildup of the chemical in the environment. Triclosan in hand soap is directly introduced into wastewater and sewage systems as a result of the handwashing process. There, its antibacterial properties can inhibit or kill bacteria responsible for the decomposition of sewage, causing septic systems to clog and back up. Eventually, triclosan in wastewater finds its way into surface waters, streams, lakes, sediments, and soils, disrupting natural populations of bacteria that carry out important environmental functions, such as inhibiting algae. Triclosan also finds its way into the bodies of amphibians and fish, where it can act as an endocrine disruptor. Detectable levels of triclosan have also been found in various human bodily fluids, including breast milk, plasma, and urine.5 In fact, a study conducted by the CDC found detectable levels of triclosan in the urine of 75% of 2,517 people tested in 2003–2004.6 This finding is even more troubling given the evidence that triclosan may affect immune function in humans.7
In December 2013, the FDA gave soap manufacturers until 2016 to prove that antibacterial soaps provide a significant benefit over traditional soaps; if unable to do so, manufacturers will be forced to remove these products from the market.
Exercise \(1\)
Why is triclosan more like an antibiotic than a traditional disinfectant?
Heavy Metals
Some of the first chemical disinfectants and antiseptics to be used were heavy metals. Heavy metals kill microbes by binding to proteins, thus inhibiting enzymatic activity (Figure \(3\)). Heavy metals are oligodynamic, meaning that very small concentrations show significant antimicrobial activity. Ions of heavy metals bind to sulfur-containing amino acids strongly and bioaccumulate within cells, allowing these metals to reach high localized concentrations. This causes proteins to denature.
Heavy metals are not selectively toxic to microbial cells. They may bioaccumulate in human or animal cells, as well, and excessive concentrations can have toxic effects on humans. If too much silver accumulates in the body, for example, it can result in a condition called argyria, in which the skin turns irreversibly blue-gray. One way to reduce the potential toxicity of heavy metals is by carefully controlling the duration of exposure and concentration of the heavy metal.
Mercury
Mercury is an example of a heavy metal that has been used for many years to control microbial growth. It was used for many centuries to treat syphilis. Mercury compounds like mercuric chloride are mainly bacteriostatic and have a very broad spectrum of activity. Various forms of mercury bind to sulfur-containing amino acids within proteins, inhibiting their functions.
In recent decades, the use of such compounds has diminished because of mercury’s toxicity. It is toxic to the central nervous, digestive, and renal systems at high concentrations, and has negative environmental effects, including bioaccumulation in fish. Topical antiseptics such as mercurochrome, which contains mercury in low concentrations, and merthiolate, a tincture (a solution of mercury dissolved in alcohol) were once commonly used. However, because of concerns about using mercury compounds, these antiseptics are no longer sold in the United States.
Silver
Silver has long been used as an antiseptic. In ancient times, drinking water was stored in silver jugs.8 Silvadene cream is commonly used to treat topical wounds and is particularly helpful in preventing infection in burn wounds. Silver nitrate drops were once routinely applied to the eyes of newborns to protect against ophthalmia neonatorum, eye infections that can occur due to exposure to pathogens in the birth canal, but antibiotic creams are more now commonly used. Silver is often combined with antibiotics, making the antibiotics thousands of times more effective.9 Silver is also commonly incorporated into catheters and bandages, rendering them antimicrobial; however, there is evidence that heavy metals may also enhance selection for antibiotic resistance.10
Copper, Nickel, and Zinc
Several other heavy metals also exhibit antimicrobial activity. Copper sulfate is a common algicide used to control algal growth in swimming pools and fish tanks. The use of metallic copper to minimize microbial growth is also becoming more widespread. Copper linings in incubators help reduce contamination of cell cultures. The use of copper pots for water storage in underdeveloped countries is being investigated as a way to combat diarrheal diseases. Copper coatings are also becoming popular for frequently handled objects such as doorknobs, cabinet hardware, and other fixtures in health-care facilities in an attempt to reduce the spread of microbes.
Nickel and zinc coatings are now being used in a similar way. Other forms of zinc, including zinc chloride and zinc oxide, are also used commercially. Zinc chloride is quite safe for humans and is commonly found in mouthwashes, substantially increasing their length of effectiveness. Zinc oxide is found in a variety of products, including topical antiseptic creams such as calamine lotion, diaper ointments, baby powder, and dandruff shampoos.
Exercise \(2\)
Why are many heavy metals both antimicrobial and toxic to humans?
Halogens
Other chemicals commonly used for disinfection are the halogens iodine, chlorine, and fluorine. Iodine works by oxidizing cellular components, including sulfur-containing amino acids, nucleotides, and fatty acids, and destabilizing the macromolecules that contain these molecules. It is often used as a topical tincture, but it may cause staining or skin irritation. An iodophor is a compound of iodine complexed with an organic molecule, thereby increasing iodine’s stability and, in turn, its efficacy. One common iodophor is povidone-iodine, which includes a wetting agent that releases iodine relatively slowly. Betadine is a brand of povidone-iodine commonly used as a hand scrub by medical personnel before surgery and for topical antisepsis of a patient’s skin before incision (Figure \(4\)).
Chlorine is another halogen commonly used for disinfection. When chlorine gas is mixed with water, it produces a strong oxidant called hypochlorous acid, which is uncharged and enters cells easily. Chlorine gas is commonly used in municipal drinking water and wastewater treatment plants, with the resulting hypochlorous acid producing the actual antimicrobial effect. Those working at water treatment facilities need to take great care to minimize personal exposure to chlorine gas. Sodium hypochlorite is the chemical component of common household bleach, and it is also used for a wide variety of disinfecting purposes. Hypochlorite salts, including sodium and calcium hypochlorites, are used to disinfect swimming pools. Chlorine gas, sodium hypochlorite, and calcium hypochlorite are also commonly used disinfectants in the food processing and restaurant industries to reduce the spread of foodborne diseases. Workers in these industries also need to take care to use these products correctly to ensure their own safety as well as the safety of consumers. A recent joint statement published by the Food and Agriculture Organization (FAO) of the United Nations and WHO indicated that none of the many beneficial uses of chlorine products in food processing to reduce the spread of foodborne illness posed risks to consumers.11
Another class of chlorinated compounds called chloramines are widely used as disinfectants. Chloramines are relatively stable, releasing chlorine over long periods time. Chloramines are derivatives of ammonia by substitution of one, two, or all three hydrogen atoms with chlorine atoms (Figure \(5\)).
Chloramines and other cholorine compounds may be used for disinfection of drinking water, and chloramine tablets are frequently used by the military for this purpose. After a natural disaster or other event that compromises the public water supply, the CDC recommends disinfecting tap water by adding small amounts of regular household bleach. Recent research suggests that sodium dichloroisocyanurate (NaDCC) may also be a good alternative for drinking water disinfection. Currently, NaDCC tablets are available for general use and for use by the military, campers, or those with emergency needs; for these uses, NaDCC is preferable to chloramine tablets. Chlorine dioxide, a gaseous agent used for fumigation and sterilization of enclosed areas, is also commonly used for the disinfection of water.
Although chlorinated compounds are relatively effective disinfectants, they have their disadvantages. Some may irritate the skin, nose, or eyes of some individuals, and they may not completely eliminate certain hardy organisms from contaminated drinking water. The fungus Cryptosporidium, for example, has a protective outer shell that makes it resistant to chlorinated disinfectants. Thus, boiling of drinking water in emergency situations is recommended when possible.
The halogen fluorine is also known to have antimicrobial properties that contribute to the prevention of dental caries(cavities).12 Fluoride is the main active ingredient of toothpaste and is also commonly added to tap water to help communities maintain oral health. Chemically, fluoride can become incorporated into the hydroxyapatite of tooth enamel, making it more resistant to corrosive acids produced by the fermentation of oral microbes. Fluoride also enhances the uptake of calcium and phosphate ions in tooth enamel, promoting remineralization. In addition to strengthening enamel, fluoride also seems to be bacteriostatic. It accumulates in plaque-forming bacteria, interfering with their metabolism and reducing their production of the acids that contribute to tooth decay.
Exercise \(3\)
What is a benefit of a chloramine over hypochlorite for disinfecting?
Alcohols
Alcohols make up another group of chemicals commonly used as disinfectants and antiseptics. They work by rapidly denaturing proteins, which inhibits cell metabolism, and by disrupting membranes, which leads to cell lysis. Once denatured, the proteins may potentially refold if enough water is present in the solution. Alcohols are typically used at concentrations of about 70% aqueous solution and, in fact, work better in aqueous solutions than 100% alcohol solutions. This is because alcohols coagulate proteins. In higher alcohol concentrations, rapid coagulation of surface proteins prevents effective penetration of cells. The most commonly used alcohols for disinfection are ethyl alcohol(ethanol) and isopropyl alcohol (isopropanol, rubbing alcohol) (Figure \(6\)).
Alcohols tend to be bactericidal and fungicidal, but may also be viricidal for enveloped viruses only. Although alcohols are not sporicidal, they do inhibit the processes of sporulation and germination. Alcohols are volatile and dry quickly, but they may also cause skin irritation because they dehydrate the skin at the site of application. One common clinical use of alcohols is swabbing the skin for degerming before needle injection. Alcohols also are the active ingredients in instant hand sanitizers, which have gained popularity in recent years. The alcohol in these hand sanitizers works both by denaturing proteins and by disrupting the microbial cell membrane, but will not work effectively in the presence of visible dirt.
Last, alcohols are used to make tinctures with other antiseptics, such as the iodine tinctures discussed previously in this chapter. All in all, alcohols are inexpensive and quite effective for the disinfection of a broad range of vegetative microbes. However, one disadvantage of alcohols is their high volatility, limiting their effectiveness to immediately after application.
Exercise \(4\)
1. Name at least three advantages of alcohols as disinfectants.
2. Describe several specific applications of alcohols used in disinfectant products.
Surfactants
Surface-active agents, or surfactants, are a group of chemical compounds that lower the surface tension of water. Surfactants are the major ingredients in soaps and detergents. Soaps are salts of long-chain fatty acids and have both polar and nonpolar regions, allowing them to interact with polar and nonpolar regions in other molecules (Figure \(7\)). They can interact with nonpolar oils and grease to create emulsions in water, loosening and lifting away dirt and microbes from surfaces and skin. Soaps do not kill or inhibit microbial growth and so are not considered antiseptics or disinfectants. However, proper use of soaps mechanically carries away microorganisms, effectively degerming a surface. Some soaps contain added bacteriostatic agents such as triclocarban or cloflucarban, compounds structurally related to triclosan, that introduce antiseptic or disinfectant properties to the soaps.
Soaps, however, often form films that are difficult to rinse away, especially in hard water, which contains high concentrations of calcium and magnesium mineral salts. Detergents contain synthetic surfactant molecules with both polar and nonpolar regions that have strong cleansing activity but are more soluble, even in hard water, and, therefore, leave behind no soapy deposits. Anionic detergents, such as those used for laundry, have a negatively charged anion at one end attached to a long hydrophobic chain, whereas cationic detergents have a positively charged cation instead. Cationic detergents include an important class of disinfectants and antiseptics called the quaternary ammonium salts (quats), named for the characteristic quaternary nitrogen atom that confers the positive charge (Figure \(8\)). Overall, quats have properties similar to phospholipids, having hydrophilic and hydrophobic ends. As such, quats have the ability to insert into the bacterial phospholipid bilayer and disrupt membrane integrity. The cationic charge of quats appears to confer their antimicrobial properties, which are diminished when neutralized. Quats have several useful properties. They are stable, nontoxic, inexpensive, colorless, odorless, and tasteless. They tend to be bactericidal by disrupting membranes. They are also active against fungi, protozoans, and enveloped viruses, but endospores are unaffected. In clinical settings, they may be used as antiseptics or to disinfect surfaces. Mixtures of quats are also commonly found in household cleaners and disinfectants, including many current formulations of Lysol brand products, which contain benzalkonium chlorides as the active ingredients. Benzalkonium chlorides, along with the quat cetylpyrimidine chloride, are also found in products such as skin antiseptics, oral rinses, and mouthwashes.
Exercise \(5\)
Why are soaps not considered disinfectants?
Handwashing the Right Way
Handwashing is critical for public health and should be emphasized in a clinical setting. For the general public, the CDC recommends handwashing before, during, and after food handling; before eating; before and after interacting with someone who is ill; before and after treating a wound; after using the toilet or changing diapers; after coughing, sneezing, or blowing the nose; after handling garbage; and after interacting with an animal, its feed, or its waste. Figure \(9\) illustrates the five steps of proper handwashing recommended by the CDC.
Handwashing is even more important for health-care workers, who should wash their hands thoroughly between every patient contact, after the removal of gloves, after contact with bodily fluids and potentially infectious fomites, and before and after assisting a surgeon with invasive procedures. Even with the use of proper surgical attire, including gloves, scrubbing for surgery is more involved than routine handwashing. The goal of surgical scrubbing is to reduce the normal microbiota on the skin’s surface to prevent the introduction of these microbes into a patient’s surgical wounds.
There is no single widely accepted protocol for surgical scrubbing. Protocols for length of time spent scrubbing may depend on the antimicrobial used; health-care workers should always check the manufacturer’s recommendations. According to the Association of Surgical Technologists (AST), surgical scrubs may be performed with or without the use of brushes (Figure \(9\)).
Link to Learning
To learn more about proper handwashing, visit the CDC’s website.
Bisbiguanides
Bisbiguanides were first synthesized in the 20th century and are cationic (positively charged) molecules known for their antiseptic properties (Figure \(10\)). One important bisbiguanide antiseptic is chlorhexidine. It has broad-spectrum activity against yeasts, gram-positive bacteria, and gram-negative bacteria, with the exception of Pseudomonas aeruginosa, which may develop resistance on repeated exposure.13 Chlorhexidine disrupts cell membranes and is bacteriostatic at lower concentrations or bactericidal at higher concentrations, in which it actually causes the cells’ cytoplasmic contents to congeal. It also has activity against enveloped viruses. However, chlorhexidine is poorly effective against Mycobacterium tuberculosis and nonenveloped viruses, and it is not sporicidal. Chlorhexidine is typically used in the clinical setting as a surgical scrub and for other handwashing needs for medical personnel, as well as for topical antisepsis for patients before surgery or needle injection. It is more persistent than iodophors, providing long-lasting antimicrobial activity. Chlorhexidine solutions may also be used as oral rinses after oral procedures or to treat gingivitis. Another bisbiguanide, alexidine, is gaining popularity as a surgical scrub and an oral rinse because it acts faster than chlorhexidine.
Exercise \(6\)
What two effects does chlorhexidine have on bacterial cells?
Alkylating Agents
The alkylating agents are a group of strong disinfecting chemicals that act by replacing a hydrogen atom within a molecule with an alkyl group (CnH2n+1), thereby inactivating enzymes and nucleic acids (Figure \(11\)). The alkylating agent formaldehyde (CH2OH) is commonly used in solution at a concentration of 37% (known as formalin) or as a gaseous disinfectant and biocide. It is a strong, broad-spectrum disinfectant and biocide that has the ability to kill bacteria, viruses, fungi, and endospores, leading to sterilization at low temperatures, which is sometimes a convenient alternative to the more labor-intensive heat sterilization methods. It also cross-links proteins and has been widely used as a chemical fixative. Because of this, it is used for the storage of tissue specimens and as an embalming fluid. It also has been used to inactivate infectious agents in vaccine preparation. Formaldehyde is very irritating to living tissues and is also carcinogenic; therefore, it is not used as an antiseptic.
Glutaraldehyde is structurally similar to formaldehyde but has two reactive aldehyde groups, allowing it to act more quickly than formaldehyde. It is commonly used as a 2% solution for sterilization and is marketed under the brand name Cidex. It is used to disinfect a variety of surfaces and surgical and medical equipment. However, similar to formaldehyde, glutaraldehyde irritates the skin and is not used as an antiseptic.
A new type of disinfectant gaining popularity for the disinfection of medical equipment is o-phthalaldehyde (OPA), which is found in some newer formulations of Cidex and similar products, replacing glutaraldehyde. o-Phthalaldehyde also has two reactive aldehyde groups, but they are linked by an aromatic bridge. o-Phthalaldehyde is thought to work similarly to glutaraldehyde and formaldehyde, but is much less irritating to skin and nasal passages, produces a minimal odor, does not require processing before use, and is more effective against mycobacteria.
Ethylene oxide is a type of alkylating agent that is used for gaseous sterilization. It is highly penetrating and can sterilize items within plastic bags such as catheters, disposable items in laboratories and clinical settings (like packaged Petri dishes), and other pieces of equipment. Ethylene oxide exposure is a form of cold sterilization, making it useful for the sterilization of heat-sensitive items. Great care needs to be taken with the use of ethylene oxide, however; it is carcinogenic, like the other alkylating agents, and is also highly explosive. With careful use and proper aeration of the products after treatment, ethylene oxide is highly effective, and ethylene oxide sterilizers are commonly found in medical settings for sterilizing packaged materials.
β-Propionolactone is an alkylating agent with a different chemical structure than the others already discussed. Like other alkylating agents, β-propionolactone binds to DNA, thereby inactivating it (Figure \(11\)). It is a clear liquid with a strong odor and has the ability to kill endospores. As such, it has been used in either liquid form or as a vapor for the sterilization of medical instruments and tissue grafts, and it is a common component of vaccines, used to maintain their sterility. It has also been used for the sterilization of nutrient broth, as well as blood plasma, milk, and water. It is quickly metabolized by animals and humans to lactic acid. It is also an irritant, however, and may lead to permanent damage of the eyes, kidneys, or liver. Additionally, it has been shown to be carcinogenic in animals; thus, precautions are necessary to minimize human exposure to β-propionolactone.14
Exercise \(7\)
1. What chemical reaction do alkylating agents participate in?
2. Why are alkylating agents not used as antiseptics?
Diehard Prions
Prions, the acellular, misfolded proteins responsible for incurable and fatal diseases such as kuru and Creutzfeldt-Jakob disease (see Viroids, Virusoids, and Prions), are notoriously difficult to destroy. Prions are extremely resistant to heat, chemicals, and radiation. They are also extremely infectious and deadly; thus, handling and disposing of prion-infected items requires extensive training and extreme caution.
Typical methods of disinfection can reduce but not eliminate the infectivity of prions. Autoclaving is not completely effective, nor are chemicals such as phenol, alcohols, formalin, and β-propiolactone. Even when fixed in formalin, affected brain and spinal cord tissues remain infectious.
Personnel who handle contaminated specimens or equipment or work with infected patients must wear a protective coat, face protection, and cut-resistant gloves. Any contact with skin must be immediately washed with detergent and warm water without scrubbing. The skin should then be washed with 1 N NaOH or a 1:10 dilution of bleach for 1 minute. Contaminated waste must be incinerated or autoclaved in a strong basic solution, and instruments must be cleaned and soaked in a strong basic solution.
Link to Learning
For more information on the handling of animals and prion-contaminated materials, visit the guidelines published on the CDC and WHO websites.
Peroxygens
Peroxygens are strong oxidizing agents that can be used as disinfectants or antiseptics. The most widely used peroxygen is hydrogen peroxide (H2O2), which is often used in solution to disinfect surfaces and may also be used as a gaseous agent. Hydrogen peroxide solutions are inexpensive skin antiseptics that break down into water and oxygen gas, both of which are environmentally safe. This decomposition is accelerated in the presence of light, so hydrogen peroxide solutions typically are sold in brown or opaque bottles. One disadvantage of using hydrogen peroxide as an antiseptic is that it also causes damage to skin that may delay healing or lead to scarring. Contact lens cleaners often include hydrogen peroxide as a disinfectant.
Hydrogen peroxide works by producing free radicals that damage cellular macromolecules. Hydrogen peroxide has broad-spectrum activity, working against gram-positive and gram-negative bacteria (with slightly greater efficacy against gram-positive bacteria), fungi, viruses, and endospores. However, bacteria that produce the oxygen-detoxifying enzymes catalase or peroxidase may have inherent tolerance to low hydrogen peroxide concentrations (Figure \(12\)). To kill endospores, the length of exposure or concentration of solutions of hydrogen peroxide must be increased. Gaseous hydrogen peroxide has greater efficacy and can be used as a sterilant for rooms or equipment.
Plasma, a hot, ionized gas, described as the fourth state of matter, is useful for sterilizing equipment because it penetrates surfaces and kills vegetative cells and endospores. Hydrogen peroxide and peracetic acid, another commonly used peroxygen, each may be introduced as a plasma. Peracetic acid can be used as a liquid or plasma sterilant insofar as it readily kills endospores, is more effective than hydrogen peroxide even at rather low concentrations, and is immune to inactivation by catalases and peroxidases. It also breaks down to environmentally innocuous compounds; in this case, acetic acid and oxygen.
Other examples of peroxygens include benzoyl peroxide and carbamide peroxide. Benzoyl peroxide is a peroxygen that used in acne medication solutions. It kills the bacterium Propionibacterium acnes, which is associated with acne. Carbamide peroxide, an ingredient used in toothpaste, is a peroxygen that combats oral biofilms that cause tooth discoloration and halitosis (bad breath).15 Last, ozone gas is a peroxygen with disinfectant qualities and is used to clean air or water supplies. Overall, peroxygens are highly effective and commonly used, with no associated environmental hazard.
Exercise \(8\)
How do peroxides kill cells?
Supercritical Fluids
Within the last 15 years, the use of supercritical fluids, especially supercritical carbon dioxide (scCO2), has gained popularity for certain sterilizing applications. When carbon dioxide is brought to approximately 10 times atmospheric pressure, it reaches a supercritical state that has physical properties between those of liquids and gases. Materials put into a chamber in which carbon dioxide is pressurized in this way can be sterilized because of the ability of scCO2 to penetrate surfaces.
Supercritical carbon dioxide works by penetrating cells and forming carbonic acid, thereby lowering the cell pH considerably. This technique is effective against vegetative cells and is also used in combination with peracetic acid to kill endospores. Its efficacy can also be augmented with increased temperature or by rapid cycles of pressurization and depressurization, which more likely produce cell lysis.
Benefits of scCO2 include the nonreactive, nontoxic, and nonflammable properties of carbon dioxide, and this protocol is effective at low temperatures. Unlike other methods, such as heat and irradiation, that can degrade the object being sterilized, the use of scCO2 preserves the object’s integrity and is commonly used for treating foods (including spices and juices) and medical devices such as endoscopes. It is also gaining popularity for disinfecting tissues such as skin, bones, tendons, and ligaments prior to transplantation. scCO2 can also be used for pest control because it can kill insect eggs and larvae within products.
Exercise \(9\)
Why is the use of supercritical carbon dioxide gaining popularity for commercial and medical uses?
Chemical Food Preservatives
Chemical preservatives are used to inhibit microbial growth and minimize spoilage in some foods. Commonly used chemical preservatives include sorbic acid, benzoic acid, and propionic acid, and their more soluble salts potassium sorbate, sodium benzoate, and calcium propionate, all of which are used to control the growth of molds in acidic foods. Each of these preservatives is nontoxic and readily metabolized by humans. They are also flavorless, so they do not compromise the flavor of the foods they preserve.
Sorbic and benzoic acids exhibit increased efficacy as the pH decreases. Sorbic acid is thought to work by inhibiting various cellular enzymes, including those in the citric acid cycle, as well as catalases and peroxidases. It is added as a preservative in a wide variety of foods, including dairy, bread, fruit, and vegetable products. Benzoic acid is found naturally in many types of fruits and berries, spices, and fermented products. It is thought to work by decreasing intracellular pH, interfering with mechanisms such as oxidative phosphorylation and the uptake of molecules such as amino acids into cells. Foods preserved with benzoic acid or sodium benzoate include fruit juices, jams, ice creams, pastries, soft drinks, chewing gum, and pickles.
Propionic acid is thought to both inhibit enzymes and decrease intracellular pH, working similarly to benzoic acid. However, propionic acid is a more effective preservative at a higher pH than either sorbic acid or benzoic acid. Propionic acid is naturally produced by some cheeses during their ripening and is added to other types of cheese and baked goods to prevent mold contamination. It is also added to raw dough to prevent contamination by the bacterium Bacillus mesentericus, which causes bread to become ropy.
Other commonly used chemical preservatives include sulfur dioxide and nitrites. Sulfur dioxide prevents browning of foods and is used for the preservation of dried fruits; it has been used in winemaking since ancient times. Sulfur dioxide gas dissolves in water readily, forming sulfites. Although sulfites can be metabolized by the body, some people have sulfite allergies, including asthmatic reactions. Additionally, sulfites degrade thiamine, an important nutrient in some foods. The mode of action of sulfites is not entirely clear, but they may interfere with the disulfide bond (see Figure 7.4.5) formation in proteins, inhibiting enzymatic activity. Alternatively, they may reduce the intracellular pH of the cell, interfering with proton motive force-driven mechanisms.
Nitrites are added to processed meats to maintain color and stop the germination of Clostridium botulinum endospores. Nitrites are reduced to nitric oxide, which reacts with heme groups and iron-sulfur groups. When nitric oxide reacts with the heme group within the myoglobin of meats, a red product forms, giving meat its red color. Alternatively, it is thought that when nitric acid reacts with the iron-sulfur enzyme ferredoxin within bacteria, this electron transport-chain carrier is destroyed, preventing ATP synthesis. Nitrosamines, however, are carcinogenic and can be produced through exposure of nitrite-preserved meats (e.g., hot dogs, lunch meat, breakfast sausage, bacon, meat in canned soups) to heat during cooking.
Natural Chemical Food Preservatives
The discovery of natural antimicrobial substances produced by other microbes has added to the arsenal of preservatives used in food. Nisin is an antimicrobial peptide produced by the bacterium Lactococcus lactis and is particularly effective against gram-positive organisms. Nisin works by disrupting cell wall production, leaving cells more prone to lysis. It is used to preserve cheeses, meats, and beverages.
Natamycin is an antifungal macrolide antibiotic produced by the bacterium Streptomyces natalensis. It was approved by the FDA in 1982 and is used to prevent fungal growth in various types of dairy products, including cottage cheese, sliced cheese, and shredded cheese. Natamycin is also used for meat preservation in countries outside the United States.
Exercise \(10\)
What are the advantages and drawbacks of using sulfites and nitrites as food preservatives?
Key Concepts and Summary
• Heavy metals, including mercury, silver, copper, and zinc, have long been used for disinfection and preservation, although some have toxicity and environmental risks associated with them.
• Halogens, including chlorine, fluorine, and iodine, are also commonly used for disinfection. Chlorine compounds, including sodium hypochlorite, chloramines, and chlorine dioxide, are commonly used for water disinfection. Iodine, in both tincture and iodophor forms, is an effective antiseptic.
• Alcohols, including ethyl alcohol and isopropyl alcohol, are commonly used antiseptics that act by denaturing proteins and disrupting membranes.
• Phenolics are stable, long-acting disinfectants that denature proteins and disrupt membranes. They are commonly found in household cleaners, mouthwashes, and hospital disinfectants, and are also used to preserve harvested crops.
• The phenolic compound triclosan, found in antibacterial soaps, plastics, and textiles is technically an antibiotic because of its specific mode of action of inhibiting bacterial fatty-acid synthesis..
• Surfactants, including soaps and detergents, lower the surface tension of water to create emulsions that mechanically carry away microbes. Soaps are long-chain fatty acids, whereas detergents are synthetic surfactants.
• Quaternary ammonium compounds (quats) are cationic detergents that disrupt membranes. They are used in household cleaners, skin disinfectants, oral rinses, and mouthwashes.
• Bisbiguanides disrupt cell membranes, causing cell contents to gel. Chlorhexidine and alexidine are commonly used for surgical scrubs, for handwashing in clinical settings, and in prescription oral rinses.
• Alkylating agents effectively sterilize materials at low temperatures but are carcinogenic and may also irritate tissue. Glutaraldehyde and o-phthalaldehyde are used as hospital disinfectants but not as antiseptics. Formaldehyde is used for the storage of tissue specimens, as an embalming fluid, and in vaccine preparation to inactivate infectious agents. Ethylene oxide is a gas sterilant that can permeate heat-sensitive packaged materials, but it is also explosive and carcinogenic.
• Peroxygens, including hydrogen peroxide, peracetic acid, benzoyl peroxide, and ozone gas, are strong oxidizing agents that produce free radicals in cells, damaging their macromolecules. They are environmentally safe and are highly effective disinfectants and antiseptics.
• Pressurized carbon dioxide in the form of a supercritical fluid easily permeates packaged materials and cells, forming carbonic acid and lowering intracellular pH. Supercritical carbon dioxide is nonreactive, nontoxic, nonflammable, and effective at low temperatures for sterilization of medical devices, implants, and transplanted tissues.
• Chemical preservatives are added to a variety of foods. Sorbic acid, benzoic acid, propionic acid, and their more soluble salts inhibit enzymes or reduce intracellular pH.
• Sulfites are used in winemaking and food processing to prevent browning of foods.
• Nitrites are used to preserve meats and maintain color, but cooking nitrite-preserved meats may produce carcinogenic nitrosamines.
• Nisin and natamycin are naturally produced preservatives used in cheeses and meats. Nisin is effective against gram-positive bacteria and natamycin against fungi.
Footnotes
1. 1 US Food and Drug Administration. “Triclosan: What Consumers Should Know.” 2015. www.fda.gov/ForConsumers/Cons.../ucm205999.htm. Accessed June 9, 2016.
2. 2 J. Stromberg. “Five Reasons Why You Should Probably Stop Using Antibacterial Soap.” Smithsonian.com January 3, 2014. www.smithsonianmag.com/scienc...948078/?no-ist. Accessed June 9, 2016.
3. 3 SP Yazdankhah et al. “Triclosan and Antimicrobial Resistance in Bacteria: An Overview.” Microbial Drug Resistance 12 no. 2 (2006):83–90.
4. 4 L. Birošová, M. Mikulášová. “Development of Triclosan and Antibiotic Resistance in Salmonella enterica serovar Typhimurium.” Journal of Medical Microbiology 58 no. 4 (2009):436–441.
5. 5 AB Dann, A. Hontela. “Triclosan: Environmental Exposure, Toxicity and Mechanisms of Action.” Journal of Applied Toxicology 31 no. 4 (2011):285–311.
6. 6 US Centers for Disease Control and Prevention. “Triclosan Fact Sheet.” 2013. www.cdc.gov/biomonitoring/Tri...FactSheet.html. Accessed June 9, 2016.
7. 7 EM Clayton et al. “The Impact of Bisphenol A and Triclosan on Immune Parameters in the US Population, NHANES 2003-2006.” Environmental Health Perspectives 119 no. 3 (2011):390.
8. 8 N. Silvestry-Rodriguez et al. “Silver as a Disinfectant.” In Reviews of Environmental Contamination and Toxicology, pp. 23-45. Edited by GW Ware and DM Whitacre. New York: Springer, 2007.
9. 9 B. Owens. “Silver Makes Antibiotics Thousands of Times More Effective.” Nature June 19 2013. http://www.nature.com/news/silver-ma...ective-1.13232
10. 10 C. Seiler, TU Berendonk. “Heavy Metal Driven Co-Selection of Antibiotic Resistance in Soil and Water Bodies Impacted by Agriculture and Aquaculture.” Frontiers in Microbiology 3 (2012):399.
11. 11 World Health Organization. “Benefits and Risks of the Use of Chlorine-Containing Disinfectants in Food Production and Food Processing: Report of a Joint FAO/WHO Expert Meeting.” Geneva, Switzerland: World Health Organization, 2009.
12. 12 RE Marquis. “Antimicrobial Actions of Fluoride for Oral Bacteria.” Canadian Journal of Microbiology 41 no. 11 (1995):955–964.
13. 13 L. Thomas et al. “Development of Resistance to Chlorhexidine Diacetate in Pseudomonas aeruginosa and the Effect of a ‘Residual’ Concentration.” Journal of Hospital Infection 46 no. 4 (2000):297–303.
14. 14 Institute of Medicine. “Long-Term Health Effects of Participation in Project SHAD (Shipboard Hazard and Defense).” Washington, DC: The National Academies Press, 2007.
15. 15 Yao, C.S. et al. “In vitro antibacterial effect of carbamide peroxide on oral biofilm.” Journal of Oral Microbiology Jun 12, 2013. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682087/. doi: 10.3402/jom.v5i0.20392. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/13%3A_Control_of_Microbial_Growth/13.03%3A_Using_Chemicals_to_Control_Microorganisms.txt |
Learning Objectives
• Describe why the phenol coefficient is used
• Compare and contrast the disk-diffusion, use-dilution, and in-use methods for testing the effectiveness of antiseptics, disinfectants, and sterilants
The effectiveness of various chemical disinfectants is reflected in the terms used to describe them. Chemical disinfectants are grouped by the power of their activity, with each category reflecting the types of microbes and viruses its component disinfectants are effective against. High-level germicides have the ability to kill vegetative cells, fungi, viruses, and endospores, leading to sterilization, with extended use. Intermediate-level germicides, as their name suggests, are less effective against endospores and certain viruses, and low-level germicides kill only vegetative cells and certain enveloped viruses, and are ineffective against endospores.
However, several environmental conditions influence the potency of an antimicrobial agent and its effectiveness. For example, length of exposure is particularly important, with longer exposure increasing efficacy. Similarly, the concentration of the chemical agent is also important, with higher concentrations being more effective than lower ones. Temperature, pH, and other factors can also affect the potency of a disinfecting agent.
One method to determine the effectiveness of a chemical agent includes swabbing surfaces before and after use to confirm whether a sterile field was maintained during use. Additional tests are described in the sections that follow. These tests allow for the maintenance of appropriate disinfection protocols in clinical settings, controlling microbial growth to protect patients, health-care workers, and the community.
Phenol Coefficient
The effectiveness of a disinfectant or antiseptic can be determined in a number of ways. Historically, a chemical agent’s effectiveness was often compared with that of phenol, the first chemical agent used by Joseph Lister. In 1903, British chemists Samuel Rideal (1863–1929) and J. T. Ainslie Walker (1868–1930) established a protocol to compare the effectiveness of a variety of chemicals with that of phenol, using as their test organisms Staphylococcus aureus (a gram-positive bacterium) and Salmonella enterica serovar Typhi (a gram-negative bacterium). They exposed the test bacteria to the antimicrobial chemical solutions diluted in water for 7.5 minutes. They then calculated a phenol coefficient for each chemical for each of the two bacteria tested. A phenol coefficient of 1.0 means that the chemical agent has about the same level of effectiveness as phenol. A chemical agent with a phenol coefficient of less than 1.0 is less effective than phenol. An example is formalin, with phenol coefficients of 0.3 (S. aureus) and 0.7 (S. enterica serovar Typhi). A chemical agent with a phenol coefficient greater than 1.0 is more effective than phenol, such as chloramine, with phenol coefficients of 133 and 100, respectively. Although the phenol coefficient was once a useful measure of effectiveness, it is no longer commonly used because the conditions and organisms used were arbitrarily chosen.
Exercise \(1\)
What are the differences between the three levels of disinfectant effectiveness?
Disk-Diffusion Method
The disk-diffusion method involves applying different chemicals to separate, sterile filter paper disks (Figure \(1\)). The disks are then placed on an agar plate that has been inoculated with the targeted bacterium and the chemicals diffuse out of the disks into the agar where the bacteria have been inoculated. As the “lawn” of bacteria grows, zones of inhibition of microbial growth are observed as clear areas around the disks. Although there are other factors that contribute to the sizes of zones of inhibition (e.g., whether the agent is water soluble and able to diffuse in the agar), larger zones typically correlate to increased inhibition effectiveness of the chemical agent. The diameter across each zone is measured in millimeters.
Exercise \(2\)
When comparing the activities of two disinfectants against the same microbe, using the disk-diffusion assay, and assuming both are water soluble and can easily diffuse in the agar, would a more effective disinfectant have a larger zone of inhibition or a smaller one?
Use-Dilution Test
Other methods are also used for measuring the effectiveness of a chemical agent in clinical settings. The use-dilution test is commonly used to determine a chemical’s disinfection effectiveness on an inanimate surface. For this test, a cylinder of stainless steel is dipped in a culture of the targeted microorganism and then dried. The cylinder is then dipped in solutions of disinfectant at various concentrations for a specified amount of time. Finally, the cylinder is transferred to a new test tube containing fresh sterile medium that does not contain disinfectant, and this test tube is incubated. Bacterial survival is demonstrated by the presence of turbidity in the medium, whereas killing of the target organism on the cylinder by the disinfectant will produce no turbidity.
The Association of Official Agricultural Chemists International (AOAC), a nonprofit group that establishes many protocol standards, has determined that a minimum of 59 of 60 replicates must show no growth in such a test to achieve a passing result, and the results must be repeatable from different batches of disinfectant and when performed on different days. Disinfectant manufacturers perform use-dilution tests to validate the efficacy claims for their products, as designated by the EPA.
Exercise \(3\)
Is the use-dilution test performed in a clinical setting? Why?
In-Use Test
An in-use test can determine whether an actively used solution of disinfectant in a clinical setting is microbially contaminated (Figure \(2\)). A 1-mL sample of the used disinfectant is diluted into 9 mL of sterile broth medium that also contains a compound to inactivate the disinfectant. Ten drops, totaling approximately 0.2 mL of this mixture, are then inoculated onto each of two agar plates. One plate is incubated at 37 °C for 3 days and the other is incubated at room temperature for 7 days. The plates are monitored for growth of microbial colonies. Growth of five or more colonies on either plate suggests that viable microbial cells existed in the disinfectant solution and that it is contaminated. Such in-use tests monitor the effectiveness of disinfectants in the clinical setting.
Exercise \(4\)
What does a positive in-use test indicate?
Clinical Focus: Resolution
Despite antibiotic treatment, Roberta’s symptoms worsened. She developed pyelonephritis, a severe kidney infection, and was rehospitalized in the intensive care unit (ICU). Her condition continued to deteriorate, and she developed symptoms of septic shock. At this point, her physician ordered a culture from her urine to determine the exact cause of her infection, as well as a drug sensitivity test to determine what antibiotics would be effective against the causative bacterium. The results of this test indicated resistance to a wide range of antibiotics, including the carbapenems, a class of antibiotics that are used as the last resort for many types of bacterial infections. This was an alarming outcome, suggesting that Roberta’s infection was caused by a so-called superbug: a bacterial strain that has developed resistance to the majority of commonly used antibiotics. In this case, the causative agent belonged to the carbapenem-resistant Enterobacteriaceae (CRE), a drug-resistant family of bacteria normally found in the digestive system (Figure \(3\)). When CRE is introduced to other body systems, as might occur through improperly cleaned surgical instruments, catheters, or endoscopes, aggressive infections can occur.
CRE infections are notoriously difficult to treat, with a 40%–50% fatality rate. To treat her kidney infection and septic shock, Roberta was treated with dialysis, intravenous fluids, and medications to maintain blood pressure and prevent blood clotting. She was also started on aggressive treatment with intravenous administration of a new drug called tigecycline, which has been successful in treating infections caused by drug-resistant bacteria.
After several weeks in the ICU, Roberta recovered from her CRE infection. However, public health officials soon noticed that Roberta’s case was not isolated. Several patients who underwent similar procedures at the same hospital also developed CRE infections, some dying as a result. Ultimately, the source of the infection was traced to the duodenoscopes used in the procedures. Despite the hospital staff meticulously following manufacturer protocols for disinfection, bacteria, including CRE, remained within the instruments and were introduced to patients during procedures.
Who Is Responsible?
Carbapenem-resistant Enterobacteriaceae infections due to contaminated endoscopes have become a high-profile problem in recent years. Several CRE outbreaks have been traced to endoscopes, including a case at Ronald Reagan UCLA Medical Center in early 2015 in which 179 patients may have been exposed to a contaminated endoscope. Seven of the patients developed infections, and two later died. Several lawsuits have been filed against Olympus, the manufacturer of the endoscopes. Some claim that Olympus did not obtain FDA approval for design changes that may have led to contamination, and others claim that the manufacturer knowingly withheld information from hospitals concerning defects in the endoscopes.
Lawsuits like these raise difficult-to-answer questions about liability. Invasive procedures are inherently risky, but negative outcomes can be minimized by strict adherence to established protocols. Who is responsible, however, when negative outcomes occur due to flawed protocols or faulty equipment? Can hospitals or health-care workers be held liable if they have strictly followed a flawed procedure? Should manufacturers be held liable—and perhaps be driven out of business—if their lifesaving equipment fails or is found defective? What is the government’s role in ensuring that use and maintenance of medical equipment and protocols are fail-safe?
Protocols for cleaning or sterilizing medical equipment are often developed by government agencies like the FDA, and other groups, like the AOAC, a nonprofit scientific organization that establishes many protocols for standard use globally. These procedures and protocols are then adopted by medical device and equipment manufacturers. Ultimately, the end-users (hospitals and their staff) are responsible for following these procedures and can be held liable if a breach occurs and patients become ill from improperly cleaned equipment.
Unfortunately, protocols are not infallible, and sometimes it takes negative outcomes to reveal their flaws. In 2008, the FDA had approved a disinfection protocol for endoscopes, using glutaraldehyde (at a lower concentration when mixed with phenol), o-phthalaldehyde, hydrogen peroxide, peracetic acid, and a mix of hydrogen peroxide with peracetic acid. However, subsequent CRE outbreaks from endoscope use showed that this protocol alone was inadequate.
As a result of CRE outbreaks, hospitals, manufacturers, and the FDA are investigating solutions. Many hospitals are instituting more rigorous cleaning procedures than those mandated by the FDA. Manufacturers are looking for ways to redesign duodenoscopes to minimize hard-to-reach crevices where bacteria can escape disinfectants, and the FDA is updating its protocols. In February 2015, the FDA added new recommendations for careful hand cleaning of the duodenoscope elevator mechanism (the location where microbes are most likely to escape disinfection), and issued more careful documentation about quality control of disinfection protocols (Figure \(4\)).
There is no guarantee that new procedures, protocols, or equipment will completely eliminate the risk for infection associated with endoscopes. Yet these devices are used successfully in 500,000–650,000 procedures annually in the United States, many of them lifesaving. At what point do the risks outweigh the benefits of these devices, and who should be held responsible when negative outcomes occur?
Key Concepts and Summary
• Chemical disinfectants are grouped by the types of microbes and infectious agents they are effective against. High-level germicides kill vegetative cells, fungi, viruses, and endospores, and can ultimately lead to sterilization. Intermediate-level germicides cannot kill all viruses and are less effective against endospores. Low-level germicides kill vegetative cells and some enveloped viruses, but are ineffective against endospores.
• The effectiveness of a disinfectant is influenced by several factors, including length of exposure, concentration of disinfectant, temperature, and pH.
• Historically, the effectiveness of a chemical disinfectant was compared with that of phenol at killing Staphylococcus aureus and Salmonella enterica serovar Typhi, and a phenol coefficient was calculated.
• The disk-diffusion method is used to test the effectiveness of a chemical disinfectant against a particular microbe.
• The use-dilution test determines the effectiveness of a disinfectant on a surface. In-use tests can determine whether disinfectant solutions are being used correctly in clinical settings. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/13%3A_Control_of_Microbial_Growth/13.04%3A_Testing_the_Effectiveness_of_Antiseptics_and_Disinfectants.txt |
13.1: Controlling Microbial Growth
Inanimate items, such as doorknobs, toys, or towels, which may harbor microbes and aid in disease transmission, are called fomites. Two factors heavily influence the level of cleanliness required for a particular fomite and, hence, the protocol chosen to achieve this level. The first factor is the application for which the item will be used and the second factor is the level of resistance to antimicrobial treatment by potential pathogens.
Multiple Choice
Which of the following types of medical items requires sterilization?
1. needles
2. bed linens
3. respiratory masks
4. blood pressure cuffs
Answer
A
Which of the following is suitable for use on tissues for microbial control to prevent infection?
1. disinfectant
2. antiseptic
3. sterilant
4. water
Answer
B
Which biosafety level is appropriate for research with microbes or infectious agents that pose moderate risk to laboratory workers and the community, and are typically indigenous?
1. BSL-1
2. BSL-2
3. BSL-3
4. BSL-4
Answer
B
Which of the following best describes a microbial control protocol that inhibits the growth of molds and yeast?
1. bacteriostatic
2. fungicidal
3. bactericidal
4. fungistatic
Answer
D
The decimal reduction time refers to the amount of time it takes to which of the following?
1. reduce a microbial population by 10%
2. reduce a microbial population by 0.1%
3. reduce a microbial population by 90%
4. completely eliminate a microbial population
Answer
C
Fill in the Blank
A medical item that comes into contact with intact skin and does not penetrate sterile tissues or come into contact with mucous membranes is called a(n) ________ item.
Answer
noncritical
The goal of ________ ________ protocols is to rid canned produce of Clostridium botulinum endospores.
Answer
commercial sterilization
True/False
Sanitization leaves an object free of microbes.
Answer
False
Short Answer
What are some characteristics of microbes and infectious agents that would require handling in a BSL-3 laboratory?
What is the purpose of degerming? Does it completely eliminate microbes?
What are some factors that alter the effectiveness of a disinfectant?
Critical Thinking
When plotting microbial death curves, how might they look different for bactericidal versus bacteriostatic treatments?
What are the benefits of cleaning something to a level of cleanliness beyond what is required? What are some possible disadvantages of doing so?
13.2: Using Physical Methods to Control Microorganisms
For thousands of years, humans have used various physical methods of microbial control for food preservation. Common control methods include the application of high temperatures, radiation, filtration, and desiccation (drying), among others. Many of these methods nonspecifically kill cells by disrupting membranes, changing membrane permeability, or damaging proteins and nucleic acids by denaturation, degradation, or chemical modification.
Multiple Choice
Which of the following methods brings about cell lysis due to cavitation induced by rapid localized pressure changes?
1. microwaving
2. gamma irradiation
3. ultraviolet radiation
4. sonication
Answer
D
Which of the following terms is used to describe the time required to kill all of the microbes within a sample at a given temperature?
1. D-value
2. thermal death point
3. thermal death time
4. decimal reduction time
Answer
C
Which of the following microbial control methods does not actually kill microbes or inhibit their growth but instead removes them physically from samples?
1. filtration
2. desiccation
3. lyophilization
4. nonionizing radiation
Answer
A
Fill in the Blank
In an autoclave, the application of pressure to ________ is increased to allow the steam to achieve temperatures above the boiling point of water.
Answer
steam
True/False
Ionizing radiation can penetrate surfaces, but nonionizing radiation cannot.
Answer
True
Moist-heat sterilization protocols require the use of higher temperatures for longer periods of time than do dry-heat sterilization protocols do.
Answer
False
Short Answer
What is the advantage of HTST pasteurization compared with sterilization? What is an advantage of UHT treatment?
How does the addition of salt or sugar help preserve food?
Which is more effective at killing microbes: autoclaving or freezing? Explain.
Critical Thinking
In 2001, endospores of Bacillus anthracis, the causative agent of anthrax, were sent to government officials and news agencies via the mail. In response, the US Postal Service began to irradiate mail with UV light. Was this an effective strategy? Why or why not?
13.3: Using Chemicals to Control Microorganisms
In addition to physical methods of microbial control, chemicals are also used to control microbial growth. A wide variety of chemicals can be used as disinfectants or antiseptics. This section describes the variety of chemicals used as disinfectants and antiseptics, including their mechanisms of action and common uses.
Multiple Choice
Which of the following refers to a disinfecting chemical dissolved in alcohol?
1. iodophor
2. tincture
3. phenolic
4. peroxygen
Answer
B
Which of the following peroxygens is widely used as a household disinfectant, is inexpensive, and breaks down into water and oxygen gas?
1. hydrogen peroxide
2. peracetic acid
3. benzoyl peroxide
4. ozone
Answer
A
Which of the following chemical food preservatives is used in the wine industry but may cause asthmatic reactions in some individuals?
1. nitrites
2. sulfites
3. propionic acid
4. benzoic acid
Answer
B
Bleach is an example of which group of chemicals used for disinfection?
1. heavy metals
2. halogens
3. quats
4. bisbiguanides
Answer
B
Which chemical disinfectant works by methylating enzymes and nucleic acids and is known for being toxic and carcinogenic?
1. sorbic acid
2. triclosan
3. formaldehyde
4. hexaclorophene
Answer
C
Fill in the Blank
Doorknobs and other surfaces in clinical settings are often coated with ________, ________, or ________ to prevent the transmission of microbes.
Answer
copper, nickel, zinc
True/False
Soaps are classified as disinfectants.
Answer
False
Mercury-based compounds have fallen out of favor for use as preservatives and antiseptics.
Answer
True
Short Answer
Which solution of ethyl alcohol is more effective at inhibiting microbial growth: a 70% solution or a 100% solution? Why?
When might a gas treatment be used to control microbial growth instead of autoclaving? What are some examples?
What is the advantage of using an iodophor rather than iodine or an iodine tincture?
Critical Thinking
Looking at Figure 13.3.11 and reviewing the functional groups in Figure 7.1.5, which alkylating agent shown lacks an aldehyde group?
Do you think naturally produced antimicrobial products like nisin and natamycin should replace sorbic acid for food preservation? Why or why not?
Why is the use of skin disinfecting compounds required for surgical scrubbing and not for everyday handwashing?
13.4: Testing the Effectiveness of Antiseptics and Disinfectants
Several environmental conditions influence the potency of an antimicrobial agent and its effectiveness. For example, length of exposure is particularly important, with longer exposure increasing efficacy. Similarly, the concentration of the chemical agent is also important, with higher concentrations being more effective than lower ones. Temperature, pH, and other factors can also affect the potency of a disinfecting agent.
Multiple Choice
Which type of test is used to determine whether disinfectant solutions actively used in a clinical setting are being used correctly?
1. disk-diffusion assay
2. phenol coefficient test
3. in-use test
4. use-dilution test
Answer
C
The effectiveness of chemical disinfectants has historically been compared to that of which of the following?
1. phenol
2. ethyl alcohol
3. bleach
4. formaldehyde
Answer
A
Which of the following refers to a germicide that can kill vegetative cells and certain enveloped viruses but not endospores?
1. high-level germicide
2. intermediate-level germicide
3. low-level germicide
4. sterilant
Answer
C
Fill in the Blank
If a chemical disinfectant is more effective than phenol, then its phenol coefficient would be ________ than 1.0.
Answer
greater
If used for extended periods of time, ________ germicides may lead to sterility.
Answer
high-level
In the disk-diffusion assay, a large zone of inhibition around a disk to which a chemical disinfectant has been applied indicates ________ of the test microbe to the chemical disinfectant.
Answer
susceptibility or sensitivity
Short Answer
Why were chemical disinfectants once commonly compared with phenol?
Why is length of exposure to a chemical disinfectant important for its activity?
Critical Thinking
What are some advantages of use-dilution and in-use tests compared with the disk-diffusion assay? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/13%3A_Control_of_Microbial_Growth/13.E%3A_Control_of_Microbial_Growth_%28Exercises%29.txt |
In nature, some microbes produce substances that inhibit or kill other microbes that might otherwise compete for the same resources. Humans have successfully exploited these abilities, using microbes to mass-produce substances that can be used as antimicrobial drugs. Since their discovery, antimicrobial drugs have saved countless lives, and they remain an essential tool for treating and controlling infectious disease. But their widespread and often unnecessary use has had an unintended side effect: the rise of multidrug-resistant microbial strains. In this chapter, we will discuss how antimicrobial drugs work, why microbes develop resistance, and what health professionals can do to encourage responsible use of antimicrobials.
• 14.1: Discovering Antimicrobial Drugs
Antimicrobial drugs produced by purposeful fermentation and/or contained in plants have been used as traditional medicines in many cultures for millennia. The purposeful and systematic search for a chemical “magic bullet” that specifically target infectious microbes was initiated by Paul Ehrlich in the early 20th century. The discovery of the natural antibiotic, penicillin, by Alexander Fleming in 1928 started the modern age of antimicrobial discovery and research.
• 14.2: Antibacterial Drugs
Antimicrobial drugs can be bacteriostatic or bactericidal, and these characteristics are important considerations when selecting the most appropriate drug. The use of narrow-spectrum antimicrobial drugs is preferred in many cases to avoid superinfection and the development of antimicrobial resistance. Broad-spectrum antimicrobial use is warranted for serious systemic infections when there is no time to determine the causative agent or when narrow-spectrum antimicrobials fail.
• 14.3: Drugs Targeting Other Microorganisms
Antibacterial compounds exhibit selective toxicity, largely due to differences between prokaryotic and eukaryotic cell structure. Cell wall synthesis inhibitors, including the β-lactams, the glycopeptides, and bacitracin, interfere with peptidoglycan synthesis, making bacterial cells more prone to osmotic lysis. There are a variety of broad-spectrum, bacterial protein synthesis inhibitors that selectively target the prokaryotic 70S ribosome, including those that bind to the 30S and 50S subunits.
• 14.4: Clinical Considerations
Because fungi, protozoans, and helminths are eukaryotic organisms like human cells, it is more challenging to develop antimicrobial drugs that specifically target them. Similarly, it is hard to target viruses because human viruses replicate inside of human cells.
• 14.5: Testing the Effectiveness of Antimicrobials
Antimicrobial resistance is on the rise and is the result of selection of drug-resistant strains in clinical environments, the overuse and misuse of antibacterials, the use of subtherapeutic doses of antibacterial drugs, and poor patient compliance with antibacterial drug therapies. Drug resistance genes are often carried on plasmids or in transposons that can undergo vertical transfer easily and between microbes through horizontal gene transfer.
• 14.6: The Emergence of Drug Resistance
The Kirby-Bauer disk diffusion test helps determine the susceptibility of a microorganism to various antimicrobial drugs. However, the zones of inhibition measured must be correlated to known standards to determine susceptibility and resistance, and do not provide information on bactericidal versus bacteriostatic activity, or allow for direct comparison of drug potencies. Antibiograms are useful for monitoring local trends in antimicrobial resistance/susceptibility.
• 14.7: Current Strategies for Antimicrobial Discovery
With the continued evolution and spread of antimicrobial resistance, and now the identification of pan-resistant bacterial pathogens, the search for new antimicrobials is essential for preventing the postantibiotic era.
• 14.E: Antimicrobial Drugs (Exercises)
Footnotes
1. 1 “Treatment of War Wounds: A Historical Review.” Clinical Orthopaedics and Related Research 467 no. 8 (2009):2168–2191.
Thumbnail: Staphylococcus aureus - Antibiotics Test plate. (Public Domain; CDC / Provider: Don Stalons).
14: Antimicrobial Drugs
Learning Objectives
• Compare and contrast natural, semisynthetic, and synthetic antimicrobial drugs
• Describe the chemotherapeutic approaches of ancient societies
• Describe the historically important individuals and events that led to the development of antimicrobial drugs
Clinical Focus: Part 1
Marisa, a 52-year-old woman, was suffering from severe abdominal pain, swollen lymph nodes, fatigue, and a fever. She had just returned home from visiting extended family in her native country of Cambodia. While abroad, she received medical care in neighboring Vietnam for a compressed spinal cord. She still had discomfort when leaving Cambodia, but the pain increased as her trip home continued and her husband drove her straight from the airport to the emergency room.
Her doctor considers whether Marisa could be suffering from appendicitis, a urinary tract infection (UTI), or pelvic inflammatory disease (PID). However, each of those conditions is typically preceded or accompanied by additional symptoms. He considers the treatment she received in Vietnam for her compressed spinal cord, but abdominal pain is not usually associated with spinal cord compression. He examines her health history further.
Exercise \(1\)
1. What type of infection or other condition may be responsible?
2. What type of lab tests might the doctor order?
Most people associate the term chemotherapy with treatments for cancer. However, chemotherapy is actually a broader term that refers to any use of chemicals or drugs to treat disease. Chemotherapy may involve drugs that target cancerous cells or tissues, or it may involve antimicrobial drugs that target infectious microorganisms. Antimicrobial drugs typically work by destroying or interfering with microbial structures and enzymes, either killing microbial cells or inhibiting of their growth. But before we examine how these drugs work, we will briefly explore the history of humans’ use of antimicrobials for the purpose of chemotherapy.
Use of Antimicrobials in Ancient Societies
Although the discovery of antimicrobials and their subsequent widespread use is commonly associated with modern medicine, there is evidence that humans have been exposed to antimicrobial compounds for millennia. Chemical analyses of the skeletal remains of people from Nubia1 (now found in present-day Sudan) dating from between 350 and 550 AD have shown residue of the antimicrobial agent tetracycline in high enough quantities to suggest the purposeful fermentation of tetracycline-producing Streptomyces during the beer-making process. The resulting beer, which was thick and gruel-like, was used to treat a variety of ailments in both adults and children, including gum disease and wounds. The antimicrobial properties of certain plants may also have been recognized by various cultures around the world, including Indian and Chinese herbalists (Figure \(1\)) who have long used plants for a wide variety of medical purposes. Healers of many cultures understood the antimicrobial properties of fungi and their use of moldy bread or other mold-containing products to treat wounds has been well documented for centuries.2 Today, while about 80% of the world’s population still relies on plant-derived medicines,3 scientists are now discovering the active compounds conferring the medicinal benefits contained in many of these traditionally used plants.
Exercise \(2\)
Give examples of how antimicrobials were used in ancient societies
The First Antimicrobial Drugs
Societies relied on traditional medicine for thousands of years; however, the first half of the 20th century brought an era of strategic drug discovery. In the early 1900s, the German physician and scientist Paul Ehrlich (1854–1915) set out to discover or synthesize chemical compounds capable of killing infectious microbes without harming the patient. In 1909, after screening more than 600 arsenic-containing compounds, Ehrlich’s assistant Sahachiro Hata (1873–1938) found one such “magic bullet.” Compound 606 targeted the bacterium Treponema pallidum, the causative agent of syphilis. Compound 606 was found to successfully cure syphilis in rabbits and soon after was marketed under the name Salvarsan as a remedy for the disease in humans (Figure \(2\)). Ehrlich’s innovative approach of systematically screening a wide variety of compounds remains a common strategy for the discovery of new antimicrobial agents even today.
A few decades later, German scientists Josef Klarer, Fritz Mietzsch, and Gerhard Domagk discovered the antibacterial activity of a synthetic dye, prontosil, that could treat streptococcal and staphylococcal infections in mice. Domagk’s own daughter was one of the first human recipients of the drug, which completely cured her of a severe streptococcal infection that had resulted from a poke with an embroidery needle. Gerhard Domagk (1895–1964) was awarded the Nobel Prize in Medicine in 1939 for his work with prontosil and sulfanilamide, the active breakdown product of prontosil in the body. Sulfanilamide, the first synthetic antimicrobial created, served as the foundation for the chemical development of a family of sulfa drugs. A synthetic antimicrobial is a drug that is developed from a chemical not found in nature. The success of the sulfa drugs led to the discovery and production of additional important classes of synthetic antimicrobials, including the quinolines and oxazolidinones.
A few years before the discovery of prontosil, scientist Alexander Fleming (1881–1955) made his own accidental discovery that turned out to be monumental. In 1928, Fleming returned from holiday and examined some old plates of staphylococci in his research laboratory at St. Mary’s Hospital in London. He observed that contaminating mold growth (subsequently identified as a strain of Penicillium notatum) inhibited staphylococcal growth on one plate. Fleming, therefore, is credited with the discovery of penicillin, the first natural antibiotic, (Figure \(3\)). Further experimentation showed that penicillin from the mold was antibacterial against streptococci, meningococci, and Corynebacterium diphtheriae, the causative agent of diphtheria.
Fleming and his colleagues were credited with discovering and identifying penicillin, but its isolation and mass production were accomplished by a team of researchers at Oxford University under the direction of Howard Florey(1898–1968) and Ernst Chain (1906–1979) (Figure \(3\)). In 1940, the research team purified penicillin and reported its success as an antimicrobial agent against streptococcal infections in mice. Their subsequent work with human subjects also showed penicillin to be very effective. Because of their important work, Fleming, Florey, and Chain were awarded the Nobel Prize in Physiology and Medicine in 1945.
In the early 1940s, scientist Dorothy Hodgkin (1910–1994), who studied crystallography at Oxford University, used X-rays to analyze the structure of a variety of natural products. In 1946, she determined the structure of penicillin, for which she was awarded the Nobel Prize in Chemistry in 1964. Once the structure was understood, scientists could modify it to produce a variety of semisynthetic penicillins. A semisynthetic antimicrobial is a chemically modified derivative of a natural antibiotic. The chemical modifications are generally designed to increase the range of bacteria targeted, increase stability, decrease toxicity, or confer other properties beneficial for treating infections.
Penicillin is only one example of a natural antibiotic. Also in the 1940s, Selman Waksman (1888–1973) (Figure \(4\)), a prominent soil microbiologist at Rutgers University, led a research team that discovered several antimicrobials, including actinomycin, streptomycin, and neomycin. The discoveries of these antimicrobials stemmed from Waksman’s study of fungi and the Actinobacteria, including soil bacteria in the genus Streptomyces, known for their natural production of a wide variety of antimicrobials. His work earned him the Nobel Prize in Physiology and Medicine in 1952. The actinomycetes are the source of more than half of all natural antibiotics4 and continue to serve as an excellent reservoir for the discovery of novel antimicrobial agents. Some researchers argue that we have not yet come close to tapping the full antimicrobial potential of this group.5
Exercise \(3\)
Why is the soil a reservoir for antimicrobial resistance genes?
Key Concepts and Summary
• Antimicrobial drugs produced by purposeful fermentation and/or contained in plants have been used as traditional medicines in many cultures for millennia.
• The purposeful and systematic search for a chemical “magic bullet” that specifically target infectious microbes was initiated by Paul Ehrlich in the early 20th century.
• The discovery of the natural antibiotic, penicillin, by Alexander Fleming in 1928 started the modern age of antimicrobial discovery and research.
• Sulfanilamide, the first synthetic antimicrobial, was discovered by Gerhard Domagk and colleagues and is a breakdown product of the synthetic dye, prontosil.
Footnotes
1. 1 M.L. Nelson et al. “Brief Communication: Mass Spectroscopic Characterization of Tetracycline in the Skeletal Remains of an Ancient Population from Sudanese Nubia 350–550 CE.” American Journal of Physical Anthropology 143 no. 1 (2010):151–154.
2. 2 M. Wainwright. “Moulds in Ancient and More Recent Medicine.” Mycologist 3 no. 1 (1989):21–23.
3. 3 S. Verma, S.P. Singh. “Current and Future Status of Herbal Medicines.” Veterinary World 1 no. 11 (2008):347–350.
4. 4 J. Berdy. “Bioactive Microbial Metabolites.” The Journal of Antibiotics 58 no. 1 (2005):1–26.
5. 5 M. Baltz. “Antimicrobials from Actinomycetes: Back to the Future.” Microbe 2 no. 3 (2007):125–131. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/14%3A_Antimicrobial_Drugs/14.01%3A_Discovering_Antimicrobial_Drugs.txt |
Learning Objectives
• Contrast bacteriostatic versus bactericidal antibacterial activities
• Contrast broad-spectrum drugs versus narrow-spectrum drugs
• Explain the significance of superinfections
• Discuss the significance of dosage and the route of administration of a drug
• Identify factors and variables that can influence the side effects of a drug
• Describe the significance of positive and negative interactions between drugs
Several factors are important in choosing the most appropriate antimicrobial drug therapy, including bacteriostatic versus bactericidal mechanisms, spectrum of activity, dosage and route of administration, the potential for side effects, and the potential interactions between drugs. The following discussion will focus primarily on antibacterial drugs, but the concepts translate to other antimicrobial classes.
Bacteriostatic Versus Bactericidal
Antibacterial drugs can be either bacteriostatic or bactericidal in their interactions with target bacteria. Bacteriostatic drugs cause a reversible inhibition of growth, with bacterial growth restarting after elimination of the drug. By contrast, bactericidal drugs kill their target bacteria. The decision of whether to use a bacteriostatic or bactericidal drugs depends on the type of infection and the immune status of the patient. In a patient with strong immune defenses, bacteriostatic and bactericidal drugs can be effective in achieving clinical cure. However, when a patient is immunocompromised, a bactericidal drug is essential for the successful treatment of infections. Regardless of the immune status of the patient, life-threatening infections such as acute endocarditis require the use of a bactericidal drug.
Spectrum of Activity
The spectrum of activity of an antibacterial drug relates to diversity of targeted bacteria. A narrow-spectrum antimicrobial targets only specific subsets of bacterial pathogens. For example, some narrow-spectrum drugs only target gram-positive bacteria, whereas others target only gram-negative bacteria. If the pathogen causing an infection has been identified, it is best to use a narrow-spectrum antimicrobial and minimize collateral damage to the normal microbiota. A broad-spectrum antimicrobial targets a wide variety of bacterial pathogens, including both gram-positive and gram-negative species, and is frequently used as empiric therapy to cover a wide range of potential pathogens while waiting on the laboratory identification of the infecting pathogen. Broad-spectrum antimicrobials are also used for polymicrobic infections (mixed infection with multiple bacterial species), or as prophylactic prevention of infections with surgery/invasive procedures. Finally, broad-spectrum antimicrobials may be selected to treat an infection when a narrow-spectrum drug fails because of development of drug resistance by the target pathogen.
The risk associated with using broad-spectrum antimicrobials is that they will also target a broad spectrum of the normal microbiota, increasing the risk of a superinfection, a secondary infection in a patient having a preexisting infection. A superinfection develops when the antibacterial intended for the preexisting infection kills the protective microbiota, allowing another pathogen resistant to the antibacterial to proliferate and cause a secondary infection (Figure \(1\)). Common examples of superinfections that develop as a result of antimicrobial usage include yeast infections (candidiasis) and pseudomembranous colitis caused by Clostridium difficile, which can be fatal.
Exercise \(1\)
What is a superinfection and how does one arise?
Dosage and Route of Administration
The amount of medication given during a certain time interval is the dosage, and it must be determined carefully to ensure that optimum therapeutic drug levels are achieved at the site of infection without causing significant toxicity(side effects) to the patient. Each drug class is associated with a variety of potential side effects, and some of these are described for specific drugs later in this chapter. Despite best efforts to optimize dosing, allergic reactions and other potentially serious side effects do occur. Therefore, the goal is to select the optimum dosage that will minimize the risk of side effects while still achieving clinical cure, and there are important factors to consider when selecting the best dose and dosage interval. For example, in children, dose is based upon the patient’s mass. However, the same is not true for adults and children 12 years of age and older, for which there is typically a single standard dose regardless of the patient’s mass. With the great variability in adult body mass, some experts have argued that mass should be considered for all patients when determining appropriate dosage.1 An additional consideration is how drugs are metabolized and eliminated from the body. In general, patients with a history of liver or kidney dysfunction may experience reduced drug metabolism or clearance from the body, resulting in increased drug levels that may lead to toxicity and make them more prone to side effects.
There are also some factors specific to the drugs themselves that influence appropriate dose and time interval between doses. For example, the half-life, or rate at which 50% of a drug is eliminated from the plasma, can vary significantly between drugs. Some drugs have a short half-life of only 1 hour and must be given multiple times a day, whereas other drugs have half-lives exceeding 12 hours and can be given as a single dose every 24 hours. Although a longer half-life can be considered an advantage for an antibacterial when it comes to convenient dosing intervals, the longer half-life can also be a concern for a drug that has serious side effects because drug levels may remain toxic for a longer time. Last, some drugs are dose dependent, meaning they are more effective when administered in large doses to provide high levels for a short time at the site of infection. Others are time dependent, meaning they are more effective when lower optimum levels are maintained over a longer period of time.
The route of administration, the method used to introduce a drug into the body, is also an important consideration for drug therapy. Drugs that can be administered orally are generally preferred because patients can more conveniently take these drugs at home. However, some drugs are not absorbed easily from the gastrointestinal (GI) tract into the bloodstream. These drugs are often useful for treating diseases of the intestinal tract, such as tapeworms treated with niclosamide, or for decontaminating the bowel, as with colistin. Some drugs that are not absorbed easily, such as bacitracin, polymyxin, and several antifungals, are available as topical preparations for treatment of superficial skin infections. Sometimes, patients may not initially be able to take oral medications because of their illness (e.g., vomiting, intubation for respirator). When this occurs, and when a chosen drug is not absorbed in the GI tract, administration of the drug by a parenteral route (intravenous or intramuscular injection) is preferred and typically is performed in health-care settings. For most drugs, the plasma levels achieved by intravenous administration is substantially higher than levels achieved by oral or intramuscular administration, and this can also be an important consideration when choosing the route of administration for treating an infection (Figure \(2\)).
Exercise \(2\)
1. List five factors to consider when determining the dosage of a drug.
2. Name some typical side effects associated with drugs and identify some factors that might contribute to these side effects.
Drug Interactions
For the optimum treatment of some infections, two antibacterial drugs may be administered together to provide a synergistic interaction that is better than the efficacy of either drug alone. A classic example of synergistic combinations is trimethoprim and sulfamethoxazole (Bactrim). Individually, these two drugs provide only bacteriostatic inhibition of bacterial growth, but combined, the drugs are bactericidal.
Whereas synergistic drug interactions provide a benefit to the patient, antagonistic interactions produce harmful effects. Antagonism can occur between two antimicrobials or between antimicrobials and nonantimicrobials being used to treat other conditions. The effects vary depending on the drugs involved, but antagonistic interactions may cause loss of drug activity, decreased therapeutic levels due to increased metabolism and elimination, or increased potential for toxicitydue to decreased metabolism and elimination. As an example, some antibacterials are absorbed most effectively from the acidic environment of the stomach. If a patient takes antacids, however, this increases the pH of the stomach and negatively impacts the absorption of these antimicrobials, decreasing their effectiveness in treating an infection. Studies have also shown an association between use of some antimicrobials and failure of oral contraceptives.2
Exercise \(3\)
Explain the difference between synergistic and antagonistic drug interactions.
Resistance Police
In the United States and many other countries, most antimicrobial drugs are self-administered by patients at home. Unfortunately, many patients stop taking antimicrobials once their symptoms dissipate and they feel better. If a 10-day course of treatment is prescribed, many patients only take the drug for 5 or 6 days, unaware of the negative consequences of not completing the full course of treatment. A shorter course of treatment not only fails to kill the target organisms to expected levels, it also selects for drug-resistant variants within the target population and within the patient’s microbiota.
Patients’ nonadherence especially amplifies drug resistance when the recommended course of treatment is long. Treatment for tuberculosis (TB) is a case in point, with the recommended treatment lasting from 6 months to a year. The CDC estimates that about one-third of the world’s population is infected with TB, most living in underdeveloped or underserved regions where antimicrobial drugs are available over the counter. In such countries, there may be even lower rates of adherence than in developed areas. Nonadherence leads to antibiotic resistance and more difficulty in controlling pathogens. As a direct result, the emergence of multidrug-resistant and extensively drug-resistant strains of TB is becoming a huge problem.
Overprescription of antimicrobials also contributes to antibiotic resistance. Patients often demand antibiotics for diseases that do not require them, like viral colds and ear infections. Pharmaceutical companies aggressively market drugs to physicians and clinics, making it easy for them to give free samples to patients, and some pharmacies even offer certain antibiotics free to low-income patients with a prescription.
In recent years, various initiatives have aimed to educate parents and clinicians about the judicious use of antibiotics. However, a recent study showed that, between 2000 and 2013, the parental expectation for antimicrobial prescriptions for children actually increased (Figure \(3\)).
One possible solution is a regimen called directly observed therapy (DOT), which involves the supervised administration of medications to patients. Patients are either required to visit a health-care facility to receive their medications, or health-care providers must administer medication in patients’ homes or another designated location. DOT has been implemented in many cases for the treatment of TB and has been shown to be effective; indeed, DOT is an integral part of WHO’s global strategy for eradicating TB.3,4 But is this a practical strategy for all antibiotics? Would patients taking penicillin, for example, be more or less likely to adhere to the full course of treatment if they had to travel to a health-care facility for each dose? And who would pay for the increased cost associated with DOT? When it comes to overprescription, should someone be policing physicians or drug companies to enforce best practices? What group should assume this responsibility, and what penalties would be effective in discouraging overprescription?
Key Concepts and Summary
• Antimicrobial drugs can be bacteriostatic or bactericidal, and these characteristics are important considerations when selecting the most appropriate drug.
• The use of narrow-spectrum antimicrobial drugs is preferred in many cases to avoid superinfection and the development of antimicrobial resistance.
• Broad-spectrum antimicrobial use is warranted for serious systemic infections when there is no time to determine the causative agent, when narrow-spectrum antimicrobials fail, or for the treatment or prevention of infections with multiple types of microbes.
• The dosage and route of administration are important considerations when selecting an antimicrobial to treat and infection. Other considerations include the patient’s age, mass, ability to take oral medications, liver and kidney function, and possible interactions with other drugs the patient may be taking.
Footnotes
1. 1 M.E. Falagas, D.E. Karageorgopoulos. “Adjustment of Dosing of Antimicrobial Agents for Bodyweight in Adults.” The Lancet 375 no. 9710 (2010):248–251.
2. 2 B.D. Dickinson et al. “Drug Interactions between Oral Contraceptives and Antibiotics.” Obstetrics & Gynecology 98, no. 5 (2001):853–860.
3. 3 Centers for Disease Control and Prevention. “Tuberculosis (TB).” www.cdc.gov/tb/education/ssmo...s9reading2.htm. Accessed June 2, 2016.
4. 4 World Health Organization. “Tuberculosis (TB): The Five Elements of DOTS.” http://www.who.int/tb/dots/whatisdots/en/. Accessed June 2, 2016.
5. 5 Vaz, L.E., et al. “Prevalence of Parental Misconceptions About Antibiotic Use.” Pediatrics 136 no.2 (August 2015). DOI: 10.1542/peds.2015-0883. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/14%3A_Antimicrobial_Drugs/14.02%3A_Antibacterial_Drugs.txt |
Learning Objectives
• Describe the mechanisms of action associated with drugs that inhibit cell wall biosynthesis, protein synthesis, membrane function, nucleic acid synthesis, and metabolic pathway.
An important quality for an antimicrobial drug is selective toxicity, meaning that it selectively kills or inhibits the growth of microbial targets while causing minimal or no harm to the host. Most antimicrobial drugs currently in clinical use are antibacterial because the prokaryotic cell provides a greater variety of unique targets for selective toxicity, in comparison to fungi, parasites, and viruses. Each class of antibacterial drugs has a unique mode of action (the way in which a drug affects microbes at the cellular level), and these are summarized in Figure \(1\) and Table \(1\).
Table \(1\): Common Antibacterial Drugs by Mode of Action
Mode of Action Target Drug Class
Inhibit cell wall biosynthesis Penicillin-binding proteins β-lactams: penicillins, cephalosporins, monobactams, carbapenems
Peptidoglycan subunits Glycopeptides
Peptidoglycan subunit transport Bacitracin
Inhibit biosynthesis of proteins 30S ribosomal subunit Aminoglycosides, tetracyclines
50S ribosomal subunit Macrolides, lincosamides, chloramphenicol, oxazolidinones
Disrupt membranes Lipopolysaccharide, inner and outer membranes Polymyxin B, colistin, daptomycin
Inhibit nucleic acid synthesis RNA Rifamycin
DNA Fluoroquinolones
Antimetabolites Folic acid synthesis enzyme Sulfonamides, trimethoprim
Mycolic acid synthesis enzyme Isonicotinic acid hydrazide
Mycobacterial adenosine triphosphate (ATP) synthase inhibitor Mycobacterial ATP synthase Diarylquinoline
Inhibitors of Cell Wall Biosynthesis
Several different classes of antibacterials block steps in the biosynthesis of peptidoglycan, making cells more susceptible to osmotic lysis (Table \(2\)). Therefore, antibacterials that target cell wall biosynthesis are bactericidal in their action. Because human cells do not make peptidoglycan, this mode of action is an excellent example of selective toxicity.
Penicillin, the first antibiotic discovered, is one of several antibacterials within a class called β-lactams. This group of compounds includes the penicillins, cephalosporins, monobactams, and carbapenems, and is characterized by the presence of a β-lactam ring found within the central structure of the drug molecule (Figure \(2\)). The β-lactam antibacterials block the crosslinking of peptide chains during the biosynthesis of new peptidoglycan in the bacterial cell wall. They are able to block this process because the β-lactam structure is similar to the structure of the peptidoglycan subunit component that is recognized by the crosslinking transpeptidase enzyme, also known as a penicillin-binding protein (PBP). Although the β-lactam ring must remain unchanged for these drugs to retain their antibacterial activity, strategic chemical changes to the R groups have allowed for development of a wide variety of semisynthetic β-lactam drugs with increased potency, expanded spectrum of activity, and longer half-lives for better dosing, among other characteristics.
Penicillin G and penicillin V are natural antibiotics from fungi and are primarily active against gram-positive bacterial pathogens, and a few gram-negative bacterial pathogens such as Pasteurella multocida. Figure \(2\) summarizes the semisynthetic development of some of the penicillins. Adding an amino group (-NH2) to penicillin G created the aminopenicillins (i.e., ampicillin and amoxicillin) that have increased spectrum of activity against more gram-negative pathogens. Furthermore, the addition of a hydroxyl group (-OH) to amoxicillin increased acid stability, which allows for improved oral absorption. Methicillin is a semisynthetic penicillin that was developed to address the spread of enzymes (penicillinases) that were inactivating the other penicillins. Changing the R group of penicillin G to the more bulky dimethoxyphenyl group provided protection of the β-lactam ring from enzymatic destruction by penicillinases, giving us the first penicillinase-resistant penicillin.
Similar to the penicillins, cephalosporins contain a β-lactam ring (Figure \(2\)) and block the transpeptidase activity of penicillin-binding proteins. However, the β-lactam ring of cephalosporins is fused to a six-member ring, rather than the five-member ring found in penicillins. This chemical difference provides cephalosporins with an increased resistance to enzymatic inactivation by β-lactamases. The drug cephalosporin C was originally isolated from the fungus Cephalosporium acremonium in the 1950s and has a similar spectrum of activity to that of penicillin against gram-positive bacteria but is active against more gram-negative bacteria than penicillin. Another important structural difference is that cephalosporin C possesses two R groups, compared with just one R group for penicillin, and this provides for greater diversity in chemical alterations and development of semisynthetic cephalosporins. The family of semisynthetic cephalosporins is much larger than the penicillins, and these drugs have been classified into generations based primarily on their spectrum of activity, increasing in spectrum from the narrow-spectrum, first-generation cephalosporins to the broad-spectrum, fourth-generation cephalosporins. A new fifth-generation cephalosporin has been developed that is active against methicillin-resistant Staphylococcus aureus (MRSA).
The carbapenems and monobactams also have a β-lactam ring as part of their core structure, and they inhibit the transpeptidase activity of penicillin-binding proteins. The only monobactam used clinically is aztreonam. It is a narrow-spectrum antibacterial with activity only against gram-negative bacteria. In contrast, the carbapenem family includes a variety of semisynthetic drugs (imipenem, meropenem, and doripenem) that provide very broad-spectrum activity against gram-positive and gram-negative bacterial pathogens.
The drug vancomycin, a member of a class of compounds called the glycopeptides, was discovered in the 1950s as a natural antibiotic from the actinomycete Amycolatopsis orientalis. Similar to the β-lactams, vancomycin inhibits cell wall biosynthesis and is bactericidal. However, in contrast to the β-lactams, the structure of vancomycin is not similar to that of cell-wall peptidoglycan subunits and does not directly inactivate penicillin-binding proteins. Rather, vancomycin is a very large, complex molecule that binds to the end of the peptide chain of cell wall precursors, creating a structural blockage that prevents the cell wall subunits from being incorporated into the growing N-acetylglucosamine and N-acetylmuramic acid (NAM-NAG) backbone of the peptidoglycan structure (transglycosylation). Vancomycin also structurally blocks transpeptidation. Vancomycin is bactericidal against gram-positive bacterial pathogens, but it is not active against gram-negative bacteria because of its inability to penetrate the protective outer membrane.
The drug bacitracin consists of a group of structurally similar peptide antibiotics originally isolated from Bacillus subtilis. Bacitracin blocks the activity of a specific cell-membrane molecule that is responsible for the movement of peptidoglycan precursors from the cytoplasm to the exterior of the cell, ultimately preventing their incorporation into the cell wall. Bacitracin is effective against a wide range of bacteria, including gram-positive organisms found on the skin, such as Staphylococcus and Streptococcus. Although it may be administered orally or intramuscularly in some circumstances, bacitracin has been shown to be nephrotoxic (damaging to the kidneys). Therefore, it is more commonly combined with neomycin and polymyxin in topical ointments such as Neosporin.
Table \(2\): Drugs that Inhibit Bacterial Cell Wall Synthesis
Mechanism of Action Drug Class Specific Drugs Natural or Semisynthetic Spectrum of Activity
Interact directly with PBPs and inhibit transpeptidase activity Penicillins Penicillin G, penicillin V Natural Narrow-spectrum against gram-positive and a few gram-negative bacteria
Ampicillin, amoxicillin Semisynthetic Narrow-spectrum against gram-positive bacteria but with increased gram-negative spectrum
Methicillin Semisynthetic Narrow-spectrum against gram-positive bacteria only, including strains producing penicillinase
Cephalosporins Cephalosporin C Natural Narrow-spectrum similar to penicillin but with increased gram-negative spectrum
First-generation cephalosporins Semisynthetic Narrow-spectrum similar to cephalosporin C
Second-generation cephalosporins Semisynthetic Narrow-spectrum but with increased gram-negative spectrum compared with first generation
Third- and fourth-generation cephalosporins Semisynthetic Broad-spectrum against gram-positive and gram-negative bacteria, including some β-lactamase producers
Fifth-generation cephalosporins Semisynthetic Broad-spectrum against gram-positive and gram-negative bacteria, including MRSA
Monobactams Aztreonam Semisynthetic Narrow-spectrum against gram-negative bacteria, including some β-lactamase producers
Carbapenems Imipenem, meropenem, doripenem Semisynthetic Broadest spectrum of the β-lactams against gram-positive and gram-negative bacteria, including many β-lactamase producers
Large molecules that bind to the peptide chain of peptidoglycan subunits, blocking transglycosylation and transpeptidation Glycopeptides Vancomycin Natural Narrow spectrum against gram-positive bacteria only, including multidrug-resistant strains
Block transport of peptidoglycan subunits across cytoplasmic membrane Bacitracin Bacitracin Natural Broad-spectrum against gram-positive and gram-negative bacteria
Exercise \(1\)
Describe the mode of action of β-lactams.
Inhibitors of Protein Biosynthesis
The cytoplasmic ribosomes found in animal cells (80S) are structurally distinct from those found in bacterial cells (70S), making protein biosynthesis a good selective target for antibacterial drugs. Several types of protein biosynthesis inhibitors are discussed in this section and are summarized in Figure \(3\).
Protein Synthesis Inhibitors That Bind the 30S Subunit
Aminoglycosides are large, highly polar antibacterial drugs that bind to the 30S subunit of bacterial ribosomes, impairing the proofreading ability of the ribosomal complex. This impairment causes mismatches between codons and anticodons, resulting in the production of proteins with incorrect amino acids and shortened proteins that insert into the cytoplasmic membrane. Disruption of the cytoplasmic membrane by the faulty proteins kills the bacterial cells. The aminoglycosides, which include drugs such as streptomycin, gentamicin, neomycin, and kanamycin, are potent broad-spectrum antibacterials. However, aminoglycosides have been shown to be nephrotoxic (damaging to kidney), neurotoxic (damaging to the nervous system), and ototoxic (damaging to the ear).
Another class of antibacterial compounds that bind to the 30S subunit is the tetracyclines. In contrast to aminoglycosides, these drugs are bacteriostatic and inhibit protein synthesis by blocking the association of tRNAs with the ribosome during translation. Naturally occurring tetracyclines produced by various strains of Streptomyces were first discovered in the 1940s, and several semisynthetic tetracyclines, including doxycycline and tigecycline have also been produced. Although the tetracyclines are broad spectrum in their coverage of bacterial pathogens, side effects that can limit their use include phototoxicity, permanent discoloration of developing teeth, and liver toxicity with high doses or in patients with kidney impairment.
Protein Synthesis Inhibitors That Bind the 50S Subunit
There are several classes of antibacterial drugs that work through binding to the 50S subunit of bacterial ribosomes. The macrolide antibacterial drugs have a large, complex ring structure and are part of a larger class of naturally produced secondary metabolites called polyketides, complex compounds produced in a stepwise fashion through the repeated addition of two-carbon units by a mechanism similar to that used for fatty acid synthesis. Macrolides are broad-spectrum, bacteriostatic drugs that block elongation of proteins by inhibiting peptide bond formation between specific combinations of amino acids. The first macrolide was erythromycin. It was isolated in 1952 from Streptomyces erythreus and prevents translocation. Semisynthetic macrolides include azithromycin and telithromycin. Compared with erythromycin, azithromycin has a broader spectrum of activity, fewer side effects, and a significantly longer half-life (1.5 hours for erythromycin versus 68 hours for azithromycin) that allows for once-daily dosing and a short 3-day course of therapy (i.e., Zpac formulation) for most infections. Telithromycin is the first semisynthetic within the class known as ketolides. Although telithromycin shows increased potency and activity against macrolide-resistant pathogens, the US Food and Drug Administration (FDA) has limited its use to treatment of community-acquired pneumonia and requires the strongest “black box warning” label for the drug because of serious hepatotoxicity.
The lincosamides include the naturally produced lincomycin and semisynthetic clindamycin. Although structurally distinct from macrolides, lincosamides are similar in their mode of action to the macrolides through binding to the 50S ribosomal subunit and preventing peptide bond formation. Lincosamides are particularly active against streptococcal and staphylococcal infections.
The drug chloramphenicol represents yet another structurally distinct class of antibacterials that also bind to the 50S ribosome, inhibiting peptide bond formation. Chloramphenicol, produced by Streptomyces venezuelae, was discovered in 1947; in 1949, it became the first broad-spectrum antibiotic that was approved by the FDA. Although it is a natural antibiotic, it is also easily synthesized and was the first antibacterial drug synthetically mass produced. As a result of its mass production, broad-spectrum coverage, and ability to penetrate into tissues efficiently, chloramphenicol was historically used to treat a wide range of infections, from meningitis to typhoid fever to conjunctivitis. Unfortunately, serious side effects, such as lethal gray baby syndrome, and suppression of bone marrow production, have limited its clinical role. Chloramphenicol also causes anemia in two different ways. One mechanism involves the targeting of mitochondrial ribosomes within hematopoietic stem cells, causing a reversible, dose-dependent suppression of blood cell production. Once chloramphenicol dosing is discontinued, blood cell production returns to normal. This mechanism highlights the similarity between 70S ribosomes of bacteria and the 70S ribosomes within our mitochondria. The second mechanism of anemia is idiosyncratic (i.e., the mechanism is not understood), and involves an irreversible lethal loss of blood cell production known as aplastic anemia. This mechanism of aplastic anemia is not dose dependent and can develop after therapy has stopped. Because of toxicity concerns, chloramphenicol usage in humans is now rare in the United States and is limited to severe infections unable to be treated by less toxic antibiotics. Because its side effects are much less severe in animals, it is used in veterinary medicine.
The oxazolidinones, including linezolid, are a new broad-spectrum class of synthetic protein synthesis inhibitors that bind to the 50S ribosomal subunit of both gram-positive and gram-negative bacteria. However, their mechanism of action seems somewhat different from that of the other 50S subunit-binding protein synthesis inhibitors already discussed. Instead, they seem to interfere with formation of the initiation complex (association of the 50S subunit, 30S subunit, and other factors) for translation, and they prevent translocation of the growing protein from the ribosomal A site to the P site. Table \(3\) summarizes the protein synthesis inhibitors.
Table \(3\): Drugs That Inhibit Bacterial Protein Synthesis
Molecular Target Mechanism of Action Drug Class Specific Drugs Bacteriostatic or Bactericidal Spectrum of Activity
30S subunit Causes mismatches between codons and anticodons, leading to faulty proteins that insert into and disrupt cytoplasmic membrane Aminoglycosides Streptomycin, gentamicin, neomycin, kanamycin Bactericidal Broad spectrum
Blocks association of tRNAs with ribosome Tetracyclines Tetracycline, doxycycline, tigecycline Bacteriostatic Broad spectrum
50S subunit Blocks peptide bond formation between amino acids Macrolides Erythromycin, azithromycin, telithromycin Bacteriostatic Broad spectrum
Lincosamides Lincomycin, clindamycin Bacteriostatic Narrow spectrum
Not applicable Chloramphenicol Bacteriostatic Broad spectrum
Interferes with the formation of the initiation complex between 50S and 30S subunits and other factors. Oxazolidinones Linezolid Bacteriostatic Broad spectrum
Exercise \(2\)
Compare and contrast the different types of protein synthesis inhibitors.
Inhibitors of Membrane Function
A small group of antibacterials target the bacterial membrane as their mode of action (Table \(4\)). The polymyxins are natural polypeptide antibiotics that were first discovered in 1947 as products of Bacillus polymyxa; only polymyxin B and polymyxin E (colistin) have been used clinically. They are lipophilic with detergent-like properties and interact with the lipopolysaccharide component of the outer membrane of gram-negative bacteria, ultimately disrupting both their outer and inner membranes and killing the bacterial cells. Unfortunately, the membrane-targeting mechanism is not a selective toxicity, and these drugs also target and damage the membrane of cells in the kidney and nervous system when administered systemically. Because of these serious side effects and their poor absorption from the digestive tract, polymyxin B is used in over-the-counter topical antibiotic ointments (e.g., Neosporin), and oral colistin was historically used only for bowel decontamination to prevent infections originating from bowel microbes in immunocompromised patients or for those undergoing certain abdominal surgeries. However, the emergence and spread of multidrug-resistant pathogens has led to increased use of intravenous colistin in hospitals, often as a drug of last resort to treat serious infections. The antibacterial daptomycin is a cyclic lipopeptide produced by Streptomyces roseosporus that seems to work like the polymyxins, inserting in the bacterial cell membrane and disrupting it. However, in contrast to polymyxin B and colistin, which target only gram-negative bacteria, daptomycin specifically targets gram-positive bacteria. It is typically administered intravenously and seems to be well tolerated, showing reversible toxicity in skeletal muscles.
Table \(4\): Drugs That Inhibit Bacterial Membrane Function
Mechanism of Action Drug Class Specific Drugs Spectrum of Activity Clinical Use
Interacts with lipopolysaccharide in the outer membrane of gram-negative bacteria, killing the cell through the eventual disruption of the outer membrane and cytoplasmic membrane Polymyxins Polymyxin B Narrow spectrum against gram-negative bacteria, including multidrug-resistant strains Topical preparations to prevent infections in wounds
Polymyxin E (colistin) Narrow spectrum against gram-negative bacteria, including multidrug-resistant strains Oral dosing to decontaminate bowels to prevent infections in immunocompromised patients or patients undergoing invasive surgery/procedures.
Intravenous dosing to treat serious systemic infections caused by multidrug-resistant pathogens
Inserts into the cytoplasmic membrane of gram-positive bacteria, disrupting the membrane and killing the cell Lipopeptide Daptomycin Narrow spectrum against gram-positive bacteria, including multidrug-resistant strains Complicated skin and skin-structure infections and bacteremia caused by gram-positive pathogens, including MRSA
Exercise \(3\)
How do polymyxins inhibit membrane function?
Inhibitors of Nucleic Acid Synthesis
Some antibacterial drugs work by inhibiting nucleic acid synthesis (Table \(5\)). For example, metronidazole is a semisynthetic member of the nitroimidazole family that is also an antiprotozoan. It interferes with DNA replication in target cells. The drug rifampin is a semisynthetic member of the rifamycin family and functions by blocking RNA polymerase activity in bacteria. The RNA polymerase enzymes in bacteria are structurally different from those in eukaryotes, providing for selective toxicity against bacterial cells. It is used for the treatment of a variety of infections, but its primary use, often in a cocktail with other antibacterial drugs, is against mycobacteria that cause tuberculosis. Despite the selectivity of its mechanism, rifampin can induce liver enzymes to increase metabolism of other drugs being administered (antagonism), leading to hepatotoxicity (liver toxicity) and negatively influencing the bioavailability and therapeutic effect of the companion drugs.
One member of the quinolone family, a group of synthetic antimicrobials, is nalidixic acid. It was discovered in 1962 as a byproduct during the synthesis of chloroquine, an antimalarial drug. Nalidixic acid selectively inhibits the activity of bacterial DNA gyrase, blocking DNA replication. Chemical modifications to the original quinolone backbone have resulted in the production of fluoroquinolones, like ciprofloxacin and levofloxacin, which also inhibit the activity of DNA gyrase. Ciprofloxacin and levofloxacin are effective against a broad spectrum of gram-positive or gram-negative bacteria, and are among the most commonly prescribed antibiotics used to treat a wide range of infections, including urinary tract infections, respiratory infections, abdominal infections, and skin infections. However, despite their selective toxicity against DNA gyrase, side effects associated with different fluoroquinolones include phototoxicity, neurotoxicity, cardiotoxicity, glucose metabolism dysfunction, and increased risk for tendon rupture.
Table \(5\): Drugs That Inhibit Bacterial Nucleic Acid Synthesis
Mechanisms of Action Drug Class Specific Drugs Spectrum of activity Clinical Use
Inhibits bacterial RNA polymerase activity and blocks transcription, killing the cell Rifamycin Rifampin Narrow spectrum with activity against gram-positive and limited numbers of gram-negative bacteria. Also active against Mycobacterium tuberculosis. Combination therapy for treatment of tuberculosis
Inhibits the activity of DNA gyrase and blocks DNA replication, killing the cell Fluoroquinolones Ciprofloxacin, ofloxacin, moxifloxacin Broad spectrum against gram-positive and gram-negative bacteria Wide variety of skin and systemic infections
Exercise \(4\)
Why do inhibitors of bacterial nucleic acid synthesis not target host cells?
Inhibitors of Metabolic Pathways
Some synthetic drugs control bacterial infections by functioning as antimetabolites, competitive inhibitors for bacterial metabolic enzymes (Table \(6\)). The sulfonamides (sulfa drugs) are the oldest synthetic antibacterial agents and are structural analogues of para-aminobenzoic acid (PABA), an early intermediate in folic acid synthesis (Figure \(4\)). By inhibiting the enzyme involved in the production of dihydrofolic acid, sulfonamides block bacterial biosynthesis of folic acid and, subsequently, pyrimidines and purines required for nucleic acid synthesis. This mechanism of action provides bacteriostatic inhibition of growth against a wide spectrum of gram-positive and gram-negative pathogens. Because humans obtain folic acid from food instead of synthesizing it intracellularly, sulfonamides are selectively toxic for bacteria. However, allergic reactions to sulfa drugs are common. The sulfones are structurally similar to sulfonamides but are not commonly used today except for the treatment of Hansen’s disease (leprosy).
Trimethoprim is a synthetic antimicrobial compound that serves as an antimetabolite within the same folic acid synthesis pathway as sulfonamides. However, trimethoprim is a structural analogue of dihydrofolic acid and inhibits a later step in the metabolic pathway (Figure \(4\)). Trimethoprim is used in combination with the sulfa drug sulfamethoxazole to treat urinary tract infections, ear infections, and bronchitis. As discussed, the combination of trimethoprim and sulfamethoxazole is an example of antibacterial synergy. When used alone, each antimetabolite only decreases production of folic acid to a level where bacteriostatic inhibition of growth occurs. However, when used in combination, inhibition of both steps in the metabolic pathway decreases folic acid synthesis to a level that is lethal to the bacterial cell. Because of the importance of folic acid during fetal development, sulfa drugs and trimethoprim use should be carefully considered during early pregnancy.
The drug isoniazid is an antimetabolite with specific toxicity for mycobacteria and has long been used in combination with rifampin or streptomycin in the treatment of tuberculosis. It is administered as a prodrug, requiring activation through the action of an intracellular bacterial peroxidase enzyme, forming isoniazid-nicotinamide adenine dinucleotide (NAD) and isoniazid-nicotinamide adenine dinucleotide phosphate (NADP), ultimately preventing the synthesis of mycolic acid, which is essential for mycobacterial cell walls. Possible side effects of isoniazid use include hepatotoxicity, neurotoxicity, and hematologic toxicity (anemia).
Table \(6\): Antimetabolite Drugs
Metabolic Pathway Target Mechanism of Action Drug Class Specific Drugs Spectrum of Activity
Folic acid synthesis Inhibits the enzyme involved in production of dihydrofolic acid Sulfonamides Sulfamethoxazole Broad spectrum against gram-positive and gram-negative bacteria
Sulfones Dapsone
Inhibits the enzyme involved in the production of tetrahydrofolic acid Not applicable Trimethoprim Broad spectrum against gram-positive and gram-negative bacteria
Mycolic acid synthesis Interferes with the synthesis of mycolic acid Not applicable Isoniazid Narrow spectrum against Mycobacterium spp., including M. tuberculosis
Exercise \(5\)
How do sulfonamides and trimethoprim selectively target bacteria?
Inhibitor of ATP Synthase
Bedaquiline, representing the synthetic antibacterial class of compounds called the diarylquinolones, uses a novel mode of action that specifically inhibits mycobacterial growth. Although the specific mechanism has yet to be elucidated, this compound appears to interfere with the function of ATP synthases, perhaps by interfering with the use of the hydrogen ion gradient for ATP synthesis by oxidative phosphorylation, leading to reduced ATP production. Due to its side effects, including hepatotoxicity and potentially lethal heart arrhythmia, its use is reserved for serious, otherwise untreatable cases of tuberculosis.
Clinical Focus: Part 2
Reading thorough Marisa’s health history, the doctor noticed that during her hospitalization in Vietnam, she was catheterized and received the antimicrobial drugs ceftazidime and metronidazole. Upon learning this, the doctor ordered a CT scan of Marisa’s abdomen to rule out appendicitis; the doctor also requested blood work to see if she had an elevated white blood cell count, and ordered a urine analysis test and urine culture to look for the presence of white blood cells, red blood cells, and bacteria.
Marisa’s urine sample came back positive for the presence of bacteria, indicating a urinary tract infection (UTI). The doctor prescribed ciprofloxacin. In the meantime, her urine was cultured to grow the bacterium for further testing.
Exercise \(6\)
1. What types of antimicrobials are typically prescribed for UTIs?
2. Based upon the antimicrobial drugs she was given in Vietnam, which of the antimicrobials for treatment of a UTI would you predict to be ineffective?
Key Concepts and Summary
• Antibacterial compounds exhibit selective toxicity, largely due to differences between prokaryotic and eukaryotic cell structure.
• Cell wall synthesis inhibitors, including the β-lactams, the glycopeptides, and bacitracin, interfere with peptidoglycan synthesis, making bacterial cells more prone to osmotic lysis.
• There are a variety of broad-spectrum, bacterial protein synthesis inhibitors that selectively target the prokaryotic 70S ribosome, including those that bind to the 30S subunit (aminoglycosides and tetracyclines) and others that bind to the 50S subunit (macrolides, lincosamides, chloramphenicol, and oxazolidinones).
• Polymyxins are lipophilic polypeptide antibiotics that target the lipopolysaccharide component of gram-negative bacteria and ultimately disrupt the integrity of the outer and inner membranes of these bacteria.
• The nucleic acid synthesis inhibitors rifamycins and fluoroquinolones target bacterial RNA transcription and DNA replication, respectively.
• Some antibacterial drugs are antimetabolites, acting as competitive inhibitors for bacterial metabolic enzymes. Sulfonamides and trimethoprim are antimetabolites that interfere with bacterial folic acid synthesis. Isoniazid is an antimetabolite that interferes with mycolic acid synthesis in mycobacteria. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/14%3A_Antimicrobial_Drugs/14.03%3A_Drugs_Targeting_Other_Microorganisms.txt |
Learning Objectives
• Explain the differences between modes of action of drugs that target fungi, protozoa, helminths, and viruses
Because fungi, protozoa, and helminths are eukaryotic, their cells are very similar to human cells, making it more difficult to develop drugs with selective toxicity. Additionally, viruses replicate within human host cells, making it difficult to develop drugs that are selectively toxic to viruses or virus-infected cells. Despite these challenges, there are antimicrobial drugs that target fungi, protozoa, helminths, and viruses, and some even target more than one type of microbe. Table \(1\), Table \(2\), Table \(3\), and Table \(4\) provide examples for antimicrobial drugs in these various classes.
Antifungal Drugs
The most common mode of action for antifungal drugs is the disruption of the cell membrane. Antifungals take advantage of small differences between fungi and humans in the biochemical pathways that synthesize sterols. The sterols are important in maintaining proper membrane fluidity and, hence, proper function of the cell membrane. For most fungi, the predominant membrane sterol is ergosterol. Because human cell membranes use cholesterol, instead of ergosterol, antifungal drugs that target ergosterol synthesis are selectively toxic (Figure \(1\)).
The imidazoles are synthetic fungicides that disrupt ergosterol biosynthesis; they are commonly used in medical applications and also in agriculture to keep seeds and harvested crops from molding. Examples include miconazole, ketoconazole, and clotrimazole, which are used to treat fungal skin infections such as ringworm, specifically tinea pedis (athlete’s foot), tinea cruris (jock itch), and tinea corporis. These infections are commonly caused by dermatophytes of the genera Trichophyton, Epidermophyton, and Microsporum. Miconazole is also used predominantly for the treatment of vaginal yeast infections caused by the fungus Candida, and ketoconazole is used for the treatment of tinea versicolor and dandruff, which both can be caused by the fungus Malassezia.
The triazole drugs, including fluconazole, also inhibit ergosterol biosynthesis. However, they can be administered orally or intravenously for the treatment of several types of systemic yeast infections, including oral thrush and cryptococcal meningitis, both of which are prevalent in patients with AIDS. The triazoles also exhibit more selective toxicity, compared with the imidazoles, and are associated with fewer side effects.
The allylamines, a structurally different class of synthetic antifungal drugs, inhibit an earlier step in ergosterol biosynthesis. The most commonly used allylamine is terbinafine (marketed under the brand name Lamisil), which is used topically for the treatment of dermatophytic skin infections like athlete’s foot, ringworm, and jock itch. Oral treatment with terbinafine is also used for the treatment of fingernail and toenail fungus, but it can be associated with the rare side effect of hepatotoxicity.
The polyenes are a class of antifungal agents naturally produced by certain actinomycete soil bacteria and are structurally related to macrolides. These large, lipophilic molecules bind to ergosterol in fungal cytoplasmic membranes, thus creating pores. Common examples include nystatin and amphotericin B. Nystatin is typically used as a topical treatment for yeast infections of the skin, mouth, and vagina, but may also be used for intestinal fungal infections. The drug amphotericin B is used for systemic fungal infections like aspergillosis, cryptococcal meningitis, histoplasmosis, blastomycosis, and candidiasis. Amphotericin B was the only antifungal drug available for several decades, but its use is associated with some serious side effects, including nephrotoxicity (kidney toxicity).
Amphotericin B is often used in combination with flucytosine, a fluorinated pyrimidine analog that is converted by a fungal-specific enzyme into a toxic product that interferes with both DNA replication and protein synthesis in fungi. Flucytosine is also associated with hepatotoxicity (liver toxicity) and bone marrow depression.
Beyond targeting ergosterol in fungal cell membranes, there are a few antifungal drugs that target other fungal structures (Figure \(2\)). The echinocandins, including caspofungin, are a group of naturally produced antifungal compounds that block the synthesis of β(1→3) glucan found in fungal cell walls but not found in human cells. This drug class has the nickname “penicillin for fungi.” Caspofungin is used for the treatment of aspergillosis as well as systemic yeast infections.
Although chitin is only a minor constituent of fungal cell walls, it is also absent in human cells, making it a selective target. The polyoxins and nikkomycins are naturally produced antifungals that target chitin synthesis. Polyoxins are used to control fungi for agricultural purposes, and nikkomycin Z is currently under development for use in humans to treat yeast infections and Valley fever (coccidioidomycosis), a fungal disease prevalent in the southwestern US.1
The naturally produced antifungal griseofulvin is thought to specifically disrupt fungal cell division by interfering with microtubules involved in spindle formation during mitosis. It was one of the first antifungals, but its use is associated with hepatotoxicity. It is typically administered orally to treat various types of dermatophytic skin infections when other topical antifungal treatments are ineffective.
There are a few drugs that act as antimetabolites against fungal processes. For example, atovaquone, a representative of the naphthoquinone drug class, is a semisynthetic antimetabolite for fungal and protozoal versions of a mitochondrial cytochrome important in electron transport. Structurally, it is an analog of coenzyme Q, with which it competes for electron binding. It is particularly useful for the treatment of Pneumocystis pneumonia caused by Pneumocystis jirovecii. The antibacterial sulfamethoxazole-trimethoprim combination also acts as an antimetabolite against P. jirovecii.
Table \(1\) shows the various therapeutic classes of antifungal drugs, categorized by mode of action, with examples of each.
Table \(1\): Common Antifungal Drugs
Mechanism of Action Drug Class Specific Drugs Clinical Uses
Inhibit ergosterol synthesis Imidazoles Miconazole, ketoconazole, clotrimazole Fungal skin infections and vaginal yeast infections
Triazoles Fluconazole Systemic yeast infections, oral thrush, and cryptococcal meningitis
Allylamines Terbinafine Dermatophytic skin infections (athlete’s foot, ring worm, jock itch), and infections of fingernails and toenails
Bind ergosterol in the cell membrane and create pores that disrupt the membrane Polyenes Nystatin Used topically for yeast infections of skin, mouth, and vagina; also used for fungal infections of the intestine
Amphotericin B Variety systemic fungal infections
Inhibit cell wall synthesis Echinocandins Caspofungin Aspergillosis and systemic yeast infections
Not applicable Nikkomycin Z Coccidioidomycosis (Valley fever) and yeast infections
Inhibit microtubules and cell division Not applicable Griseofulvin Dermatophytic skin infections
Exercise \(1\)
How is disruption of ergosterol biosynthesis an effective mode of action for antifungals?
Treating a Fungal Infection of the Lungs
Jack, a 48-year-old engineer, is HIV positive but generally healthy thanks to antiretroviral therapy (ART). However, after a particularly intense week at work, he developed a fever and a dry cough. He assumed that he just had a cold or mild flu due to overexertion and didn’t think much of it. However, after about a week, he began to experience fatigue, weight loss, and shortness of breath. He decided to visit his physician, who found that Jack had a low level of blood oxygenation. The physician ordered blood testing, a chest X-ray, and the collection of an induced sputum sample for analysis. His X-ray showed a fine cloudiness and several pneumatoceles (thin-walled pockets of air), which indicated Pneumocystis pneumonia (PCP), a type of pneumonia caused by the fungus Pneumocystis jirovecii. Jack’s physician admitted him to the hospital and prescribed Bactrim, a combination of sulfamethoxazole and trimethoprim, to be administered intravenously.
P. jirovecii is a yeast-like fungus with a life cycle similar to that of protozoans. As such, it was classified as a protozoan until the 1980s. It lives only in the lung tissue of infected persons and is transmitted from person to person, with many people exposed as children. Typically, P. jirovecii only causes pneumonia in immunocompromised individuals. Healthy people may carry the fungus in their lungs with no symptoms of disease. PCP is particularly problematic among HIV patients with compromised immune systems.
PCP is usually treated with oral or intravenous Bactrim, but atovaquone or pentamidine(another antiparasitic drug) are alternatives. If not treated, PCP can progress, leading to a collapsed lung and nearly 100% mortality. Even with antimicrobial drug therapy, PCP still is responsible for 10% of HIV-related deaths.
The cytological examination, using direct immunofluorescence assay (DFA), of a smear from Jack’s sputum sample confirmed the presence of P. jirovecii (Figure \(3\)). Additionally, the results of Jack’s blood tests revealed that his white blood cell count had dipped, making him more susceptible to the fungus. His physician reviewed his ART regimen and made adjustments. After a few days of hospitalization, Jack was released to continue his antimicrobial therapy at home. With the adjustments to his ART therapy, Jack’s CD4 counts began to increase and he was able to go back to work.
Antiprotozoan Drugs
There are a few mechanisms by which antiprotozoan drugs target infectious protozoans (Table \(3\)). Some are antimetabolites, such as atovaquone, proguanil, and artemisinins. Atovaquone, in addition to being antifungal, blocks electron transport in protozoans and is used for the treatment of protozoan infections including malaria, babesiosis, and toxoplasmosis. Proguanil is another synthetic antimetabolite that is processed in parasitic cells into its active form, which inhibits protozoan folic acid synthesis. It is often used in combination with atovaquone, and the combination is marketed as Malarone for both malaria treatment and prevention.
Artemisinin, a plant-derived antifungal first discovered by Chinese scientists in the 1970s, is quite effective against malaria. Semisynthetic derivatives of artemisinin are more water soluble than the natural version, which makes them more bioavailable. Although the exact mechanism of action is unclear, artemisinins appear to act as prodrugs that are metabolized by target cells to produce reactive oxygen species (ROS) that damage target cells. Due to the rise in resistance to antimalarial drugs, artemisinins are also commonly used in combination with other antimalarial compounds in artemisinin-based combination therapy (ACT).
Several antimetabolites are used for the treatment of toxoplasmosis caused by the parasite Toxoplasma gondii. The synthetic sulfa drug sulfadiazine competitively inhibits an enzyme in folic acid production in parasites and can be used to treat malaria and toxoplasmosis. Pyrimethamine is a synthetic drug that inhibits a different enzyme in the folic acid production pathway and is often used in combination with sulfadoxine (another sulfa drug) for the treatment of malariaor in combination with sulfadiazine for the treatment of toxoplasmosis. Side effects of pyrimethamine include decreased bone marrow activity that may cause increased bruising and low red blood cell counts. When toxicity is a concern, spiramycin, a macrolide protein synthesis inhibitor, is typically administered for the treatment of toxoplasmosis.
Two classes of antiprotozoan drugs interfere with nucleic acid synthesis: nitroimidazoles and quinolines. Nitroimidazoles, including semisynthetic metronidazole, which was discussed previously as an antibacterial drug, and synthetic tinidazole, are useful in combating a wide variety of protozoan pathogens, such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis. Upon introduction into these cells in low-oxygen environments, nitroimidazoles become activated and introduce DNA strand breakage, interfering with DNA replication in target cells. Unfortunately, metronidazole is associated with carcinogenesis (the development of cancer) in humans.
Another type of synthetic antiprotozoan drug that has long been thought to specifically interfere with DNA replication in certain pathogens is pentamidine. It has historically been used for the treatment of African sleeping sickness (caused by the protozoan Trypanosoma brucei) and leishmaniasis (caused by protozoa of the genus Leishmania), but it is also an alternative treatment for the fungus Pneumocystis. Some studies indicate that it specifically binds to the DNA found within kinetoplasts (kDNA; long mitochondrion-like structures unique to trypanosomes), leading to the cleavage of kDNA. However, nuclear DNA of both the parasite and host remain unaffected. It also appears to bind to tRNA, inhibiting the addition of amino acids to tRNA, thus preventing protein synthesis. Possible side effects of pentamidine use include pancreatic dysfunction and liver damage.
The quinolines are a class of synthetic compounds related to quinine, which has a long history of use against malaria. Quinolines are thought to interfere with heme detoxification, which is necessary for the parasite’s effective breakdown of hemoglobin into amino acids inside red blood cells. The synthetic derivatives chloroquine, quinacrine (also called mepacrine), and mefloquine are commonly used as antimalarials, and chloroquine is also used to treat amebiasis typically caused by Entamoeba histolytica. Long-term prophylactic use of chloroquine or mefloquine may result in serious side effects, including hallucinations or cardiac issues. Patients with glucose-6-phosphate dehydrogenase deficiency experience severe anemia when treated with chloroquine.
Table \(2\): Common Antiprotozoan Drugs
Mechanism of Action Drug Class Specific Drugs Clinical Uses
Inhibit electron transport in mitochondria Naphthoquinone Atovaquone Malaria, babesiosis, and toxoplasmosis
Inhibit folic acid synthesis Not applicable Proquanil Combination therapy with atovaquone for malaria treatment and prevention
Sulfonamide Sulfadiazine Malaria and toxoplasmosis
Not applicable Pyrimethamine Combination therapy with sulfadoxine (sulfa drug) for malaria
Produces damaging reactive oxygen species Not applicable Artemisinin Combination therapy to treat malaria
Inhibit DNA synthesis Nitroimidazoles Metronidazole, tinidazole Infections caused by Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis
Not applicable Pentamidine African sleeping sickness and leishmaniasis
Inhibit heme detoxification Quinolines Chloroquine Malaria and infections with E. histolytica
Mepacrine, mefloquine Malaria
Exercise \(2\)
List two modes of action for antiprotozoan drugs.
Antihelminthic Drugs
Because helminths are multicellular eukaryotes like humans, developing drugs with selective toxicity against them is extremely challenging. Despite this, several effective classes have been developed (Table \(3\)). Synthetic benzimidazoles, like mebendazole and albendazole, bind to helminthic β-tubulin, preventing microtubule formation. Microtubules in the intestinal cells of the worms seem to be particularly affected, leading to a reduction in glucose uptake. Besides their activity against a broad range of helminths, benzimidazoles are also active against many protozoans, fungi, and viruses, and their use for inhibiting mitosis and cell cycle progression in cancer cells is under study.2 Possible side effects of their use include liver damage and bone marrow suppression.
The avermectins are members of the macrolide family that were first discovered from a Japanese soil isolate, Streptomyces avermectinius. A more potent semisynthetic derivative of avermectin is ivermectin, which binds to glutamate-gated chloride channels specific to invertebrates including helminths, blocking neuronal transmission and causing starvation, paralysis, and death of the worms. Ivermectin is used to treat roundworm diseases, including onchocerciasis (also called river blindness, caused by the worm Onchocerca volvulus) and strongyloidiasis (caused by the worm Strongyloides stercoralis or S. fuelleborni). Ivermectin also can also treat parasitic insects like mites, lice, and bed bugs, and is nontoxic to humans.
Niclosamide is a synthetic drug that has been used for over 50 years to treat tapeworm infections. Although its mode of action is not entirely clear, niclosamide appears to inhibit ATP formation under anaerobic conditions and inhibit oxidative phosphorylation in the mitochondria of its target pathogens. Niclosamide is not absorbed from the gastrointestinal tract, thus it can achieve high localized intestinal concentrations in patients. Recently, it has been shown to also have antibacterial, antiviral, and antitumor activities.345
Another synthetic antihelminthic drug is praziquantel, which used for the treatment of parasitic tapeworms and liver flukes, and is particularly useful for the treatment of schistosomiasis (caused by blood flukes from three genera of Schistosoma). Its mode of action remains unclear, but it appears to cause the influx of calcium into the worm, resulting in intense spasm and paralysis of the worm. It is often used as a preferred alternative to niclosamide in the treatment of tapeworms when gastrointestinal discomfort limits niclosamide use.
The thioxanthenones, another class of synthetic drugs structurally related to quinine, exhibit antischistosomal activity by inhibiting RNA synthesis. The thioxanthenone lucanthone and its metabolite hycanthone were the first used clinically, but serious neurological, gastrointestinal, cardiovascular, and hepatic side effects led to their discontinuation. Oxamniquine, a less toxic derivative of hycanthone, is only effective against S. mansoni, one of the three species known to cause schistosomiasis in humans. Praziquantel was developed to target the other two schistosome species, but concerns about increasing resistance have renewed interest in developing additional derivatives of oxamniquine to target all three clinically important schistosome species.
Table \(3\): Common Antihelminthic Drugs
Mechanism of Action Drug Class Specific Drugs Clinical Uses
Inhibit microtubule formation, reducing glucose uptake Benzimidazoles Mebendazole, albendazole Variety of helminth infections
Block neuronal transmission, causing paralysis and starvation Avermectins Ivermectin Roundworm diseases, including river blindness and strongyloidiasis, and treatment of parasitic insects
Inhibit ATP production Not applicable Niclosamide Intestinal tapeworm infections
Induce calcium influx Not applicable Praziquantel Schistosomiasis (blood flukes)
Inhibit RNA synthesis Thioxanthenones Lucanthone, hycanthone, oxamniquine Schistosomiasis (blood flukes)
Exercise \(3\)
Why are antihelminthic drugs difficult to develop?
Antiviral Drugs
Unlike the complex structure of fungi, protozoa, and helminths, viral structure is simple, consisting of nucleic acid, a protein coat, viral enzymes, and, sometimes, a lipid envelope. Furthermore, viruses are obligate intracellular pathogens that use the host’s cellular machinery to replicate. These characteristics make it difficult to develop drugs with selective toxicity against viruses.
Many antiviral drugs are nucleoside analogs and function by inhibiting nucleic acid biosynthesis. For example, acyclovir(marketed as Zovirax) is a synthetic analog of the nucleoside guanosine (Figure \(4\)). It is activated by the herpes simplex viral enzyme thymidine kinase and, when added to a growing DNA strand during replication, causes chain termination. Its specificity for virus-infected cells comes from both the need for a viral enzyme to activate it and the increased affinity of the activated form for viral DNA polymerase compared to host cell DNA polymerase. Acyclovir and its derivatives are frequently used for the treatment of herpes virus infections, including genital herpes, chickenpox, shingles, Epstein-Barr virus infections, and cytomegalovirus infections. Acyclovir can be administered either topically or systemically, depending on the infection. One possible side effect of its use includes nephrotoxicity. The drug adenine-arabinoside, marketed as vidarabine, is a synthetic analog to deoxyadenosine that has a mechanism of action similar to that of acyclovir. It is also effective for the treatment of various human herpes viruses. However, because of possible side effects involving low white blood cell counts and neurotoxicity, treatment with acyclovir is now preferred.
Ribavirin, another synthetic guanosine analog, works by a mechanism of action that is not entirely clear. It appears to interfere with both DNA and RNA synthesis, perhaps by reducing intracellular pools of guanosine triphosphate (GTP). Ribavarin also appears to inhibit the RNA polymerase of hepatitis C virus. It is primarily used for the treatment of the RNA viruses like hepatitis C (in combination therapy with interferon) and respiratory syncytial virus. Possible side effects of ribavirin use include anemia and developmental effects on unborn children in pregnant patients. In recent years, another nucleotide analog, sofosbuvir (Solvaldi), has also been developed for the treatment of hepatitis C. Sofosbuvir is a uridine analog that interferes with viral polymerase activity. It is commonly coadministered with ribavirin, with and without interferon.
Inhibition of nucleic acid synthesis is not the only target of synthetic antivirals. Although the mode of action of amantadine and its relative rimantadine are not entirely clear, these drugs appear to bind to a transmembrane protein that is involved in the escape of the influenza virus from endosomes. Blocking escape of the virus also prevents viral RNA release into host cells and subsequent viral replication. Increasing resistance has limited the use of amantadine and rimantadine in the treatment of influenza A. Use of amantadine can result in neurological side effects, but the side effects of rimantadine seem less severe. Interestingly, because of their effects on brain chemicals such as dopamine and NMDA (N-methyl D-aspartate), amantadine and rimantadine are also used for the treatment of Parkinson’s disease.
Neuraminidase inhibitors, including olsetamivir (Tamiflu), zanamivir (Relenza), and peramivir (Rapivab), specifically target influenza viruses by blocking the activity of influenza virus neuraminidase, preventing the release of the virus from infected cells. These three antivirals can decrease flu symptoms and shorten the duration of illness, but they differ in their modes of administration: olsetamivir is administered orally, zanamivir is inhaled, and peramivir is administered intravenously. Resistance to these neuraminidase inhibitors still seems to be minimal.
Pleconaril is a synthetic antiviral under development that showed promise for the treatment of picornaviruses. Use of pleconaril for the treatment of the common cold caused by rhinoviruses was not approved by the FDA in 2002 because of lack of proven effectiveness, lack of stability, and association with irregular menstruation. Its further development for this purpose was halted in 2007. However, pleconaril is still being investigated for use in the treatment of life-threatening complications of enteroviruses, such as meningitis and sepsis. It is also being investigated for use in the global eradication of a specific enterovirus, polio.6 Pleconaril seems to work by binding to the viral capsid and preventing the uncoating of viral particles inside host cells during viral infection.
Viruses with complex life cycles, such as HIV, can be more difficult to treat. First, HIV targets CD4-positive white blood cells, which are necessary for a normal immune response to infection. Second, HIV is a retrovirus, meaning that it converts its RNA genome into a DNA copy that integrates into the host cell’s genome, thus hiding within host cell DNA. Third, the HIV reverse transcriptase lacks proofreading activity and introduces mutations that allow for rapid development of antiviral drug resistance. To help prevent the emergence of resistance, a combination of specific synthetic antiviral drugs is typically used in ART for HIV (Figure \(5\)).
The reverse transcriptase inhibitors block the early step of converting viral RNA genome into DNA, and can include competitive nucleoside analog inhibitors (e.g., azidothymidine/zidovudine, or AZT) and non-nucleoside noncompetitive inhibitors (e.g., etravirine) that bind reverse transcriptase and cause an inactivating conformational change. Drugs called protease inhibitors (e.g., ritonavir) block the processing of viral proteins and prevent viral maturation. Protease inhibitors are also being developed for the treatment of other viral types.7 For example, simeprevir (Olysio) has been approved for the treatment of hepatitis C and is administered with ribavirin and interferon in combination therapy. The integrase inhibitors (e.g., raltegravir), block the activity of the HIV integrase responsible for the recombination of a DNA copy of the viral genome into the host cell chromosome. Additional drug classes for HIV treatment include the CCR5 antagonists and the fusion inhibitors (e.g., enfuviritide), which prevent the binding of HIV to the host cell coreceptor (chemokine receptor type 5 [CCR5]) and the merging of the viral envelope with the host cell membrane, respectively. Table \(4\) shows the various therapeutic classes of antiviral drugs, categorized by mode of action, with examples of each.
Table \(4\): Common Antiviral Drugs
Mechanism of Action Drug Clinical Uses
Nucleoside analog inhibition of nucleic acid synthesis Acyclovir Herpes virus infections
Azidothymidine/zidovudine (AZT) HIV infections
Ribavirin Hepatitis C virus and respiratory syncytial virus infections
Vidarabine Herpes virus infections
Sofosbuvir Hepatitis C virus infections
Non-nucleoside noncompetitive inhibition Etravirine HIV infections
Inhibit escape of virus from endosomes Amantadine, rimantadine Infections with influenza virus
Inhibit neuraminadase Olsetamivir, zanamivir, peramivir Infections with influenza virus
Inhibit viral uncoating Pleconaril Serious enterovirus infections
Inhibition of protease Ritonavir HIV infections
Simeprevir Hepatitis C virus infections
Inhibition of integrase Raltegravir HIV infections
Inhibition of membrane fusion Enfuviritide HIV infections
Exercise \(4\)
Why is HIV difficult to treat with antivirals?
Link to Learning
To learn more about the various classes of antiretroviral drugs used in the ART of HIV infection, explore each of the drugs in the HIV drug classes provided by US Department of Health and Human Services at this website.
Key Concepts and Summary
• Because fungi, protozoans, and helminths are eukaryotic organisms like human cells, it is more challenging to develop antimicrobial drugs that specifically target them. Similarly, it is hard to target viruses because human viruses replicate inside of human cells.
• Antifungal drugs interfere with ergosterol synthesis, bind to ergosterol to disrupt fungal cell membrane integrity, or target cell wall-specific components or other cellular proteins.
• Antiprotozoan drugs increase cellular levels of reactive oxygen species, interfere with protozoal DNA replication (nuclear versus kDNA, respectively), and disrupt heme detoxification.
• Antihelminthic drugs disrupt helminthic and protozoan microtubule formation; block neuronal transmissions; inhibit anaerobic ATP formation and/or oxidative phosphorylation; induce a calcium influx in tapeworms, leading to spasms and paralysis; and interfere with RNA synthesis in schistosomes.
• Antiviral drugs inhibit viral entry, inhibit viral uncoating, inhibit nucleic acid biosynthesis, prevent viral escape from endosomes in host cells, and prevent viral release from infected cells.
• Because it can easily mutate to become drug resistant, HIV is typically treated with a combination of several antiretroviral drugs, which may include reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and drugs that interfere with viral binding and fusion to initiate infection.
Footnotes
1. 1 Centers for Disease Control and Prevention. “Valley Fever: Awareness Is Key.” www.cdc.gov/features/valleyfever/. Accessed June 1, 2016.
2. 2 B. Chu et al. “A Benzimidazole Derivative Exhibiting Antitumor Activity Blocks EGFR and HER2 Activity and Upregulates DR5 in Breast Cancer Cells.” Cell Death and Disease 6 (2015):e1686
3. 3 J.-X. Pan et al. “Niclosamide, An Old Antihelminthic Agent, Demonstrates Antitumor Activity by Blocking Multiple Signaling Pathways of Cancer Stem Cells.” Chinese Journal of Cancer 31 no. 4 (2012):178–184.
4. 4 F. Imperi et al. “New Life for an Old Drug: The Anthelmintic Drug Niclosamide Inhibits Pseudomonas aeruginosa Quorum Sensing.” Antimicrobial Agents and Chemotherapy 57 no. 2 (2013):996-1005.
5. 5 A. Jurgeit et al. “Niclosamide Is a Proton Carrier and Targets Acidic Endosomes with Broad Antiviral Effects.” PLoS Pathogens 8 no. 10 (2012):e1002976.
6. 6 M.J. Abzug. “The Enteroviruses: Problems in Need of Treatments.” Journal of Infection 68 no. S1 (2014):108–14.
7. 7 B.L. Pearlman. “Protease Inhibitors for the Treatment of Chronic Hepatitis C Genotype-1 Infection: The New Standard of Care.” Lancet Infectious Diseases 12 no. 9 (2012):717–728. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/14%3A_Antimicrobial_Drugs/14.04%3A_Clinical_Considerations.txt |
Learning Objectives
• Explain the concept of drug resistance
• Describe how microorganisms develop or acquire drug resistance
• Describe the different mechanisms of antimicrobial drug resistance
Antimicrobial resistance is not a new phenomenon. In nature, microbes are constantly evolving in order to overcome the antimicrobial compounds produced by other microorganisms. Human development of antimicrobial drugs and their widespread clinical use has simply provided another selective pressure that promotes further evolution. Several important factors can accelerate the evolution of drug resistance. These include the overuse and misuse of antimicrobials, inappropriate use of antimicrobials, subtherapeutic dosing, and patient noncompliance with the recommended course of treatment.
Exposure of a pathogen to an antimicrobial compound can select for chromosomal mutations conferring resistance, which can be transferred vertically to subsequent microbial generations and eventually become predominant in a microbial population that is repeatedly exposed to the antimicrobial. Alternatively, many genes responsible for drug resistance are found on plasmids or in transposons that can be transferred easily between microbes through horizontal gene transfer (see How Asexual Prokaryotes Achieve Genetic Diversity). Transposons also have the ability to move resistance genes between plasmids and chromosomes to further promote the spread of resistance.
Mechanisms for Drug Resistance
There are several common mechanisms for drug resistance, which are summarized in Figure \(1\). These mechanisms include enzymatic modification of the drug, modification of the antimicrobial target, and prevention of drug penetration or accumulation.
Drug Modification or Inactivation
Resistance genes may code for enzymes that chemically modify an antimicrobial, thereby inactivating it, or destroy an antimicrobial through hydrolysis. Resistance to many types of antimicrobials occurs through this mechanism. For example, aminoglycoside resistance can occur through enzymatic transfer of chemical groups to the drug molecule, impairing the binding of the drug to its bacterial target. For β-lactams, bacterial resistance can involve the enzymatic hydrolysis of the β-lactam bond within the β-lactam ring of the drug molecule. Once the β-lactam bond is broken, the drug loses its antibacterial activity. This mechanism of resistance is mediated by β-lactamases, which are the most common mechanism of β-lactam resistance. Inactivation of rifampin commonly occurs through glycosylation, phosphorylation, or adenosine diphosphate (ADP) ribosylation, and resistance to macrolides and lincosamides can also occur due to enzymatic inactivation of the drug or modification.
Prevention of Cellular Uptake or Efflux
Microbes may develop resistance mechanisms that involve inhibiting the accumulation of an antimicrobial drug, which then prevents the drug from reaching its cellular target. This strategy is common among gram-negative pathogens and can involve changes in outer membrane lipid composition, porin channel selectivity, and/or porin channel concentrations. For example, a common mechanism of carbapenem resistance among Pseudomonas aeruginosa is to decrease the amount of its OprD porin, which is the primary portal of entry for carbapenems through the outer membrane of this pathogen. Additionally, many gram-positive and gram-negative pathogenic bacteria produce efflux pumps that actively transport an antimicrobial drug out of the cell and prevent the accumulation of drug to a level that would be antibacterial. For example, resistance to β-lactams, tetracyclines, and fluoroquinolones commonly occurs through active efflux out of the cell, and it is rather common for a single efflux pump to have the ability to translocate multiple types of antimicrobials.
Target Modification
Because antimicrobial drugs have very specific targets, structural changes to those targets can prevent drug binding, rendering the drug ineffective. Through spontaneous mutations in the genes encoding antibacterial drug targets, bacteria have an evolutionary advantage that allows them to develop resistance to drugs. This mechanism of resistance development is quite common. Genetic changes impacting the active site of penicillin-binding proteins (PBPs) can inhibit the binding of β-lactam drugs and provide resistance to multiple drugs within this class. This mechanism is very common among strains of Streptococcus pneumoniae, which alter their own PBPs through genetic mechanisms. In contrast, strains of Staphylococcus aureus develop resistance to methicillin (MRSA) through the acquisition of a new low-affinity PBP, rather than structurally alter their existing PBPs. Not only does this new low-affinity PBP provide resistance to methicillin but it provides resistance to virtually all β-lactam drugs, with the exception of the newer fifth-generation cephalosporins designed specifically to kill MRSA. Other examples of this resistance strategy include alterations in
• ribosome subunits, providing resistance to macrolides, tetracyclines, and aminoglycosides;
• lipopolysaccharide (LPS) structure, providing resistance to polymyxins;
• RNA polymerase, providing resistance to rifampin;
• DNA gyrase, providing resistance to fluoroquinolones;
• metabolic enzymes, providing resistance to sulfa drugs, sulfones, and trimethoprim; and
• peptidoglycan subunit peptide chains, providing resistance to glycopeptides.
Target Overproduction or Enzymatic Bypass
When an antimicrobial drug functions as an antimetabolite, targeting a specific enzyme to inhibit its activity, there are additional ways that microbial resistance may occur. First, the microbe may overproduce the target enzyme such that there is a sufficient amount of antimicrobial-free enzyme to carry out the proper enzymatic reaction. Second, the bacterial cell may develop a bypass that circumvents the need for the functional target enzyme. Both of these strategies have been found as mechanisms of sulfonamide resistance. Vancomycin resistance among S. aureus has been shown to involve the decreased cross-linkage of peptide chains in the bacterial cell wall, which provides an increase in targets for vancomycin to bind to in the outer cell wall. Increased binding of vancomycin in the outer cell wall provides a blockage that prevents free drug molecules from penetrating to where they can block new cell wall synthesis.
Target Mimicry
A recently discovered mechanism of resistance called target mimicry involves the production of proteins that bind and sequester drugs, preventing the drugs from binding to their target. For example, Mycobacterium tuberculosisproduces a protein with regular pentapeptide repeats that appears to mimic the structure of DNA. This protein binds fluoroquinolones, sequestering them and keeping them from binding to DNA, providing M. tuberculosis resistance to fluoroquinolones. Proteins that mimic the A-site of the bacterial ribosome have been found to contribute to aminoglycoside resistance as well.1
Exercise \(1\)
List several mechanisms for drug resistance.
Multidrug-Resistant Microbes and Cross Resistance
From a clinical perspective, our greatest concerns are multidrug-resistant microbes (MDRs) and cross resistance. MDRs are colloquially known as “superbugs” and carry one or more resistance mechanism(s), making them resistant to multiple antimicrobials. In cross-resistance, a single resistance mechanism confers resistance to multiple antimicrobial drugs. For example, having an efflux pump that can export multiple antimicrobial drugs is a common way for microbes to be resistant to multiple drugs by using a single resistance mechanism. In recent years, several clinically important superbugs have emerged, and the CDC reports that superbugs are responsible for more than 2 million infections in the US annually, resulting in at least 23,000 fatalities.2 Several of the superbugs discussed in the following sections have been dubbed the ESKAPE pathogens. This acronym refers to the names of the pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) but it is also fitting in that these pathogens are able to “escape” many conventional forms of antimicrobial therapy. As such, infections by ESKAPE pathogens can be difficult to treat and they cause a large number of nosocomial infections.
Methicillin-Resistant Staphylococcus aureus (MRSA)
Methicillin, a semisynthetic penicillin, was designed to resist inactivation by β-lactamases. Unfortunately, soon after the introduction of methicillin to clinical practice, methicillin-resistant strains of S. aureus appeared and started to spread. The mechanism of resistance, acquisition of a new low-affinity PBP, provided S. aureus with resistance to all available β-lactams. Strains of methicillin-resistant S. aureus (MRSA) are widespread opportunistic pathogens and a particular concern for skin and other wound infections, but may also cause pneumonia and septicemia. Although originally a problem in health-care settings (hospital-acquired MRSA [HA-MRSA]), MRSA infections are now also acquired through contact with contaminated members of the general public, called community-associated MRSA (CA-MRSA). Approximately one-third of the population carries S. aureus as a member of their normal nasal microbiota without illness, and about 6% of these strains are methicillin resistant.34
Clavulanic Acid: Penicillin’s Little Helper
With the introduction of penicillin in the early 1940s, and its subsequent mass production, society began to think of antibiotics as miracle cures for a wide range of infectious diseases. Unfortunately, as early as 1945, penicillin resistance was first documented and started to spread. Greater than 90% of current S. aureus clinical isolates are resistant to penicillin.5
Although developing new antimicrobial drugs is one solution to this problem, scientists have explored new approaches, including the development of compounds that inactivate resistance mechanisms. The development of clavulanic acid represents an early example of this strategy. Clavulanic acid is a molecule produced by the bacterium Streptococcus clavuligerus. It contains a β-lactam ring, making it structurally similar to penicillin and other β-lactams, but shows no clinical effectiveness when administered on its own. Instead, clavulanic acid binds irreversibly within the active site of β-lactamases and prevents them from inactivating a coadministered penicillin.
Clavulanic acid was first developed in the 1970s and was mass marketed in combination with amoxicillin beginning in the 1980s under the brand name Augmentin. As is typically the case, resistance to the amoxicillin-clavulanic acid combination soon appeared. Resistance most commonly results from bacteria increasing production of their β-lactamase and overwhelming the inhibitory effects of clavulanic acid, mutating their β-lactamase so it is no longer inhibited by clavulanic acid, or from acquiring a new β-lactamase that is not inhibited by clavulanic acid. Despite increasing resistance concerns, clavulanic acid and related β-lactamase inhibitors (sulbactam and tazobactam) represent an important new strategy: the development of compounds that directly inhibit antimicrobial resistance-conferring enzymes.
Vancomycin-Resistant Enterococci and Staphylococcus aureus
Vancomycin is only effective against gram-positive organisms, and it is used to treat wound infections, septic infections, endocarditis, and meningitis that are caused by pathogens resistant to other antibiotics. It is considered one of the last lines of defense against such resistant infections, including MRSA. With the rise of antibiotic resistance in the 1970s and 1980s, vancomycin use increased, and it is not surprising that we saw the emergence and spread of vancomycin-resistant enterococci (VRE), vancomycin-resistant S. aureus (VRSA), and vancomycin-intermediate S. aureus(VISA). The mechanism of vancomycin resistance among enterococci is target modification involving a structural change to the peptide component of the peptidoglycan subunits, preventing vancomycin from binding. These strains are typically spread among patients in clinical settings by contact with health-care workers and contaminated surfaces and medical equipment.
VISA and VRSA strains differ from each other in the mechanism of resistance and the degree of resistance each mechanism confers. VISA strains exhibit intermediate resistance, with a minimum inhibitory concentration (MIC) of 4–8 μg/mL, and the mechanism involves an increase in vancomycin targets. VISA strains decrease the crosslinking of peptide chains in the cell wall, providing an increase in vancomycin targets that trap vancomycin in the outer cell wall. In contrast, VRSA strains acquire vancomycin resistance through horizontal transfer of resistance genes from VRE, an opportunity provided in individuals coinfected with both VRE and MRSA. VRSA exhibit a higher level of resistance, with MICs of 16 μg/mL or higher.6 In the case of all three types of vancomycin-resistant bacteria, rapid clinical identification is necessary so proper procedures to limit spread can be implemented. The oxazolidinones like linezolid are useful for the treatment of these vancomycin-resistant, opportunistic pathogens, as well as MRSA.
Extended-Spectrum β-Lactamase–Producing Gram-Negative Pathogens
Gram-negative pathogens that produce extended-spectrum β-lactamases (ESBLs) show resistance well beyond just penicillins. The spectrum of β-lactams inactivated by ESBLs provides for resistance to all penicillins, cephalosporins, monobactams, and the β-lactamase-inhibitor combinations, but not the carbapenems. An even greater concern is that the genes encoding for ESBLs are usually found on mobile plasmids that also contain genes for resistance to other drug classes (e.g., fluoroquinolones, aminoglycosides, tetracyclines), and may be readily spread to other bacteria by horizontal gene transfer. These multidrug-resistant bacteria are members of the intestinal microbiota of some individuals, but they are also important causes of opportunistic infections in hospitalized patients, from whom they can be spread to other people.
Carbapenem-Resistant Gram-Negative Bacteria
The occurrence of carbapenem-resistant Enterobacteriaceae (CRE) and carbapenem resistance among other gram-negative bacteria (e.g., P. aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophila) is a growing health-care concern. These pathogens develop resistance to carbapenems through a variety of mechanisms, including production of carbapenemases (broad-spectrum β-lactamases that inactivate all β-lactams, including carbapenems), active efflux of carbapenems out of the cell, and/or prevention of carbapenem entry through porin channels. Similar to concerns with ESBLs, carbapenem-resistant, gram-negative pathogens are usually resistant to multiple classes of antibacterials, and some have even developed pan-resistance (resistance to all available antibacterials). Infections with carbapenem-resistant, gram-negative pathogens commonly occur in health-care settings through interaction with contaminated individuals or medical devices, or as a result of surgery.
Multidrug-Resistant Mycobacterium tuberculosis
The emergence of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) and extensively drug-resistantMycobacterium tuberculosis (XDR-TB) is also of significant global concern. MDR-TB strains are resistant to both rifampin and isoniazid, the drug combination typically prescribed for treatment of tuberculosis. XDR-TB strains are additionally resistant to any fluoroquinolone and at least one of three other drugs (amikacin, kanamycin, or capreomycin) used as a second line of treatment, leaving these patients very few treatment options. Both types of pathogens are particularly problematic in immunocompromised persons, including those suffering from HIV infection. The development of resistance in these strains often results from the incorrect use of antimicrobials for tuberculosistreatment, selecting for resistance.
Exercise \(2\)
How does drug resistance lead to superbugs?
Link to Learning
To learn more about the top 18 drug-resistant threats to the US, visit the CDC’s website.
Factory Farming and Drug Resistance
Although animal husbandry has long been a major part of agriculture in America, the rise of concentrated animal feeding operations (CAFOs) since the 1950s has brought about some new environmental issues, including the contamination of water and air with biological waste, and ethical issues regarding animal rights also are associated with growing animals in this way. Additionally, the increase in CAFOs involves the extensive use of antimicrobial drugs in raising livestock. Antimicrobials are used to prevent the development of infectious disease in the close quarters of CAFOs; however, the majority of antimicrobials used in factory farming are for the promotion of growth—in other words, to grow larger animals.
The mechanism underlying this enhanced growth remains unclear. These antibiotics may not necessarily be the same as those used clinically for humans, but they are structurally related to drugs used for humans. As a result, use of antimicrobial drugs in animals can select for antimicrobial resistance, with these resistant bacteria becoming cross-resistant to drugs typically used in humans. For example, tylosin use in animals appears to select for bacteria also cross-resistant to other macrolides, including erythromycin, commonly used in humans.
Concentrations of the drug-resistant bacterial strains generated by CAFOs become increased in water and soil surrounding these farms. If not directly pathogenic in humans, these resistant bacteria may serve as a reservoir of mobile genetic elements that can then pass resistance genes to human pathogens. Fortunately, the cooking process typically inactivates any antimicrobials remaining in meat, so humans typically are not directly ingesting these drugs. Nevertheless, many people are calling for more judicious use of these drugs, perhaps charging farmers user fees to reduce indiscriminate use. In fact, in 2012, the FDA published guidelines for farmers who voluntarily phase out the use of antimicrobial drugs except under veterinary supervision and when necessary to ensure animal health. Although following the guidelines is voluntary at this time, the FDA does recommend what it calls “judicious” use of antimicrobial drugs in food-producing animals in an effort to decrease antimicrobial resistance.
Clinical Focus: Part 3
Unfortunately, Marisa’s urinary tract infection did not resolve with ciprofloxacin treatment. Laboratory testing showed that her infection was caused by a strain of Klebsiella pneumoniae with significant antimicrobial resistance. The resistance profile of this K. pneumoniae included resistance to the carbapenem class of antibacterials, a group of β-lactams that is typically reserved for the treatment of highly resistant bacteria. K. pneumoniae is an opportunistic, capsulated, gram-negative rod that may be a member of the normal microbiota of the intestinal tract, but may also cause a number of diseases, including pneumonia and UTIs.
Specific laboratory tests looking for carbapenemase production were performed on Marisa’s samples and came back positive. Based upon this result, in combination with her health history, production of a carbapenemase known as the New Delhi Metallo-β-lactamase (NDM) was suspected. Although the origin of the NDM carbapenemase is not completely known, many patients infected with NDM-containing strains have travel histories involving hospitalizations in India or surrounding countries.
Exercise \(1\)
How would doctors determine which types of antimicrobial drugs should be administered?
Key Concepts and Summary
• Antimicrobial resistance is on the rise and is the result of selection of drug-resistant strains in clinical environments, the overuse and misuse of antibacterials, the use of subtherapeutic doses of antibacterial drugs, and poor patient compliance with antibacterial drug therapies.
• Drug resistance genes are often carried on plasmids or in transposons that can undergo vertical transfer easily and between microbes through horizontal gene transfer.
• Common modes of antimicrobial drug resistance include drug modification or inactivation, prevention of cellular uptake or efflux, target modification, target overproduction or enzymatic bypass, and target mimicry.
• Problematic microbial strains showing extensive antimicrobial resistance are emerging; many of these strains can reside as members of the normal microbiota in individuals but also can cause opportunistic infection. The transmission of many of these highly resistant microbial strains often occurs in clinical settings, but can also be community-acquired.
Footnotes
1. 1 D.H. Fong, A.M. Berghuis. “Substrate Promiscuity of an Aminoglycoside Antibiotic Resistance Enzyme Via Target Mimicry.” EMBO Journal 21 no. 10 (2002):2323–2331.
2. 2 Centers for Disease Control and Prevention. “Antibiotic/Antimicrobial Resistance.” http://www.cdc.gov/drugresistance/index.html. Accessed June 2, 2016.
3. 3 A.S. Kalokhe et al. “Multidrug-Resistant Tuberculosis Drug Susceptibility and Molecular Diagnostic Testing: A Review of the Literature. American Journal of the Medical Sciences 345 no. 2 (2013):143–148.
4. 4 Centers for Disease Control and Prevention. “Methicillin-Resistant Staphylococcus aureus (MRSA): General Information About MRSA in the Community.” http://www.cdc.gov/mrsa/community/index.html. Accessed June 2, 2016
5. 5 F.D. Lowy. “Antimicrobial Resistance: The Example of Staphylococcus aureus.” Journal of Clinical Investigation 111 no. 9 (2003):1265–1273.
6. 6 Centers for Disease Control and Prevention. “Healthcare-Associated Infections (HIA): General Information about VISA/VRSA.” www.cdc.gov/HAI/organisms/vis...visa_vrsa.html. Accessed June 2, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/14%3A_Antimicrobial_Drugs/14.05%3A_Testing_the_Effectiveness_of_Antimicrobials.txt |
Learning Objectives
• Describe how the Kirby-Bauer disk diffusion test determines the susceptibility of a microbe to an antibacterial drug.
• Explain the significance of the minimal inhibitory concentration and the minimal bactericidal concentration relative to the effectiveness of an antimicrobial drug.
Testing the effectiveness of antimicrobial drugs against specific organisms is important in identifying their spectrum of activity and the therapeutic dosage. This type of test, generally described as antimicrobial susceptibility testing (AST), is commonly performed in a clinical laboratory. In this section, we will discuss common methods of testing the effectiveness of antimicrobials.
The Kirby-Bauer Disk Diffusion Test
The Kirby-Bauer disk diffusion test has long been used as a starting point for determining the susceptibility of specific microbes to various antimicrobial drugs. The Kirby-Bauer assay starts with a Mueller-Hinton agar plate on which a confluent lawn is inoculated with a patient’s isolated bacterial pathogen. Filter paper disks impregnated with known amounts of antibacterial drugs to be tested are then placed on the agar plate. As the bacterial inoculum grows, antibiotic diffuses from the circular disk into the agar and interacts with the growing bacteria. Antibacterial activity is observed as a clear circular zone of inhibition around the drug-impregnated disk, similar to the disk-diffusion assay. The diameter of the zone of inhibition, measured in millimeters and compared to a standardized chart, determines the susceptibility or resistance of the bacterial pathogen to the drug.
There are multiple factors that determine the size of a zone of inhibition in this assay, including drug solubility, rate of drug diffusion through agar, the thickness of the agar medium, and the drug concentration impregnated into the disk. Due to a lack of standardization of these factors, interpretation of the Kirby-Bauer disk diffusion assay provides only limited information on susceptibility and resistance to the drugs tested. The assay cannot distinguish between bacteriostatic and bactericidal activities, and differences in zone sizes cannot be used to compare drug potencies or efficacies. Comparison of zone sizes to a standardized chart will only provide information on the antibacterials to which a bacterial pathogen is susceptible or resistant.
Exercise \(1\)
How does one use the information from a Kirby-Bauer assay to predict the therapeutic effectiveness of an antimicrobial drug in a patient?
Antibiograms: Taking Some of the Guesswork Out of Prescriptions
Unfortunately, infectious diseases don’t take a time-out for lab work. As a result, physicians rarely have the luxury of conducting susceptibility testing before they write a prescription. Instead, they rely primarily on the empirical evidence (i.e., the signs and symptoms of disease) and their professional experience to make an educated guess as to the diagnosis, causative agent(s), and drug most likely to be effective. This approach allows treatment to begin sooner so the patient does not have to wait for lab test results. In many cases, the prescription is effective; however, in an age of increased antimicrobial resistance, it is becoming increasingly more difficult to select the most appropriate empiric therapy. Selecting an inappropriate empiric therapy not only puts the patient at risk but may promote greater resistance to the drug prescribed.
Recently, studies have shown that antibiograms are useful tools in the decision-making process of selecting appropriate empiric therapy. An antibiogram is a compilation of local antibiotic susceptibility data broken down by bacterial pathogen. In a November 2014 study published in the journal Infection Control and Hospital Epidemiology, researchers determined that 85% of the prescriptions ordered in skilled nursing facilities were decided upon empirically, but only 35% of those prescriptions were deemed appropriate when compared with the eventual pathogen identification and susceptibility profile obtained from the clinical laboratory. However, in one nursing facility where use of antibiograms was implemented to direct selection of empiric therapy, appropriateness of empiric therapy increased from 32% before antibiogram implementation to 45% after implementation of antibiograms.1 Although these data are preliminary, they do suggest that health-care facilities can reduce the number of inappropriate prescriptions by using antibiograms to select empiric therapy, thus benefiting patients and minimizing opportunities for antimicrobial resistance to develop.
Dilution Tests
As discussed, the limitations of the Kirby-Bauer disk diffusion test do not allow for a direct comparison of antibacterial potencies to guide selection of the best therapeutic choice. However, antibacterial dilution tests can be used to determine a particular drug’s minimal inhibitory concentration (MIC), the lowest concentration of drug that inhibits visible bacterial growth, and minimal bactericidal concentration (MBC), the lowest drug concentration that kills ≥99.9% of the starting inoculum. Determining these concentrations helps identify the correct drug for a particular pathogen. For the macrobroth dilution assay, a dilution series of the drug in broth is made in test tubes and the same number of cells of a test bacterial strain is added to each tube (Figure \(1\)). The MIC is determined by examining the tubes to find the lowest drug concentration that inhibits visible growth; this is observed as turbidity (cloudiness) in the broth. Tubes with no visible growth are then inoculated onto agar media without antibiotic to determine the MBC. Generally, serum levels of an antibacterial should be at least three to five times above the MIC for treatment of an infection.
The MIC assay can also be performed using 96-well microdilution trays, which allow for the use of small volumes and automated dispensing devices, as well as the testing of multiple antimicrobials and/or microorganisms in one tray (Figure \(2\)). MICs are interpreted as the lowest concentration that inhibits visible growth, the same as for the macrobroth dilution in test tubes. Growth may also be interpreted visually or by using a spectrophotometer or similar device to detect turbidity or a color change if an appropriate biochemical substrate that changes color in the presence of bacterial growth is also included in each well.
The Etest is an alternative method used to determine MIC, and is a combination of the Kirby-Bauer disk diffusion testand dilution methods. Similar to the Kirby-Bauer assay, a confluent lawn of a bacterial isolate is inoculated onto the surface of an agar plate. Rather than using circular disks impregnated with one concentration of drug, however, commercially available plastic strips that contain a gradient of an antibacterial are placed on the surface of the inoculated agar plate (Figure \(3\)). As the bacterial inoculum grows, antibiotic diffuses from the plastic strips into the agar and interacts with the bacterial cells. Because the rate of drug diffusion is directly related to concentration, an elliptical zone of inhibition is observed with the Etest drug gradient, rather than a circular zone of inhibition observed with the Kirby-Bauer assay. To interpret the results, the intersection of the elliptical zone with the gradient on the drug-containing strip indicates the MIC. Because multiple strips containing different antimicrobials can be placed on the same plate, the MIC of multiple antimicrobials can be determined concurrently and directly compared. However, unlike the macrobroth and microbroth dilution methods, the MBC cannot be determined with the Etest.
Exercise \(2\)
Compare and contrast MIC and MBC.
Clinical Focus: Resolution
Marisa’s UTI was likely caused by the catheterizations she had in Vietnam. Most bacteria that cause UTIs are members of the normal gut microbiota, but they can cause infections when introduced to the urinary tract, as might have occurred when the catheter was inserted. Alternatively, if the catheter itself was not sterile, bacteria on its surface could have been introduced into Marisa’s body. The antimicrobial therapy Marisa received in Cambodia may also have been a complicating factor because it may have selected for antimicrobial-resistant strains already present in her body. These bacteria would have already contained genes for antimicrobial resistance, either acquired by spontaneous mutation or through horizontal gene transfer, and, therefore, had the best evolutionary advantage for adaptation and growth in the presence of the antimicrobial therapy. As a result, one of these resistant strains may have been subsequently introduced into her urinary tract.
Laboratory testing at the CDC confirmed that the strain of Klebsiella pneumoniae from Marisa’s urine sample was positive for the presence of NDM, a very active carbapenemasethat is beginning to emerge as a new problem in antimicrobial resistance. While NDM-positive strains are resistant to a wide range of antimicrobials, they have shown susceptibility to tigecycline (structurally related to tetracycline) and the polymyxins B and E (colistin).
To prevent her infection from spreading, Marisa was isolated from the other patients in a separate room. All hospital staff interacting with her were advised to follow strict protocols to prevent surface and equipment contamination. This would include especially stringent hand hygiene practices and careful disinfection of all items coming into contact with her.
Marisa’s infection finally responded to tigecycline and eventually cleared. She was discharged a few weeks after admission, and a follow-up stool sample showed her stool to be free of NDM-containing K. pneumoniae, meaning that she was no longer harboring the highly resistant bacterium.
Key Concepts and Summary
• The Kirby-Bauer disk diffusion test helps determine the susceptibility of a microorganism to various antimicrobial drugs. However, the zones of inhibition measured must be correlated to known standards to determine susceptibility and resistance, and do not provide information on bactericidal versus bacteriostatic activity, or allow for direct comparison of drug potencies.
• Antibiograms are useful for monitoring local trends in antimicrobial resistance/susceptibility and for directing appropriate selection of empiric antibacterial therapy.
• There are several laboratory methods available for determining the minimum inhibitory concentration (MIC) of an antimicrobial drug against a specific microbe. The minimal bactericidal concentration (MBC) can also be determined, typically as a follow-up experiment to MIC determination using the tube dilution method.
Footnotes
1. 1 J.P. Furuno et al. “Using Antibiograms to Improve Antibiotic Prescribing in Skilled Nursing Facilities.” Infection Control and Hospital Epidemiology 35 no. Suppl S3 (2014):S56–61. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/14%3A_Antimicrobial_Drugs/14.06%3A_The_Emergence_of_Drug_Resistance.txt |
Learning Objectives
• Describe the methods and strategies used for discovery of new antimicrobial agents.
With the continued evolution and spread of antimicrobial resistance, and now the identification of pan-resistant bacterial pathogens, the search for new antimicrobials is essential for preventing the postantibiotic era. Although development of more effective semisynthetic derivatives is one strategy, resistance to them develops rapidly because bacterial pathogens are already resistant to earlier-generation drugs in the family and can easily mutate and develop resistance to the new semisynthetic drugs. Today, scientists continue to hunt for new antimicrobial compounds and explore new avenues of antimicrobial discovery and synthesis. They check large numbers of soils and microbial products for antimicrobial activity by using high-throughput screening methods, which use automation to test large numbers of samples simultaneously. The recent development of the iChip1 allows researchers to investigate the antimicrobial-producing capabilities of soil microbes that are difficult to grow by standard cultivation techniques in the laboratory. Rather than grow the microbes in the laboratory, they are grown in situ—right in the soil. Use of the iChip has resulted in the discovery of teixobactin, a novel antimicrobial from Mount Ararat, Turkey. Teixobactin targets two distinct steps in gram-positive cell wall synthesis and for which antimicrobial resistance appears not yet to have evolved.
Although soils have been widely examined, other environmental niches have not been tested as fully. Since 70% of the earth is covered with water, marine environments could be mined more fully for the presence of antimicrobial-producing microbes. In addition, researchers are using combinatorial chemistry, a method for making a very large number of related compounds from simple precursors, and testing them for antimicrobial activity. An additional strategy that needs to be explored further is the development of compounds that inhibit resistance mechanisms and restore the activity of older drugs, such as the strategy described earlier for β-lactamase inhibitors like clavulanic acid. Finally, developing inhibitors of virulence factor production and function could be a very important avenue. Although this strategy would not be directly antibacterial, drugs that slow the progression of an infection could provide an advantage for the immune system and could be used successfully in combination with antimicrobial drugs.
Exercise \(1\)
What are new sources and strategies for developing drugs to fight infectious diseases?
The (Free?) Market for New Antimicrobials
There used to be plenty of antimicrobial drugs on the market to treat infectious diseases. However, the spread of antimicrobial resistance has created a need for new antibiotics to replace those that are no longer as effective as they once were. Unfortunately, pharmaceutical companies are not particularly motivated to fill this need. As of 2009, all but five pharmaceutical companies had moved away from antimicrobial drug development.2 As a result, the number of FDA approvals of new antimicrobials has fallen drastically in recent decades (Figure \(1\)).
Given that demand usually encourages supply, one might expect pharmaceutical companies to be rushing to get back in the business of developing new antibiotics. But developing new drugs is a lengthy process and requires large investments in research and development. Pharmaceutical companies can typically get a higher return on their investment by developing products for chronic, nonmicrobial diseases like diabetes; such drugs must be taken for life, and therefore generate more long-term revenue than an antibiotic that does its job in a week or two. But what will happen when drugs like vancomycin, a superantimicrobial reserved for use as a last resort, begin to lose their effectiveness against ever more drug-resistant superbugs? Will drug companies wait until all antibiotics have become useless before beginning to look for new ones?
Recently, it has been suggested that large pharmaceutical companies should be given financial incentives to pursue such research. In September 2014, the White House released an executive order entitled “Combating Antibiotic Resistant Bacteria,” calling upon various government agencies and the private sector to work together to “accelerate basic and applied research and development for new antimicrobials, other therapeutics, and vaccines.”3 As a result, as of March 2015, President Obama’s proposed fiscal year 2016 budget doubled the amount of federal funding to \$1.2 billion for “combating and preventing antibiotic resistance,” which includes money for antimicrobial research and development.4 Similar suggestions have also been made on a global scale. In December 2014, a report chaired by former Goldman Sachs economist Jim O’Neill was published in The Review on Antimicrobial Resistance.5
These developments reflect the growing belief that for-profit pharmaceutical companies must be subsidized to encourage development of new antimicrobials. But some ask whether pharmaceutical development should be motivated by profit at all. Given that millions of lives may hang in the balance, some might argue that drug companies have an ethical obligation to devote their research and development efforts to high-utility drugs, as opposed to highly profitable ones. Yet this obligation conflicts with the fundamental goals of a for-profit company. Are government subsidies enough to ensure that drug companies make the public interest a priority, or should government agencies assume responsibility for developing critical drugs that may have little or no return on investment?
Key Concepts and Summary
• Current research into the development of antimicrobial drugs involves the use of high-throughput screening and combinatorial chemistry technologies.
• New technologies are being developed to discover novel antibiotics from soil microorganisms that cannot be cultured by standard laboratory methods.
• Additional strategies include searching for antibiotics from sources other than soil, identifying new antibacterial targets, using combinatorial chemistry to develop novel drugs, developing drugs that inhibit resistance mechanisms, and developing drugs that target virulence factors and hold infections in check.
Footnotes
1. 1 L. Losee et al. “A New Antibiotic Kills Pathogens Without Detectable Resistance.” Nature 517 no. 7535 (2015):455–459.
2. 2 H.W. Boucher et al. “Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America.” Clinical Infectious Diseases 48 no. 1 (2009):1–12.
3. 3 The White House. National Action Plan for Combating Antibiotic-Resistant Bacteria. Washington, DC: The White House, 2015.
4. 4 White House Office of the Press Secretary. “Fact Sheet: Obama Administration Releases National Action Plan to Combat Antibiotic-Resistant Bacteria.” March 27, 2015. https://www.whitehouse.gov/the-press...lan-combat-ant
5. 5 Review on Antimicrobial Resistance. http://amr-review.org. Accessed June 1, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/14%3A_Antimicrobial_Drugs/14.07%3A_Current_Strategies_for_Antimicrobial_Discovery.txt |
14.1: Discovering Antimicrobial Drugs
Antimicrobial drugs produced by purposeful fermentation and/or contained in plants have been used as traditional medicines in many cultures for millennia. The purposeful and systematic search for a chemical “magic bullet” that specifically target infectious microbes was initiated by Paul Ehrlich in the early 20th century. The discovery of the natural antibiotic, penicillin, by Alexander Fleming in 1928 started the modern age of antimicrobial discovery and research.
Multiple Choice
A scientist discovers that a soil bacterium he has been studying produces an antimicrobial that kills gram-negative bacteria. She isolates and purifies the antimicrobial compound, then chemically converts a chemical side chain to a hydroxyl group. When she tests the antimicrobial properties of this new version, she finds that this antimicrobial drug can now also kill gram-positive bacteria. The new antimicrobial drug with broad-spectrum activity is considered to be which of the following?
1. resistant
2. semisynthetic
3. synthetic
4. natural
Answer
B
Which of the following antimicrobial drugs is synthetic?
1. sulfanilamide
2. penicillin
3. actinomycin
4. neomycin
Answer
A
Fill in the Blank
The group of soil bacteria known for their ability to produce a wide variety of antimicrobials is called the ________.
Answer
actinomycetes
Short Answer
Where do antimicrobials come from naturally? Why?
Why was Salvarsan considered to be a “magic bullet” for the treatment of syphilis?
Critical Thinking
In nature, why do antimicrobial-producing microbes commonly also have antimicrobial resistance genes?
14.2: Antibacterial Drugs
Antimicrobial drugs can be bacteriostatic or bactericidal, and these characteristics are important considerations when selecting the most appropriate drug. The use of narrow-spectrum antimicrobial drugs is preferred in many cases to avoid superinfection and the development of antimicrobial resistance. Broad-spectrum antimicrobial use is warranted for serious systemic infections when there is no time to determine the causative agent or when narrow-spectrum antimicrobials fail.
Multiple Choice
Which of the following combinations would most likely contribute to the development of a superinfection?
1. long-term use of narrow-spectrum antimicrobials
2. long-term use of broad-spectrum antimicrobials
3. short-term use of narrow-spectrum antimicrobials
4. short-term use of broad-spectrum antimicrobials
Answer
B
Which of the following routes of administration would be appropriate and convenient for home administration of an antimicrobial to treat a systemic infection?
1. oral
2. intravenous
3. topical
4. parenteral
Answer
A
Which clinical situation would be appropriate for treatment with a narrow-spectrum antimicrobial drug?
1. treatment of a polymicrobic mixed infection in the intestine
2. prophylaxis against infection after a surgical procedure
3. treatment of strep throat caused by culture identified Streptococcus pyogenes
4. empiric therapy of pneumonia while waiting for culture results
Answer
C
Fill in the Blank
The bacterium known for causing pseudomembranous colitis, a potentially deadly superinfection, is ________.
Answer
Clostridium difficile
True/False
Narrow-spectrum antimicrobials are commonly used for prophylaxis following surgery.
Answer
False
Short Answer
When prescribing antibiotics, what aspects of the patient’s health history should the clinician ask about and why?
When is using a broad-spectrum antimicrobial drug warranted?
Critical Thinking
Why are yeast infections a common type of superinfection that results from long-term use of broad-spectrum antimicrobials?
Too often patients will stop taking antimicrobial drugs before the prescription is finished. What are factors that cause a patient to stop too soon, and what negative impacts could this have?
14.3: Drugs Targeting Other Microorganisms
Antibacterial compounds exhibit selective toxicity, largely due to differences between prokaryotic and eukaryotic cell structure. Cell wall synthesis inhibitors, including the β-lactams, the glycopeptides, and bacitracin, interfere with peptidoglycan synthesis, making bacterial cells more prone to osmotic lysis. There are a variety of broad-spectrum, bacterial protein synthesis inhibitors that selectively target the prokaryotic 70S ribosome, including those that bind to the 30S and 50S subunits.
Multiple Choice
Which of the following terms refers to the ability of an antimicrobial drug to harm the target microbe without harming the host?
1. mode of action
2. therapeutic level
3. spectrum of activity
4. selective toxicity
Answer
D
Which of the following is not a type of β-lactam antimicrobial?
1. penicillins
2. glycopeptides
3. cephalosporins
4. monobactams
Answer
B
Which of the following does not bind to the 50S ribosomal subunit?
1. tetracyclines
2. lincosamides
3. macrolides
4. chloramphenicol
Answer
A
Which of the following antimicrobials inhibits the activity of DNA gyrase?
1. polymyxin B
2. clindamycin
3. nalidixic acid
4. rifampin
Answer
C
Fill in the Blank
Selective toxicity antimicrobials are easier to develop against bacteria because they are ________ cells, whereas human cells are eukaryotic.
Answer
prokaryotic
True/False
β-lactamases can degrade vancomycin.
Answer
false
Short Answer
If human cells and bacterial cells perform transcription, how are the rifamycins specific for bacterial infections?
What bacterial structural target would make an antibacterial drug selective for gram-negative bacteria? Provide one example of an antimicrobial compound that targets this structure.
Critical Thinking
In considering the cell structure of prokaryotes compared with that of eukaryotes, propose one possible reason for side effects in humans due to treatment of bacterial infections with protein synthesis inhibitors.
14.4: Clinical Considerations
Because fungi, protozoans, and helminths are eukaryotic organisms like human cells, it is more challenging to develop antimicrobial drugs that specifically target them. Similarly, it is hard to target viruses because human viruses replicate inside of human cells.
Multiple Choice
Which of the following is not an appropriate target for antifungal drugs?
1. ergosterol
2. chitin
3. cholesterol
4. β(1→3) glucan
Answer
C
Which of the following drug classes specifically inhibits neuronal transmission in helminths?
1. quinolines
2. avermectins
3. amantadines
4. imidazoles
Answer
B
Which of the following is a nucleoside analog commonly used as a reverse transcriptase inhibitor in the treatment of HIV?
1. acyclovir
2. ribavirin
3. adenine-arabinoside
4. azidothymidine
Answer
D
Which of the following is an antimalarial drug that is thought to increase ROS levels in target cells?
1. artemisinin
2. amphotericin b
3. praziquantel
4. pleconaril
Answer
A
Fill in the Blank
Antiviral drugs, like Tamiflu and Relenza, that are effective against the influenza virus by preventing viral escape from host cells are called ________.
Answer
neuraminidase inhibitors
True/False
Echinocandins, known as “penicillin for fungi,” target β(1→3) glucan in fungal cell walls.
Answer
true
Short Answer
How does the biology of HIV necessitate the need to treat HIV infections with multiple drugs?
Niclosamide is insoluble and thus is not readily absorbed from the stomach into the bloodstream. How does the insolubility of niclosamide aid its effectiveness as a treatment for tapeworm infection?
Critical Thinking
Which of the following molecules is an example of a nucleoside analog?
Why can’t drugs used to treat influenza, like amantadines and neuraminidase inhibitors, be used to treat a wider variety of viral infections?
14.5: Testing the Effectiveness of Antimicrobials
Antimicrobial resistance is on the rise and is the result of selection of drug-resistant strains in clinical environments, the overuse and misuse of antibacterials, the use of subtherapeutic doses of antibacterial drugs, and poor patient compliance with antibacterial drug therapies. Drug resistance genes are often carried on plasmids or in transposons that can undergo vertical transfer easily and between microbes through horizontal gene transfer.
Multiple Choice
Which of the following resistance mechanisms describes the function of β-lactamase?
1. efflux pump
2. target mimicry
3. drug inactivation
4. target overproduction
Answer
C
Which of the following resistance mechanisms is commonly effective against a wide range of antimicrobials in multiple classes?
1. efflux pump
2. target mimicry
3. target modification
4. target overproduction
Answer
A
Which of the following resistance mechanisms is the most nonspecific to a particular class of antimicrobials?
1. drug modification
2. target mimicry
3. target modification
4. efflux pump
Answer
D
Which of the following types of drug-resistant bacteria do not typically persist in individuals as a member of their intestinal microbiota?
1. MRSA
2. VRE
3. CRE
4. ESBL-producing bacteria
Answer
A
Fill in the Blank
Staphylococcus aureus, including MRSA strains, may commonly be carried as a normal member of the ________ microbiota in some people.
Answer
nasal
Short Answer
Why does the length of time of antimicrobial treatment for tuberculosis contribute to the rise of resistant strains?
What is the difference between multidrug resistance and cross-resistance?
14.6: The Emergence of Drug Resistance
The Kirby-Bauer disk diffusion test helps determine the susceptibility of a microorganism to various antimicrobial drugs. However, the zones of inhibition measured must be correlated to known standards to determine susceptibility and resistance, and do not provide information on bactericidal versus bacteriostatic activity, or allow for direct comparison of drug potencies. Antibiograms are useful for monitoring local trends in antimicrobial resistance/susceptibility.
Multiple Choice
In the Kirby-Bauer disk diffusion test, the _______ of the zone of inhibition is measured and used for interpretation.
1. diameter
2. microbial population
3. circumference
4. depth
Answer
A
Which of the following techniques cannot be used to determine the minimum inhibitory concentration of an antimicrobial drug against a particular microbe?
1. Etest
2. microbroth dilution test
3. Kirby-Bauer disk diffusion test
4. macrobroth dilution test
Answer
C
The utility of an antibiogram is that it shows antimicrobial susceptibility trends
1. over a large geographic area.
2. for an individual patient.
3. in research laboratory strains.
4. in a localized population.
Answer
D
Fill in the Blank
The method that can determine the MICs of multiple antimicrobial drugs against a microbial strain using a single agar plate is called the ________.
Answer
Etest
True/False
If drug A produces a larger zone of inhibition than drug B on the Kirby-Bauer disk diffusion test, drug A should always be prescribed.
Answer
false
Short Answer
How is the information from a Kirby-Bauer disk diffusion test used for the recommendation of the clinical use of an antimicrobial drug?
What is the difference between MIC and MBC?
Critical Thinking
Can an Etest be used to find the MBC of a drug? Explain.
14.7: Current Strategies for Antimicrobial Discovery
With the continued evolution and spread of antimicrobial resistance, and now the identification of pan-resistant bacterial pathogens, the search for new antimicrobials is essential for preventing the postantibiotic era.
Multiple Choice
Which of the following has yielded compounds with the most antimicrobial activity?
1. water
2. air
3. volcanoes
4. soil
Answer
D
True/False
The rate of discovery of antimicrobial drugs has decreased significantly in recent decades.
Answer
true
Critical Thinking
Who should be responsible for discovering and developing new antibiotics? Support your answer with reasoning. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/14%3A_Antimicrobial_Drugs/14.E%3A_Antimicrobial_Drugs_%28Exercises%29.txt |
Jane woke up one spring morning feeling not quite herself. Her throat felt a bit dry and she was sniffling. She wondered why she felt so lousy. Was it because of a change in the weather? The pollen count? Was she coming down with something? Did she catch a bug from her coworker who sneezed on her in the elevator yesterday?
The signs and symptoms we associate with illness can have many different causes. Sometimes they are the direct result of a pathogenic infection, but in other cases they result from a response by our immune system to a pathogen or another perceived threat. For example, in response to certain pathogens, the immune system may release pyrogens, chemicals that cause the body temperature to rise, resulting in a fever. This response creates a less-than-favorable environment for the pathogen, but it also makes us feel sick.
Medical professionals rely heavily on analysis of signs and symptoms to determine the cause of an ailment and prescribe treatment. In some cases, signs and symptoms alone are enough to correctly identify the causative agent of a disease, but since few diseases produce truly unique symptoms, it is often necessary to confirm the identity of the infectious agent by other direct and indirect diagnostic methods.
• 15.1: Characteristics of Infectious Diseases
In an infection, a microorganism enters a host and begins to multiply. Some infections cause disease, which is any deviation from the normal function or structure of the host. Signs of a disease are objective and are measured. Symptoms of a disease are subjective and are reported by the patient. Diseases can either be noninfectious (due to genetics and environment) or infectious (due to pathogens).
• 15.2: How Pathogens Cause Disease
Koch’s postulates are used to determine whether a particular microorganism is a pathogen. Molecular Koch’s postulates are used to determine what genes contribute to a pathogen’s ability to cause disease. Virulence, the degree to which a pathogen can cause disease, can be quantified by calculating either the ID50 or LD50 of a pathogen on a given population. Primary pathogens are capable of causing pathological changes associated with disease in a healthy individual.
• 15.3: Virulence Factors
Virulence factors contribute to a pathogen’s ability to cause disease. Exoenzymes and toxins allow pathogens to invade host tissue and cause tissue damage. Exoenzymes are classified according to the macromolecule they target and exotoxins are classified based on their mechanism of action. Bacterial toxins include endotoxin and exotoxins. Endotoxin is the lipid A component of the LPS of the gram-negative cell envelope. Exotoxins are proteins secreted mainly by gram-positive bacteria.
• 15.4: Aseptic Techniques
Fungal and parasitic pathogens use pathogenic mechanisms and virulence factors that are similar to those of bacterial pathogens. Fungi initiate infections through the interaction of adhesins with receptors on host cells. Some fungi produce toxins and exoenzymes involved in disease production and capsules that provide protection of phagocytosis. Protozoa adhere to target cells through complex mechanisms and can cause cellular damage through release of cytopathic substances.
• 15.E: Microbial Mechanisms of Pathogenicity (Exercises)
These are exercises for Chapter 15 "Microbial Mechanisms of Pathogenicity" in OpenStax's Microbiology Textmap.
Thumbnail: Ulcer-causing bacterium (H. Pylori) crossing mucus layer of stomach. (Public Domain/modified from original; National Science Foundation via Wikimedia Commons).
15: Microbial Mechanisms of Pathogenicity
Learning Objectives
• Distinguish between signs and symptoms of disease
• Explain the difference between a communicable disease and a noncommunicable disease
• Compare different types of infectious diseases, including iatrogenic, nosocomial, and zoonotic diseases
• Identify and describe the stages of an acute infectious disease in terms of number of pathogens present and severity of signs and symptoms
A disease is any condition in which the normal structure or functions of the body are damaged or impaired. Physical injuries or disabilities are not classified as disease, but there can be several causes for disease, including infection by a pathogen, genetics (as in many cancers or deficiencies), noninfectious environmental causes, or inappropriate immune responses. Our focus in this chapter will be on infectious diseases, although when diagnosing infectious diseases, it is always important to consider possible noninfectious causes.
Clinical Focus: Part 1
Michael, a 10-year-old boy in generally good health, went to a birthday party on Sunday with his family. He ate many different foods but was the only one in the family to eat the undercooked hot dogs served by the hosts. Monday morning, he woke up feeling achy and nauseous, and he was running a fever of 38 °C (100.4 °F). His parents, assuming Michael had caught the flu, made him stay home from school and limited his activities. But after 4 days, Michael began to experience severe headaches, and his fever spiked to 40 °C (104 °F). Growing worried, his parents finally decide to take Michael to a nearby clinic.
Exercise \(1\)
1. What signs and symptoms is Michael experiencing?
2. What do these signs and symptoms tell us about the stage of Michael’s disease?
Signs and Symptoms of Disease
An infection is the successful colonization of a host by a microorganism. Infections can lead to disease, which causes signs and symptoms resulting in a deviation from the normal structure or functioning of the host. Microorganisms that can cause disease are known as pathogens.
The signs of disease are objective and measurable, and can be directly observed by a clinician. Vital signs, which are used to measure the body’s basic functions, include body temperature (normally 37 °C [98.6 °F]), heart rate (normally 60–100 beats per minute), breathing rate (normally 12–18 breaths per minute), and blood pressure (normally between 90/60 and 120/80 mm Hg). Changes in any of the body’s vital signs may be indicative of disease. For example, having a fever (a body temperature significantly higher than 37 °C or 98.6 °F) is a sign of disease because it can be measured.
In addition to changes in vital signs, other observable conditions may be considered signs of disease. For example, the presence of antibodies in a patient’s serum (the liquid portion of blood that lacks clotting factors) can be observed and measured through blood tests and, therefore, can be considered a sign. However, it is important to note that the presence of antibodies is not always a sign of an active disease. Antibodies can remain in the body long after an infection has resolved; also, they may develop in response to a pathogen that is in the body but not currently causing disease.
Unlike signs, symptoms of disease are subjective. Symptoms are felt or experienced by the patient, but they cannot be clinically confirmed or objectively measured. Examples of symptoms include nausea, loss of appetite, and pain. Such symptoms are important to consider when diagnosing disease, but they are subject to memory bias and are difficult to measure precisely. Some clinicians attempt to quantify symptoms by asking patients to assign a numerical value to their symptoms. For example, the Wong-Baker Faces pain-rating scale asks patients to rate their pain on a scale of 0–10. An alternative method of quantifying pain is measuring skin conductance fluctuations. These fluctuations reflect sweating due to skin sympathetic nerve activity resulting from the stressor of pain.1
A specific group of signs and symptoms characteristic of a particular disease is called a syndrome. Many syndromes are named using a nomenclature based on signs and symptoms or the location of the disease. Table \(1\) lists some of the prefixes and suffixes commonly used in naming syndromes.
Table \(1\): Nomenclature of Symptoms
Affix Meaning Example
cyto- cell cytopenia: reduction in the number of blood cells
hepat- of the liver hepatitis: inflammation of the liver
-pathy disease neuropathy: a disease affecting nerves
-emia of the blood bacteremia: presence of bacteria in blood
-itis inflammation colitis: inflammation of the colon
-lysis destruction hemolysis: destruction of red blood cells
-oma tumor lymphoma: cancer of the lymphatic system
-osis diseased or abnormal condition leukocytosis: abnormally high number of white blood cells
-derma of the skin keratoderma: a thickening of the skin
Clinicians must rely on signs and on asking questions about symptoms, medical history, and the patient’s recent activities to identify a particular disease and the potential causative agent. Diagnosis is complicated by the fact that different microorganisms can cause similar signs and symptoms in a patient. For example, an individual presenting with symptoms of diarrhea may have been infected by one of a wide variety of pathogenic microorganisms. Bacterial pathogens associated with diarrheal disease include Vibrio cholerae, Listeria monocytogenes, Campylobacter jejuni, and enteropathogenic Escherichia coli (EPEC). Viral pathogens associated with diarrheal disease include norovirus and rotavirus. Parasitic pathogens associated with diarrhea include Giardia lamblia and Cryptosporidium parvum. Likewise, fever is indicative of many types of infection, from the common cold to the deadly Ebola hemorrhagic fever.
Finally, some diseases may be asymptomatic or subclinical, meaning they do not present any noticeable signs or symptoms. For example, most individual infected with herpes simplex virus remain asymptomatic and are unaware that they have been infected.
Exercise \(2\)
Explain the difference between signs and symptoms.
Classifications of Disease
The World Health Organization’s (WHO) International Classification of Diseases (ICD) is used in clinical fields to classify diseases and monitor morbidity (the number of cases of a disease) and mortality (the number of deaths due to a disease). In this section, we will introduce terminology used by the ICD (and in health-care professions in general) to describe and categorize various types of disease.
An infectious disease is any disease caused by the direct effect of a pathogen. A pathogen may be cellular (bacteria, parasites, and fungi) or acellular (viruses, viroids, and prions). Some infectious diseases are also communicable, meaning they are capable of being spread from person to person through either direct or indirect mechanisms. Some infectious communicable diseases are also considered contagious diseases, meaning they are easily spread from person to person. Not all contagious diseases are equally so; the degree to which a disease is contagious usually depends on how the pathogen is transmitted. For example, measles is a highly contagious viral disease that can be transmitted when an infected person coughs or sneezes and an uninfected person breathes in droplets containing the virus. Gonorrhea is not as contagious as measles because transmission of the pathogen (Neisseria gonorrhoeae) requires close intimate contact (usually sexual) between an infected person and an uninfected person.
Diseases that are contracted as the result of a medical procedure are known as iatrogenic diseases. Iatrogenic diseases can occur after procedures involving wound treatments, catheterization, or surgery if the wound or surgical site becomes contaminated. For example, an individual treated for a skin wound might acquire necrotizing fasciitis (an aggressive, “flesh-eating” disease) if bandages or other dressings became contaminated by Clostridium perfringens or one of several other bacteria that can cause this condition.
Diseases acquired in hospital settings are known as nosocomial diseases. Several factors contribute to the prevalence and severity of nosocomial diseases. First, sick patients bring numerous pathogens into hospitals, and some of these pathogens can be transmitted easily via improperly sterilized medical equipment, bed sheets, call buttons, door handles, or by clinicians, nurses, or therapists who do not wash their hands before touching a patient. Second, many hospital patients have weakened immune systems, making them more susceptible to infections. Compounding this, the prevalence of antibiotics in hospital settings can select for drug-resistant bacteria that can cause very serious infections that are difficult to treat.
Certain infectious diseases are not transmitted between humans directly but can be transmitted from animals to humans. Such a disease is called zoonotic disease (or zoonosis). According to WHO, a zoonosis is a disease that occurs when a pathogen is transferred from a vertebrate animal to a human; however, sometimes the term is defined more broadly to include diseases transmitted by all animals (including invertebrates). For example, rabies is a viral zoonotic disease spread from animals to humans through bites and contact with infected saliva. Many other zoonotic diseases rely on insects or other arthropods for transmission. Examples include yellow fever (transmitted through the bite of mosquitoes infected with yellow fever virus) and Rocky Mountain spotted fever (transmitted through the bite of ticks infected with Rickettsia rickettsii).
In contrast to communicable infectious diseases, a noncommunicable infectious disease is not spread from one person to another. One example is tetanus, caused by Clostridium tetani, a bacterium that produces endospores that can survive in the soil for many years. This disease is typically only transmitted through contact with a skin wound; it cannot be passed from an infected person to another person. Similarly, Legionnaires disease is caused by Legionella pneumophila, a bacterium that lives within amoebae in moist locations like water-cooling towers. An individual may contract Legionnaires disease via contact with the contaminated water, but once infected, the individual cannot pass the pathogen to other individuals.
In addition to the wide variety of noncommunicable infectious diseases, noninfectious diseases (those not caused by pathogens) are an important cause of morbidity and mortality worldwide. Noninfectious diseases can be caused by a wide variety factors, including genetics, the environment, or immune system dysfunction, to name a few. For example, sickle cell anemia is an inherited disease caused by a genetic mutation that can be passed from parent to offspring (Figure \(1\)). Other types of noninfectious diseases are listed in Table \(2\).
Table \(2\): Types of Noninfectious Diseases
Type Definition Example
Inherited A genetic disease Sickle cell anemia
Congenital Disease that is present at or before birth Down syndrome
Degenerative Progressive, irreversible loss of function Parkinson disease (affecting central nervous system)
Nutritional deficiency Impaired body function due to lack of nutrients Scurvy (vitamin C deficiency)
Endocrine Disease involving malfunction of glands that release hormones to regulate body functions Hypothyroidism – thyroid does not produce enough thyroid hormone, which is important for metabolism
Neoplastic Abnormal growth (benign or malignant) Some forms of cancer
Idiopathic Disease for which the cause is unknown Idiopathic juxtafoveal retinal telangiectasia (dilated, twisted blood vessels in the retina of the eye)
Link to Learning
Lists of common infectious diseases can be found at the following Centers for Disease Control and Prevention (CDC), World Health Organization (WHO), and International Classification of Diseases websites.
Exercise \(3\)
1. Describe how a disease can be infectious but not contagious.
2. Explain the difference between iatrogenic disease and nosocomial disease.
Periods of Disease
The five periods of disease (sometimes referred to as stages or phases) include the incubation, prodromal, illness, decline, and convalescence periods (Figure \(2\)). The incubation period occurs in an acute disease after the initial entry of the pathogen into the host (patient). It is during this time the pathogen begins multiplying in the host. However, there are insufficient numbers of pathogen particles (cells or viruses) present to cause signs and symptoms of disease. Incubation periods can vary from a day or two in acute disease to months or years in chronic disease, depending upon the pathogen. Factors involved in determining the length of the incubation period are diverse, and can include strength of the pathogen, strength of the host immune defenses, site of infection, type of infection, and the size infectious dose received. During this incubation period, the patient is unaware that a disease is beginning to develop.
The prodromal period occurs after the incubation period. During this phase, the pathogen continues to multiply and the host begins to experience general signs and symptoms of illness, which typically result from activation of the immune system, such as fever, pain, soreness, swelling, or inflammation. Usually, such signs and symptoms are too general to indicate a particular disease. Following the prodromal period is the period of illness, during which the signs and symptoms of disease are most obvious and severe.
The period of illness is followed by the period of decline, during which the number of pathogen particles begins to decrease, and the signs and symptoms of illness begin to decline. However, during the decline period, patients may become susceptible to developing secondary infections because their immune systems have been weakened by the primary infection. The final period is known as the period of convalescence. During this stage, the patient generally returns to normal functions, although some diseases may inflict permanent damage that the body cannot fully repair.
Infectious diseases can be contagious during all five of the periods of disease. Which periods of disease are more likely to associated with transmissibility of an infection depends upon the disease, the pathogen, and the mechanisms by which the disease develops and progresses. For example, with meningitis (infection of the lining of brain), the periods of infectivity depend on the type of pathogen causing the infection. Patients with bacterial meningitis are contagious during the incubation period for up to a week before the onset of the prodromal period, whereas patients with viral meningitis become contagious when the first signs and symptoms of the prodromal period appear. With many viral diseases associated with rashes (e.g., chickenpox, measles, rubella, roseola), patients are contagious during the incubation period up to a week before the rash develops. In contrast, with many respiratory infections (e.g., colds, influenza, diphtheria, strep throat, and pertussis) the patient becomes contagious with the onset of the prodromal period. Depending upon the pathogen, the disease, and the individual infected, transmission can still occur during the periods of decline, convalescence, and even long after signs and symptoms of the disease disappear. For example, an individual recovering from a diarrheal disease may continue to carry and shed the pathogen in feces for some time, posing a risk of transmission to others through direct contact or indirect contact (e.g., through contaminated objects or food).
Exercise \(4\)
Name some of the factors that can affect the length of the incubation period of a particular disease.
Acute and Chronic Diseases
The duration of the period of illness can vary greatly, depending on the pathogen, effectiveness of the immune response in the host, and any medical treatment received. For an acute disease, pathologic changes occur over a relatively short time (e.g., hours, days, or a few weeks) and involve a rapid onset of disease conditions. For example, influenza (caused by Influenzavirus) is considered an acute disease because the incubation period is approximately 1–2 days. Infected individuals can spread influenza to others for approximately 5 days after becoming ill. After approximately 1 week, individuals enter the period of decline.
For a chronic disease, pathologic changes can occur over longer time spans (e.g., months, years, or a lifetime). For example, chronic gastritis (inflammation of the lining of the stomach) is caused by the gram-negative bacterium Helicobacter pylori. H. pylori is able to colonize the stomach and persist in its highly acidic environment by producing the enzyme urease, which modifies the local acidity, allowing the bacteria to survive indefinitely.2 Consequently, H. pylori infections can recur indefinitely unless the infection is cleared using antibiotics.3 Hepatitis B virus can cause a chronic infection in some patients who do not eliminate the virus after the acute illness. A chronic infection with hepatitis B virus is characterized by the continued production of infectious virus for 6 months or longer after the acute infection, as measured by the presence of viral antigen in blood samples.
In latent diseases, as opposed to chronic infections, the causal pathogen goes dormant for extended periods of time with no active replication. Examples of diseases that go into a latent state after the acute infection include herpes(herpes simplex viruses [HSV-1 and HSV-2]), chickenpox (varicella-zoster virus [VZV]), and mononucleosis (Epstein-Barr virus [EBV]). HSV-1, HSV-2, and VZV evade the host immune system by residing in a latent form within cells of the nervous system for long periods of time, but they can reactivate to become active infections during times of stress and immunosuppression. For example, an initial infection by VZV may result in a case of childhood chickenpox, followed by a long period of latency. The virus may reactivate decades later, causing episodes of shingles in adulthood. EBV goes into latency in B cells of the immune system and possibly epithelial cells; it can reactivate years later to produce B-cell lymphoma.
Exercise \(5\)
Explain the difference between latent disease and chronic disease.
Key Concepts and Summary
• In an infection, a microorganism enters a host and begins to multiply. Some infections cause disease, which is any deviation from the normal function or structure of the host.
• Signs of a disease are objective and are measured. Symptoms of a disease are subjective and are reported by the patient.
• Diseases can either be noninfectious (due to genetics and environment) or infectious (due to pathogens). Some infectious diseases are communicable (transmissible between individuals) or contagious (easily transmissible between individuals); others are noncommunicable, but may be contracted via contact with environmental reservoirs or animals (zoonoses)
• Nosocomial diseases are contracted in hospital settings, whereas iatrogenic disease are the direct result of a medical procedure
• An acute disease is short in duration, whereas a chronic disease lasts for months or years. Latent diseases last for years, but are distinguished from chronic diseases by the lack of active replication during extended dormant periods.
• The periods of disease include the incubation period, the prodromal period, the period of illness, the period of decline, and the period of convalescence. These periods are marked by changes in the number of infectious agents and the severity of signs and symptoms.
Footnotes
1. 1 F. Savino et al. “Pain Assessment in Children Undergoing Venipuncture: The Wong–Baker Faces Scale Versus Skin Conductance Fluctuations.” PeerJ 1 (2013):e37; https://peerj.com/articles/37/
2. 2 J.G. Kusters et al. Pathogenesis of Helicobacter pylori Infection. Clinical Microbiology Reviews 19 no. 3 (2006):449–490.
3. 3 N.R. Salama et al. “Life in the Human Stomach: Persistence Strategies of the Bacterial Pathogen Helicobacter pylori.” Nature Reviews Microbiology 11 (2013):385–399. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/15%3A_Microbial_Mechanisms_of_Pathogenicity/15.01%3A_Characteristics_of_Infectious_Diseases.txt |
Learning Objectives
• Summarize Koch’s postulates and molecular Koch’s postulates, respectively, and explain their significance and limitations
• Explain the concept of pathogenicity (virulence) in terms of infectious and lethal dose
• Distinguish between primary and opportunistic pathogens and identify specific examples of each
• Summarize the stages of pathogenesis
• Explain the roles of portals of entry and exit in the transmission of disease and identify specific examples of these portals
For most infectious diseases, the ability to accurately identify the causative pathogen is a critical step in finding or prescribing effective treatments. Today’s physicians, patients, and researchers owe a sizable debt to the physician Robert Koch (1843–1910), who devised a systematic approach for confirming causative relationships between diseases and specific pathogens.
Koch’s Postulates
In 1884, Koch published four postulates that summarized his method for determining whether a particular microorganism was the cause of a particular disease. Each of Koch’s postulates represents a criterion that must be met before a disease can be positively linked with a pathogen. In order to determine whether the criteria are met, tests are performed on laboratory animals and cultures from healthy and diseased animals are compared (Figure \(1\)).
Koch’s Postulates
1. The suspected pathogen must be found in every case of disease and not be found in healthy individuals.
2. The suspected pathogen can be isolated and grown in pure culture.
3. A healthy test subject infected with the suspected pathogen must develop the same signs and symptoms of disease as seen in postulate
4. The pathogen must be re-isolated from the new host and must be identical to the pathogen from postulate 2.
In many ways, Koch’s postulates are still central to our current understanding of the causes of disease. However, advances in microbiology have revealed some important limitations in Koch’s criteria. Koch made several assumptions that we now know are untrue in many cases. The first relates to postulate 1, which assumes that pathogens are only found in diseased, not healthy, individuals. This is not true for many pathogens. For example, H. pylori, described earlier in this chapter as a pathogen causing chronic gastritis, is also part of the normal microbiota of the stomach in many healthy humans who never develop gastritis. It is estimated that upwards of 50% of the human population acquires H. pylori early in life, with most maintaining it as part of the normal microbiota for the rest of their life without ever developing disease.
Koch’s second faulty assumption was that all healthy test subjects are equally susceptible to disease. We now know that individuals are not equally susceptible to disease. Individuals are unique in terms of their microbiota and the state of their immune system at any given time. The makeup of the resident microbiota can influence an individual’s susceptibility to an infection. Members of the normal microbiota play an important role in immunity by inhibiting the growth of transient pathogens. In some cases, the microbiota may prevent a pathogen from establishing an infection; in others, it may not prevent an infection altogether but may influence the severity or type of signs and symptoms. As a result, two individuals with the same disease may not always present with the same signs and symptoms. In addition, some individuals have stronger immune systems than others. Individuals with immune systems weakened by age or an unrelated illness are much more susceptible to certain infections than individuals with strong immune systems.
Koch also assumed that all pathogens are microorganisms that can be grown in pure culture (postulate 2) and that animals could serve as reliable models for human disease. However, we now know that not all pathogens can be grown in pure culture, and many human diseases cannot be reliably replicated in animal hosts. Viruses and certain bacteria, including Rickettsia and Chlamydia, are obligate intracellular pathogens that can grow only when inside a host cell. If a microbe cannot be cultured, a researcher cannot move past postulate 2. Likewise, without a suitable nonhuman host, a researcher cannot evaluate postulate 2 without deliberately infecting humans, which presents obvious ethical concerns. AIDS is an example of such a disease because the human immunodeficiency virus (HIV) only causes disease in humans.
Exercise \(1\)
Briefly summarize the limitations of Koch’s postulates.
Molecular Koch’s Postulates
In 1988, Stanley Falkow (1934–) proposed a revised form of Koch’s postulates known as molecular Koch’s postulates. These are listed in the left column of Table \(1\). The premise for molecular Koch’s postulates is not in the ability to isolate a particular pathogen but rather to identify a gene that may cause the organism to be pathogenic.
Falkow’s modifications to Koch’s original postulates explain not only infections caused by intracellular pathogens but also the existence of pathogenic strains of organisms that are usually nonpathogenic. For example, the predominant form of the bacterium Escherichia coli is a member of the normal microbiota of the human intestine and is generally considered harmless. However, there are pathogenic strains of E. coli such as enterotoxigenic E. coli (ETEC) and enterohemorrhagic E. coli (O157:H7) (EHEC). We now know ETEC and EHEC exist because of the acquisition of new genes by the once-harmless E. coli, which, in the form of these pathogenic strains, is now capable of producing toxins and causing illness. The pathogenic forms resulted from minor genetic changes. The right-side column of Table \(1\) illustrates how molecular Koch’s postulates can be applied to identify EHEC as a pathogenic bacterium.
Table \(1\): Molecular Koch’s Postulates Applied to EHEC
Molecular Koch’s Postulates Application to EHEC
(1) The phenotype (sign or symptom of disease) should be associated only with pathogenic strains of a species. EHEC causes intestinal inflammation and diarrhea, whereas nonpathogenic strains of E. coli do not.
(2) Inactivation of the suspected gene(s) associated with pathogenicity should result in a measurable loss of pathogenicity. One of the genes in EHEC encodes for Shiga toxin, a bacterial toxin (poison) that inhibits protein synthesis. Inactivating this gene reduces the bacteria’s ability to cause disease.
(3) Reversion of the inactive gene should restore the disease phenotype. By adding the gene that encodes the toxin back into the genome (e.g., with a phage or plasmid), EHEC’s ability to cause disease is restored.
As with Koch’s original postulates, the molecular Koch’s postulates have limitations. For example, genetic manipulation of some pathogens is not possible using current methods of molecular genetics. In a similar vein, some diseases do not have suitable animal models, which limits the utility of both the original and molecular postulates.
Exercise \(2\)
Explain the differences between Koch’s original postulates and the molecular Koch’s postulates.
Pathogenicity and Virulence
The ability of a microbial agent to cause disease is called pathogenicity, and the degree to which an organism is pathogenic is called virulence. Virulence is a continuum. On one end of the spectrum are organisms that are avirulent (not harmful) and on the other are organisms that are highly virulent. Highly virulent pathogens will almost always lead to a disease state when introduced to the body, and some may even cause multi-organ and body system failure in healthy individuals. Less virulent pathogens may cause an initial infection, but may not always cause severe illness. Pathogens with low virulence would more likely result in mild signs and symptoms of disease, such as low-grade fever, headache, or muscle aches. Some individuals might even be asymptomatic.
An example of a highly virulent microorganism is Bacillus anthracis, the pathogen responsible for anthrax. B. anthracis can produce different forms of disease, depending on the route of transmission (e.g., cutaneous injection, inhalation, ingestion). The most serious form of anthrax is inhalation anthrax. After B. anthracis spores are inhaled, they germinate. An active infection develops and the bacteria release potent toxins that cause edema (fluid buildup in tissues), hypoxia (a condition preventing oxygen from reaching tissues), and necrosis (cell death and inflammation). Signs and symptoms of inhalation anthrax include high fever, difficulty breathing, vomiting and coughing up blood, and severe chest pains suggestive of a heart attack. With inhalation anthrax, the toxins and bacteria enter the bloodstream, which can lead to multi-organ failure and death of the patient. If a gene (or genes) involved in pathogenesis is inactivated, the bacteria become less virulent or nonpathogenic.
Virulence of a pathogen can be quantified using controlled experiments with laboratory animals. Two important indicators of virulence are the median infectious dose (ID50) and the median lethal dose (LD50), both of which are typically determined experimentally using animal models. The ID50 is the number of pathogen cells or virions required to cause active infection in 50% of inoculated animals. The LD50 is the number of pathogenic cells, virions, or amount of toxin required to kill 50% of infected animals. To calculate these values, each group of animals is inoculated with one of a range of known numbers of pathogen cells or virions. In graphs like the one shown in Figure \(2\), the percentage of animals that have been infected (for ID50) or killed (for LD50) is plotted against the concentration of pathogen inoculated. Figure \(2\) represents data graphed from a hypothetical experiment measuring the LD50 of a pathogen. Interpretation of the data from this graph indicates that the LD50 of the pathogen for the test animals is 104 pathogen cells or virions (depending upon the pathogen studied).
Table \(2\) lists selected foodborne pathogens and their ID50 values in humans (as determined from epidemiologic data and studies on human volunteers). Keep in mind that these are median values. The actual infective dose for an individual can vary widely, depending on factors such as route of entry; the age, health, and immune status of the host; and environmental and pathogen-specific factors such as susceptibility to the acidic pH of the stomach. It is also important to note that a pathogen’s infective dose does not necessarily correlate with disease severity. For example, just a single cell of Salmonella enterica serotype Typhimurium can result in an active infection. The resultant disease, Salmonella gastroenteritis or salmonellosis, can cause nausea, vomiting, and diarrhea, but has a mortality rate of less than 1% in healthy adults. In contrast, S. enterica serotype Typhi has a much higher ID50, typically requiring as many as 1,000 cells to produce infection. However, this serotype causes typhoid fever, a much more systemic and severe disease that has a mortality rate as high as 10% in untreated individuals.
Table \(2\): ID50 for Selected Foodborne Diseases1
Pathogen ID50
Viruses
Hepatitis A virus 10–100
Norovirus 1–10
Rotavirus 10–100
Bacteria
Escherichia coli, enterohemorrhagic (EHEC, serotype O157) 10–100
E. coli, enteroinvasive (EIEC) 200–5,000
E. coli, enteropathogenic (EPEC) 10,000,000–10,000,000,000
E. coli, enterotoxigenic (ETEC) 10,000,000–10,000,000,000
Salmonella enterica serovar Typhi <1,000
S. enterica serovar Typhimurium ≥1
Shigella dysenteriae 10–200
Vibrio cholerae (serotypes O139, O1) 1,000,000
V. parahemolyticus 100,000,000
Protozoa
Giardia lamblia 1
Cryptosporidium parvum 10–100
Exercise \(3\)
1. What is the difference between a pathogen’s infective dose and lethal dose?
2. Which is more closely related to the severity of a disease?
Primary Pathogens versus Opportunistic Pathogens
Pathogens can be classified as either primary pathogens or opportunistic pathogens. A primary pathogen can cause disease in a host regardless of the host’s resident microbiota or immune system. An opportunistic pathogen, by contrast, can only cause disease in situations that compromise the host’s defenses, such as the body’s protective barriers, immune system, or normal microbiota. Individuals susceptible to opportunistic infections include the very young, the elderly, women who are pregnant, patients undergoing chemotherapy, people with immunodeficiencies (such as acquired immunodeficiency syndrome [AIDS]), patients who are recovering from surgery, and those who have had a breach of protective barriers (such as a severe wound or burn).
An example of a primary pathogen is enterohemorrhagic E. coli (EHEC), which produces a virulence factor known as Shiga toxin. This toxin inhibits protein synthesis, leading to severe and bloody diarrhea, inflammation, and renal failure, even in patients with healthy immune systems. Staphylococcus epidermidis, on the other hand, is an opportunistic pathogen that is among the most frequent causes of nosocomial disease.2 S. epidermidis is a member of the normal microbiota of the skin, where it is generally avirulent. However, in hospitals, it can also grow in biofilms that form on catheters, implants, or other devices that are inserted into the body during surgical procedures. Once inside the body, S. epidermidis can cause serious infections such as endocarditis, and it produces virulence factors that promote the persistence of such infections.
Other members of the normal microbiota can also cause opportunistic infections under certain conditions. This often occurs when microbes that reside harmlessly in one body location end up in a different body system, where they cause disease. For example, E. coli normally found in the large intestine can cause a urinary tract infection if it enters the bladder. This is the leading cause of urinary tract infections among women.
Members of the normal microbiota may also cause disease when a shift in the environment of the body leads to overgrowth of a particular microorganism. For example, the yeast Candida is part of the normal microbiota of the skin, mouth, intestine, and vagina, but its population is kept in check by other organisms of the microbiota. If an individual is taking antibacterial medications, however, bacteria that would normally inhibit the growth of Candida can be killed off, leading to a sudden growth in the population of Candida, which is not affected by antibacterial medications because it is a fungus. An overgrowth of Candida can manifest as oral thrush (growth on mouth, throat, and tongue), a vaginal yeast infection, or cutaneous candidiasis. Other scenarios can also provide opportunities for Candida infections. Untreated diabetes can result in a high concentration of glucose in the saliva, which provides an optimal environment for the growth of Candida, resulting in thrush. Immunodeficiencies such as those seen in patients with HIV, AIDS, and cancer also lead to higher incidence of thrush. Vaginal yeast infections can result from decreases in estrogen levels during the menstruation or menopause. The amount of glycogen available to lactobacilli in the vagina is controlled by levels of estrogen; when estrogen levels are low, lactobacilli produce less lactic acid. The resultant increase in vaginal pH allows overgrowth of Candida in the vagina.
Exercise \(4\)
1. Explain the difference between a primary pathogen and an opportunistic pathogen.
2. Describe some conditions under which an opportunistic infection can occur.
Stages of Pathogenesis
To cause disease, a pathogen must successfully achieve four steps or stages of pathogenesis: exposure (contact), adhesion (colonization), invasion, and infection. The pathogen must be able to gain entry to the host, travel to the location where it can establish an infection, evade or overcome the host’s immune response, and cause damage (i.e., disease) to the host. In many cases, the cycle is completed when the pathogen exits the host and is transmitted to a new host.
Exposure
An encounter with a potential pathogen is known as exposure or contact. The food we eat and the objects we handle are all ways that we can come into contact with potential pathogens. Yet, not all contacts result in infection and disease. For a pathogen to cause disease, it needs to be able to gain access into host tissue. An anatomic site through which pathogens can pass into host tissue is called a portal of entry. These are locations where the host cells are in direct contact with the external environment. Major portals of entry are identified in Figure \(3\) and include the skin, mucous membranes, and parenteral routes.
Mucosal surfaces are the most important portals of entry for microbes; these include the mucous membranes of the respiratory tract, the gastrointestinal tract, and the genitourinary tract. Although most mucosal surfaces are in the interior of the body, some are contiguous with the external skin at various body openings, including the eyes, nose, mouth, urethra, and anus.
Most pathogens are suited to a particular portal of entry. A pathogen’s portal specificity is determined by the organism’s environmental adaptions and by the enzymes and toxins they secrete. The respiratory and gastrointestinal tracts are particularly vulnerable portals of entry because particles that include microorganisms are constantly inhaled or ingested, respectively.
Pathogens can also enter through a breach in the protective barriers of the skin and mucous membranes. Pathogens that enter the body in this way are said to enter by the parenteral route. For example, the skin is a good natural barrier to pathogens, but breaks in the skin (e.g., wounds, insect bites, animal bites, needle pricks) can provide a parenteral portal of entry for microorganisms.
In pregnant women, the placenta normally prevents microorganisms from passing from the mother to the fetus. However, a few pathogens are capable of crossing the blood-placental barrier. The gram-positive bacterium Listeria monocytogenes, which causes the foodborne disease listeriosis, is one example that poses a serious risk to the fetus and can sometimes lead to spontaneous abortion. Other pathogens that can pass the placental barrier to infect the fetus are known collectively by the acronym TORCH (Table \(3\)).
Transmission of infectious diseases from mother to baby is also a concern at the time of birth when the baby passes through the birth canal. Babies whose mothers have active chlamydia or gonorrhea infections may be exposed to the causative pathogens in the vagina, which can result in eye infections that lead to blindness. To prevent this, it is standard practice to administer antibiotic drops to infants’ eyes shortly after birth.
Table \(3\): Pathogens Capable of Crossing the Placental Barrier (TORCH Infections)
Disease Pathogen
T Toxoplasmosis Toxoplasma gondii (protozoan)
O3
Syphilis
Chickenpox
Hepatitis B
HIV
Fifth disease (erythema infectiosum)
Treponema pallidum (bacterium)
Varicella-zoster virus (human herpesvirus 3)
Hepatitis B virus (hepadnavirus)
Retrovirus
Parvovirus B19
R Rubella (German measles) Togavirus
C Cytomegalovirus Human herpesvirus 5
H Herpes Herpes simplex viruses (HSV) 1 and 2
Clinical Focus: Part 2
At the clinic, a physician takes down Michael’s medical history and asks about his activities and diet over the past week. Upon learning that Michael became sick the day after the party, the physician orders a blood test to check for pathogens associated with foodborne diseases. After tests confirm that presence of a gram-positive rod in Michael’s blood, he is given an injection of a broad-spectrum antibiotic and sent to a nearby hospital, where he is admitted as a patient. There he is to receive additional intravenous antibiotic therapy and fluids.
Exercise \(5\)
1. Is this bacterium in Michael’s blood part of normal microbiota?
2. What portal of entry did the bacteria use to cause this infection?
Adhesion
Following the initial exposure, the pathogen adheres at the portal of entry. The term adhesion refers to the capability of pathogenic microbes to attach to the cells of the body using adhesion factors, and different pathogens use various mechanisms to adhere to the cells of host tissues.
Molecules (either proteins or carbohydrates) called adhesins are found on the surface of certain pathogens and bind to specific receptors (glycoproteins) on host cells. Adhesins are present on the fimbriae and flagella of bacteria, the cilia of protozoa, and the capsids or membranes of viruses. Protozoans can also use hooks and barbs for adhesion; spike proteins on viruses also enhance viral adhesion. The production of glycocalyces (slime layers and capsules) (Figure \(4\)), with their high sugar and protein content, can also allow certain bacterial pathogens to attach to cells.
Biofilm growth can also act as an adhesion factor. A biofilm is a community of bacteria that produce a glycocalyx, known as extrapolymeric substance (EPS), that allows the biofilm to attach to a surface. Persistent Pseudomonas aeruginosa infections are common in patients suffering from cystic fibrosis, burn wounds, and middle-ear infections (otitis media) because P. aeruginosa produces a biofilm. The EPS allows the bacteria to adhere to the host cells and makes it harder for the host to physically remove the pathogen. The EPS not only allows for attachment but provides protection against the immune system and antibiotic treatments, preventing antibiotics from reaching the bacterial cells within the biofilm. In addition, not all bacteria in a biofilm are rapidly growing; some are in stationary phase. Since antibiotics are most effective against rapidly growing bacteria, portions of bacteria in a biofilm are protected against antibiotics.4
Invasion
Once adhesion is successful, invasion can proceed. Invasion involves the dissemination of a pathogen throughout local tissues or the body. Pathogens may produce exoenzymes or toxins, which serve as virulence factors that allow them to colonize and damage host tissues as they spread deeper into the body. Pathogens may also produce virulence factors that protect them against immune system defenses. A pathogen’s specific virulence factors determine the degree of tissue damage that occurs. Figure \(5\) shows the invasion of H. pylori into the tissues of the stomach, causing damage as it progresses.
Intracellular pathogens achieve invasion by entering the host’s cells and reproducing. Some are obligate intracellular pathogens (meaning they can only reproduce inside of host cells) and others are facultative intracellular pathogens(meaning they can reproduce either inside or outside of host cells). By entering the host cells, intracellular pathogens are able to evade some mechanisms of the immune system while also exploiting the nutrients in the host cell.
Entry to a cell can occur by endocytosis. For most kinds of host cells, pathogens use one of two different mechanisms for endocytosis and entry. One mechanism relies on effector proteins secreted by the pathogen; these effector proteins trigger entry into the host cell. This is the method that Salmonella and Shigella use when invading intestinal epithelial cells. When these pathogens come in contact with epithelial cells in the intestine, they secrete effector molecules that cause protrusions of membrane ruffles that bring the bacterial cell in. This process is called membrane ruffling. The second mechanism relies on surface proteins expressed on the pathogen that bind to receptors on the host cell, resulting in entry. For example, Yersinia pseudotuberculosis produces a surface protein known as invasin that binds to beta-1 integrins expressed on the surface of host cells.
Some host cells, such as white blood cells and other phagocytes of the immune system, actively endocytose pathogens in a process called phagocytosis. Although phagocytosis allows the pathogen to gain entry to the host cell, in most cases, the host cell kills and degrades the pathogen by using digestive enzymes. Normally, when a pathogen is ingested by a phagocyte, it is enclosed within a phagosome in the cytoplasm; the phagosome fuses with a lysosome to form a phagolysosome, where digestive enzymes kill the pathogen (see Pathogen Recognition and Phagocytosis). However, some intracellular pathogens have the ability to survive and multiply within phagocytes. Examples include Listeria monocytogenes and Shigella; these bacteria produce proteins that lyse the phagosome before it fuses with the lysosome, allowing the bacteria to escape into the phagocyte’s cytoplasm where they can multiply. Bacteria such as Mycobacterium tuberculosis, Legionella pneumophila, and Salmonella species use a slightly different mechanism to evade being digested by the phagocyte. These bacteria prevent the fusion of the phagosome with the lysosome, thus remaining alive and dividing within the phagosome.
Infection
Following invasion, successful multiplication of the pathogen leads to infection. Infections can be described as local, focal, or systemic, depending on the extent of the infection. A local infection is confined to a small area of the body, typically near the portal of entry. For example, a hair follicle infected by Staphylococcus aureus infection may result in a boil around the site of infection, but the bacterium is largely contained to this small location. Other examples of local infections that involve more extensive tissue involvement include urinary tract infections confined to the bladder or pneumonia confined to the lungs.
In a focal infection, a localized pathogen, or the toxins it produces, can spread to a secondary location. For example, a dental hygienist nicking the gum with a sharp tool can lead to a local infection in the gum by Streptococcus bacteria of the normal oral microbiota. These Streptococcus spp. may then gain access to the bloodstream and make their way to other locations in the body, resulting in a secondary infection.
When an infection becomes disseminated throughout the body, we call it a systemic infection. For example, infection by the varicella-zoster virus typically gains entry through a mucous membrane of the upper respiratory system. It then spreads throughout the body, resulting in the classic red skin lesions associated with chickenpox. Since these lesions are not sites of initial infection, they are signs of a systemic infection.
Sometimes a primary infection, the initial infection caused by one pathogen, can lead to a secondary infection by another pathogen. For example, the immune system of a patient with a primary infection by HIV becomes compromised, making the patient more susceptible to secondary diseases like oral thrush and others caused by opportunistic pathogens. Similarly, a primary infection by Influenzavirus damages and decreases the defense mechanisms of the lungs, making patients more susceptible to a secondary pneumonia by a bacterial pathogen like Haemophilus influenzae or Streptococcus pneumoniae. Some secondary infections can even develop as a result of treatment for a primary infection. Antibiotic therapy targeting the primary pathogen can cause collateral damage to the normal microbiota, creating an opening for opportunistic pathogens (see Case in Point: A Secondary Yeast Infection below).
A Secondary Yeast Infection
Anita, a 36-year-old mother of three, goes to an urgent care center complaining of pelvic pressure, frequent and painful urination, abdominal cramps, and occasional blood-tinged urine. Suspecting a urinary tract infection (UTI), the physician requests a urine sample and sends it to the lab for a urinalysis. Since it will take approximately 24 hours to get the results of the culturing, the physician immediately starts Anita on the antibiotic ciprofloxacin. The next day, the microbiology lab confirms the presence of E. coli in Anita’s urine, which is consistent with the presumptive diagnosis. However, the antimicrobial susceptibility test indicates that ciprofloxacin would not effectively treat Anita’s UTI, so the physician prescribes a different antibiotic.
After taking her antibiotics for 1 week, Anita returns to the clinic complaining that the prescription is not working. Although the painful urination has subsided, she is now experiencing vaginal itching, burning, and discharge. After a brief examination, the physician explains to Anita that the antibiotics were likely successful in killing the E. coli responsible for her UTI; however, in the process, they also wiped out many of the “good” bacteria in Anita’s normal microbiota. The new symptoms that Anita has reported are consistent with a secondary yeast infection by Candida albicans, an opportunistic fungus that normally resides in the vagina but is inhibited by the bacteria that normally reside in the same environment.
To confirm this diagnosis, a microscope slide of a direct vaginal smear is prepared from the discharge to check for the presence of yeast. A sample of the discharge accompanies this slide to the microbiology lab to determine if there has been an increase in the population of yeast causing vaginitis. After the microbiology lab confirms the diagnosis, the physician prescribes an antifungal drug for Anita to use to eliminate her secondary yeast infection.
Exercise \(6\)
1. Why was Candida not killed by the antibiotics prescribed for the UTI?
2. List three conditions that could lead to a secondary infection.
Transmission of Disease
For a pathogen to persist, it must put itself in a position to be transmitted to a new host, leaving the infected host through a portal of exit (Figure \(6\)). As with portals of entry, many pathogens are adapted to use a particular portal of exit. Similar to portals of entry, the most common portals of exit include the skin and the respiratory, urogenital, and gastrointestinal tracts. Coughing and sneezing can expel pathogens from the respiratory tract. A single sneeze can send thousands of virus particles into the air. Secretions and excretions can transport pathogens out of other portals of exit. Feces, urine, semen, vaginal secretions, tears, sweat, and shed skin cells can all serve as vehicles for a pathogen to leave the body. Pathogens that rely on insect vectors for transmission exit the body in the blood extracted by a biting insect. Similarly, some pathogens exit the body in blood extracted by needles.
Key Concepts and Summary
• Koch’s postulates are used to determine whether a particular microorganism is a pathogen. Molecular Koch’s postulates are used to determine what genes contribute to a pathogen’s ability to cause disease.
• Virulence, the degree to which a pathogen can cause disease, can be quantified by calculating either the ID50 or LD50 of a pathogen on a given population.
• Primary pathogens are capable of causing pathological changes associated with disease in a healthy individual, whereas opportunistic pathogens can only cause disease when the individual is compromised by a break in protective barriers or immunosuppression.
• Infections and disease can be caused by pathogens in the environment or microbes in an individual’s resident microbiota.
• Infections can be classified as local, focal, or systemic depending on the extent to which the pathogen spreads in the body.
• A secondary infection can sometimes occur after the host’s defenses or normal microbiota are compromised by a primary infection or antibiotic treatment.
• Pathogens enter the body through portals of entry and leave through portals of exit. The stages of pathogenesis include exposure, adhesion, invasion, infection, and transmission.
Footnotes
1. 1 Food and Drug Administration. “Bad Bug Book, Foodborne Pathogenic Microorganisms and Natural Toxins.” 2nd ed. Silver Spring, MD: US Food and Drug Administration; 2012.
2. 2 M. Otto. “Staphylococcus epidermidis--The ‘Accidental’ Pathogen.” Nature Reviews Microbiology 7 no. 8 (2009):555–567.
3. 3 The O in TORCH stands for “other.”
4. 4 D. Davies. “Understanding Biofilm Resistance to Antibacterial Agents.” Nature Reviews Drug Discovery 2 (2003):114–122. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/15%3A_Microbial_Mechanisms_of_Pathogenicity/15.02%3A_How_Pathogens_Cause_Disease.txt |
Learning Objectives
• Explain how virulence factors contribute to signs and symptoms of infectious disease
• Differentiate between endotoxins and exotoxins
• Describe and differentiate between various types of exotoxins
• Describe the mechanisms viruses use for adhesion and antigenic variation
In the previous section, we explained that some pathogens are more virulent than others. This is due to the unique virulence factors produced by individual pathogens, which determine the extent and severity of disease they may cause. A pathogen’s virulence factors are encoded by genes that can be identified using molecular Koch’s postulates. When genes encoding virulence factors are inactivated, virulence in the pathogen is diminished. In this section, we examine various types and specific examples of virulence factors and how they contribute to each step of pathogenesis.
Virulence Factors for Adhesion
As discussed in the previous section, the first two steps in pathogenesis are exposure and adhesion. Recall that an adhesin is a protein or glycoprotein found on the surface of a pathogen that attaches to receptors on the host cell. Adhesins are found on bacterial, viral, fungal, and protozoan pathogens. One example of a bacterial adhesin is type 1 fimbrial adhesin, a molecule found on the tips of fimbriae of enterotoxigenic E. coli (ETEC). Recall that fimbriae are hairlike protein bristles on the cell surface. Type 1 fimbrial adhesin allows the fimbriae of ETEC cells to attach to the mannose glycans expressed on intestinal epithelial cells. Table \(1\) lists common adhesins found in some of the pathogens we have discussed or will be seeing later in this chapter.
Table \(1\): Some Bacterial Adhesins and Their Host Attachment Sites
Pathogen Disease Adhesin Attachment Site
Streptococcus pyogenes Strep throat Protein F Respiratory epithelial cells
Streptococcus mutans Dental caries Adhesin P1 Teeth
Neisseria gonorrhoeae Gonorrhea Type IV pili Urethral epithelial cells
Enterotoxigenic E. coli (ETEC) Traveler’s diarrhea Type 1 fimbriae Intestinal epithelial cells
Vibrio cholerae Cholera N-methylphenylalanine pili Intestinal epithelial cells
Clinical Focus: Part 3
The presence of bacteria in Michael’s blood is a sign of infection, since blood is normally sterile. There is no indication that the bacteria entered the blood through an injury. Instead, it appears the portal of entry was the gastrointestinal route. Based on Michael’s symptoms, the results of his blood test, and the fact that Michael was the only one in the family to partake of the hot dogs, the physician suspects that Michael is suffering from a case of listeriosis.
Listeria monocytogenes, the facultative intracellular pathogen that causes listeriosis, is a common contaminant in ready-to-eat foods such as lunch meats and dairy products. Once ingested, these bacteria invade intestinal epithelial cells and translocate to the liver, where they grow inside hepatic cells. Listeriosis is fatal in about one in five normal healthy people, and mortality rates are slightly higher in patients with pre-existing conditions that weaken the immune response. A cluster of virulence genes encoded on a pathogenicity island is responsible for the pathogenicity of L. monocytogenes. These genes are regulated by a transcriptional factor known as peptide chain release factor 1 (PrfA). One of the genes regulated by PrfA is hyl, which encodes a toxin known as listeriolysin O (LLO), which allows the bacterium to escape vacuoles upon entry into a host cell. A second gene regulated by PrfA is actA, which encodes for a surface protein known as actin assembly-inducing protein (ActA). ActA is expressed on the surface of Listeria and polymerizes host actin. This enables the bacterium to produce actin tails, move around the cell’s cytoplasm, and spread from cell to cell without exiting into the extracellular compartment.
Michael’s condition has begun to worsen. He is now experiencing a stiff neck and hemiparesis (weakness of one side of the body). Concerned that the infection is spreading, the physician decides to conduct additional tests to determine what is causing these new symptoms.
Exercise \(1\)
1. What kind of pathogen causes listeriosis, and what virulence factors contribute to the signs and symptoms Michael is experiencing?
2. Is it likely that the infection will spread from Michael’s blood? If so, how might this explain his new symptoms?
Bacterial Exoenzymes and Toxins as Virulence Factors
After exposure and adhesion, the next step in pathogenesis is invasion, which can involve enzymes and toxins. Many pathogens achieve invasion by entering the bloodstream, an effective means of dissemination because blood vessels pass close to every cell in the body. The downside of this mechanism of dispersal is that the blood also includes numerous elements of the immune system. Various terms ending in –emia are used to describe the presence of pathogens in the bloodstream. The presence of bacteria in blood is called bacteremia. Bacteremia involving pyogens(pus-forming bacteria) is called pyemia. When viruses are found in the blood, it is called viremia. The term toxemiadescribes the condition when toxins are found in the blood. If bacteria are both present and multiplying in the blood, this condition is called septicemia.
Patients with septicemia are described as septic, which can lead to shock, a life-threatening decrease in blood pressure (systolic pressure <90 mm Hg) that prevents cells and organs from receiving enough oxygen and nutrients. Some bacteria can cause shock through the release of toxins (virulence factors that can cause tissue damage) and lead to low blood pressure. Gram-negative bacteria are engulfed by immune system phagocytes, which then release tumor necrosis factor, a molecule involved in inflammation and fever. Tumor necrosis factor binds to blood capillaries to increase their permeability, allowing fluids to pass out of blood vessels and into tissues, causing swelling, or edema(Figure \(1\)). With high concentrations of tumor necrosis factor, the inflammatory reaction is severe and enough fluid is lost from the circulatory system that blood pressure decreases to dangerously low levels. This can have dire consequences because the heart, lungs, and kidneys rely on normal blood pressure for proper function; thus, multi-organ failure, shock, and death can occur.
Exoenzymes
Some pathogens produce extracellular enzymes, or exoenzymes, that enable them to invade host cells and deeper tissues. Exoenzymes have a wide variety of targets. Some general classes of exoenzymes and associated pathogens are listed in Table \(2\). Each of these exoenzymes functions in the context of a particular tissue structure to facilitate invasion or support its own growth and defend against the immune system. For example, hyaluronidase S, an enzyme produced by pathogens like Staphylococcus aureus, Streptococcus pyogenes, and Clostridium perfringens, degrades the glycoside hylauronan (hyaluronic acid), which acts as an intercellular cement between adjacent cells in connective tissue (Figure \(2\)). This allows the pathogen to pass through the tissue layers at the portal of entry and disseminate elsewhere in the body (Figure \(2\)).
Table \(2\): Some Classes of Exoenzymes and Their Targets
Class Example Function
Glycohydrolases Hyaluronidase S in Staphylococcus aureus Degrades hyaluronic acid that cements cells together to promote spreading through tissues
Nucleases DNAse produced by S. aureus Degrades DNA released by dying cells (bacteria and host cells) that can trap the bacteria, thus promoting spread
Phospholipases Phospholipase C of Bacillus anthracis Degrades phospholipid bilayer of host cells, causing cellular lysis, and degrade membrane of phagosomes to enable escape into the cytoplasm
Proteases Collagenase in Clostridium perfringens Degrades collagen in connective tissue to promote spread
Pathogen-produced nucleases, such as DNAse produced by S. aureus, degrade extracellular DNA as a means of escape and spreading through tissue. As bacterial and host cells die at the site of infection, they lyse and release their intracellular contents. The DNA chromosome is the largest of the intracellular molecules, and masses of extracellular DNA can trap bacteria and prevent their spread. S. aureus produces a DNAse to degrade the mesh of extracellular DNA so it can escape and spread to adjacent tissues. This strategy is also used by S. aureus and other pathogens to degrade and escape webs of extracellular DNA produced by immune system phagocytes to trap the bacteria.
Enzymes that degrade the phospholipids of cell membranes are called phospholipases. Their actions are specific in regard to the type of phospholipids they act upon and where they enzymatically cleave the molecules. The pathogen responsible for anthrax, B. anthracis, produces phospholipase C. When B. anthracis is ingested by phagocytic cells of the immune system, phospholipase C degrades the membrane of the phagosome before it can fuse with the lysosome, allowing the pathogen to escape into the cytoplasm and multiply. Phospholipases can also target the membrane that encloses the phagosome within phagocytic cells. As described earlier in this chapter, this is the mechanism used by intracellular pathogens such as L. monocytogenes and Rickettsia to escape the phagosome and multiply within the cytoplasm of phagocytic cells. The role of phospholipases in bacterial virulence is not restricted to phagosomal escape. Many pathogens produce phospholipases that act to degrade cell membranes and cause lysis of target cells. These phospholipases are involved in lysis of red blood cells, white blood cells, and tissue cells.
Bacterial pathogens also produce various protein-digesting enzymes, or proteases. Proteases can be classified according to their substrate target (e.g., serine proteases target proteins with the amino acid serine) or if they contain metals in their active site (e.g., zinc metalloproteases contain a zinc ion, which is necessary for enzymatic activity).
One example of a protease that contains a metal ion is the exoenzyme collagenase. Collagenase digests collagen, the dominant protein in connective tissue. Collagen can be found in the extracellular matrix, especially near mucosal membranes, blood vessels, nerves, and in the layers of the skin. Similar to hyaluronidase, collagenase allows the pathogen to penetrate and spread through the host tissue by digesting this connective tissue protein. The collagenase produced by the gram-positive bacterium Clostridium perfringens, for example, allows the bacterium to make its way through the tissue layers and subsequently enter and multiply in the blood (septicemia). C. perfringens then uses toxins and a phospholipase to cause cellular lysis and necrosis. Once the host cells have died, the bacterium produces gas by fermenting the muscle carbohydrates. The widespread necrosis of tissue and accompanying gas are characteristic of the condition known as gas gangrene (Figure \(3\)).
Toxins
In addition to exoenzymes, certain pathogens are able to produce toxins, biological poisons that assist in their ability to invade and cause damage to tissues. The ability of a pathogen to produce toxins to cause damage to host cells is called toxigenicity.
Toxins can be categorized as endotoxins or exotoxins. The lipopolysaccharide (LPS) found on the outer membrane of gram-negative bacteria is called endotoxin (Figure \(4\)). During infection and disease, gram-negative bacterial pathogens release endotoxin either when the cell dies, resulting in the disintegration of the membrane, or when the bacterium undergoes binary fission. The lipid component of endotoxin, lipid A, is responsible for the toxic properties of the LPS molecule. Lipid A is relatively conserved across different genera of gram-negative bacteria; therefore, the toxic properties of lipid A are similar regardless of the gram-negative pathogen. In a manner similar to that of tumor necrosis factor, lipid A triggers the immune system’s inflammatory response (see Inflammation and Fever). If the concentration of endotoxin in the body is low, the inflammatory response may provide the host an effective defense against infection; on the other hand, high concentrations of endotoxin in the blood can cause an excessive inflammatory response, leading to a severe drop in blood pressure, multi-organ failure, and death.
A classic method of detecting endotoxin is by using the Limulus amebocyte lysate (LAL) test. In this procedure, the blood cells (amebocytes) of the horseshoe crab (Limulus polyphemus) is mixed with a patient’s serum. The amebocytes will react to the presence of any endotoxin. This reaction can be observed either chromogenically (color) or by looking for coagulation (clotting reaction) to occur within the serum. An alternative method that has been used is an enzyme-linked immunosorbent assay (ELISA) that uses antibodies to detect the presence of endotoxin.
Unlike the toxic lipid A of endotoxin, exotoxins are protein molecules that are produced by a wide variety of living pathogenic bacteria. Although some gram-negative pathogens produce exotoxins, the majority are produced by gram-positive pathogens. Exotoxins differ from endotoxin in several other key characteristics, summarized in Table \(3\). In contrast to endotoxin, which stimulates a general systemic inflammatory response when released, exotoxins are much more specific in their action and the cells they interact with. Each exotoxin targets specific receptors on specific cells and damages those cells through unique molecular mechanisms. Endotoxin remains stable at high temperatures, and requires heating at 121 °C (250 °F) for 45 minutes to inactivate. By contrast, most exotoxins are heat labile because of their protein structure, and many are denatured (inactivated) at temperatures above 41 °C (106 °F). As discussed earlier, endotoxin can stimulate a lethal inflammatory response at very high concentrations and has a measured LD50 of 0.24 mg/kg. By contrast, very small concentrations of exotoxins can be lethal. For example, botulinum toxin, which causes botulism, has an LD50 of 0.000001 mg/kg (240,000 times more lethal than endotoxin).
Table \(3\): Comparison of Endotoxin and Exotoxins Produced by Bacteria
Characteristic Endotoxin Exotoxin
Source Gram-negative bacteria Gram-positive (primarily) and gram-negative bacteria
Composition Lipid A component of lipopolysaccharide Protein
Effect on host General systemic symptoms of inflammation and fever Specific damage to cells dependent upon receptor-mediated targeting of cells and specific mechanisms of action
Heat stability Heat stable Most are heat labile, but some are heat stable
LD50 High Low
The exotoxins can be grouped into three categories based on their target: intracellular targeting, membrane disrupting, and superantigens. Table \(4\) provides examples of well-characterized toxins within each of these three categories.
Table \(4\): Some Common Exotoxins and Associated Bacterial Pathogens
Category Example Pathogen Mechanism and Disease
Intracellular-targeting toxins Cholera toxin Vibrio cholerae Activation of adenylate cyclase in intestinal cells, causing increased levels of cyclic adenosine monophosphate (cAMP) and secretion of fluids and electrolytes out of cell, causing diarrhea
Tetanus toxin Clostridium tetani Inhibits the release of inhibitory neurotransmitters in the central nervous system, causing spastic paralysis
Botulinum toxin Clostridium botulinum Inhibits release of the neurotransmitter acetylcholine from neurons, resulting in flaccid paralysis
Diphtheria toxin Corynebacterium diphtheriae Inhibition of protein synthesis, causing cellular death
Membrane-disrupting toxins Streptolysin Streptococcus pyogenes Proteins that assemble into pores in cell membranes, disrupting their function and killing the cell
Pneumolysin Streptococcus pneumoniae
Alpha-toxin Staphylococcus aureus
Alpha-toxin Clostridium perfringens Phospholipases that degrade cell membrane phospholipids, disrupting membrane function and killing the cell
Phospholipase C Pseudomonas aeruginosa
Beta-toxin Staphylococcus aureus
Superantigens Toxic shock syndrome toxin Staphylococcus aureus Stimulates excessive activation of immune system cells and release of cytokines (chemical mediators) from immune system cells. Life-threatening fever, inflammation, and shock are the result.
Streptococcal mitogenic exotoxin Streptococcus pyogenes
Streptococcal pyrogenic toxins Streptococcus pyogenes
The intracellular targeting toxins comprise two components: A for activity and B for binding. Thus, these types of toxins are known as A-B exotoxins (Figure \(5\)). The B component is responsible for the cellular specificity of the toxin and mediates the initial attachment of the toxin to specific cell surface receptors. Once the A-B toxin binds to the host cell, it is brought into the cell by endocytosis and entrapped in a vacuole. The A and B subunits separate as the vacuole acidifies. The A subunit then enters the cell cytoplasm and interferes with the specific internal cellular function that it targets.
Four unique examples of A-B toxins are the diphtheria, cholera, botulinum, and tetanus toxins. The diphtheria toxin is produced by the gram-positive bacterium Corynebacterium diphtheriae, the causative agent of nasopharyngeal and cutaneous diphtheria. After the A subunit of the diphtheria toxin separates and gains access to the cytoplasm, it facilitates the transfer of adenosine diphosphate (ADP)-ribose onto an elongation-factor protein (EF-2) that is needed for protein synthesis. Hence, diphtheria toxin inhibits protein synthesis in the host cell, ultimately killing the cell (Figure \(6\)).
Cholera toxin is an enterotoxin produced by the gram-negative bacterium Vibrio cholerae and is composed of one A subunit and five B subunits. The mechanism of action of the cholera toxin is complex. The B subunits bind to receptors on the intestinal epithelial cell of the small intestine. After gaining entry into the cytoplasm of the epithelial cell, the A subunit activates an intracellular G protein. The activated G protein, in turn, leads to the activation of the enzyme adenyl cyclase, which begins to produce an increase in the concentration of cyclic AMP (a secondary messenger molecule). The increased cAMP disrupts the normal physiology of the intestinal epithelial cells and causes them to secrete excessive amounts of fluid and electrolytes into the lumen of the intestinal tract, resulting in severe “rice-water stool” diarrhea characteristic of cholera.
Botulinum toxin (also known as botox) is a neurotoxin produced by the gram-positive bacterium Clostridium botulinum. It is the most acutely toxic substance known to date. The toxin is composed of a light A subunit and heavy protein chain B subunit. The B subunit binds to neurons to allow botulinum toxin to enter the neurons at the neuromuscular junction. The A subunit acts as a protease, cleaving proteins involved in the neuron’s release of acetylcholine, a neurotransmitter molecule. Normally, neurons release acetylcholine to induce muscle fiber contractions. The toxin’s ability to block acetylcholine release results in the inhibition of muscle contractions, leading to muscle relaxation. This has the potential to stop breathing and cause death. Because of its action, low concentrations of botox are used for cosmetic and medical procedures, including the removal of wrinkles and treatment of overactive bladder.
Link to Learning
Click this link to see an animation of how the cholera toxin functions.
Click this link to see an animation of how the botulinum toxin functions.
Another neurotoxin is tetanus toxin, which is produced by the gram-positive bacterium Clostridium tetani. This toxin also has a light A subunit and heavy protein chain B subunit. Unlike botulinum toxin, tetanus toxin binds to inhibitory interneurons, which are responsible for release of the inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA). Normally, these neurotransmitters bind to neurons at the neuromuscular junction, resulting in the inhibition of acetylcholine release. Tetanus toxin inhibits the release of glycine and GABA from the interneuron, resulting in permanent muscle contraction. The first symptom is typically stiffness of the jaw (lockjaw). Violent muscle spasms in other parts of the body follow, typically culminating with respiratory failure and death. Figure \(7\) shows the actions of both botulinum and tetanus toxins.
Membrane-disrupting toxins affect cell membrane function either by forming pores or by disrupting the phospholipid bilayer in host cell membranes. Two types of membrane-disrupting exotoxins are hemolysins and leukocidins, which form pores in cell membranes, causing leakage of the cytoplasmic contents and cell lysis. These toxins were originally thought to target red blood cells (erythrocytes) and white blood cells (leukocytes), respectively, but we now know they can affect other cells as well. The gram-positive bacterium Streptococcus pyogenes produces streptolysins, water-soluble hemolysins that bind to the cholesterol moieties in the host cell membrane to form a pore. The two types of streptolysins, O and S, are categorized by their ability to cause hemolysis in erythrocytes in the absence or presence of oxygen. Streptolysin O is not active in the presence of oxygen, whereas streptolysin S is active in the presence of oxygen. Other important pore-forming membrane-disrupting toxins include alpha toxin of Staphylococcus aureus and pneumolysin of Streptococcus pneumoniae.
Bacterial phospholipases are membrane-disrupting toxins that degrade the phospholipid bilayer of cell membranes rather than forming pores. We have already discussed the phospholipases associated with B. anthracis, L. pneumophila, and Rickettsia species that enable these bacteria to effect the lysis of phagosomes. These same phospholipases are also hemolysins. Other phospholipases that function as hemolysins include the alpha toxin of Clostridium perfringens, phospholipase C of P. aeruginosa, and beta toxin of Staphylococcus aureus.
Some strains of S. aureus also produce a leukocidin called Panton-Valentine leukocidin (PVL). PVL consists of two subunits, S and F. The S component acts like the B subunit of an A-B exotoxin in that it binds to glycolipids on the outer plasma membrane of animal cells. The F-component acts like the A subunit of an A-B exotoxin and carries the enzymatic activity. The toxin inserts and assembles into a pore in the membrane. Genes that encode PVL are more frequently present in S. aureus strains that cause skin infections and pneumonia.1 PVL promotes skin infections by causing edema, erythema (reddening of the skin due to blood vessel dilation), and skin necrosis. PVL has also been shown to cause necrotizing pneumonia. PVL promotes pro-inflammatory and cytotoxic effects on alveolar leukocytes. This results in the release of enzymes from the leukocytes, which, in turn, cause damage to lung tissue.
The third class of exotoxins is the superantigens. These are exotoxins that trigger an excessive, nonspecific stimulation of immune cells to secrete cytokines (chemical messengers). The excessive production of cytokines, often called a cytokine storm, elicits a strong immune and inflammatory response that can cause life-threatening high fevers, low blood pressure, multi-organ failure, shock, and death. The prototype superantigen is the toxic shock syndrome toxin of S. aureus. Most toxic shock syndrome cases are associated with vaginal colonization by toxin-producing S. aureus in menstruating women; however, colonization of other body sites can also occur. Some strains of Streptococcus pyogenes also produce superantigens; they are referred to as the streptococcal mitogenic exotoxins and the streptococcal pyrogenic toxins.
Exercise \(2\)
1. Describe how exoenzymes contribute to bacterial invasion.
2. Explain the difference between exotoxins and endotoxin.
3. Name the three classes of exotoxins.
Virulence Factors for Survival in the Host and Immune Evasion
Evading the immune system is also important to invasiveness. Bacteria use a variety of virulence factors to evade phagocytosis by cells of the immune system. For example, many bacteria produce capsules, which are used in adhesion but also aid in immune evasion by preventing ingestion by phagocytes. The composition of the capsule prevents immune cells from being able to adhere and then phagocytose the cell. In addition, the capsule makes the bacterial cell much larger, making it harder for immune cells to engulf the pathogen (Figure \(8\)). A notable capsule-producing bacterium is the gram-positive pathogen Streptococcus pneumoniae, which causes pneumococcal pneumonia, meningitis, septicemia, and other respiratory tract infections. Encapsulated strains of S. pneumoniae are more virulent than nonencapsulated strains and are more likely to invade the bloodstream and cause septicemia and meningitis.
Some pathogens can also produce proteases to protect themselves against phagocytosis. As described in Adaptive Specific Host Defenses, the human immune system produces antibodies that bind to surface molecules found on specific bacteria (e.g., capsules, fimbriae, flagella, LPS). This binding initiates phagocytosis and other mechanisms of antibacterial killing and clearance. Proteases combat antibody-mediated killing and clearance by attacking and digesting the antibody molecules (Figure \(8\)).
In addition to capsules and proteases, some bacterial pathogens produce other virulence factors that allow them to evade the immune system. The fimbriae of certain species of Streptococcus contain M protein, which alters the surface of Streptococcus and inhibits phagocytosis by blocking the binding of the complement molecules that assist phagocytes in ingesting bacterial pathogens. The acid-fast bacterium Mycobacterium tuberculosis (the causative agent of tuberculosis) produces a waxy substance known as mycolic acid in its cell envelope. When it is engulfed by phagocytes in the lung, the protective mycolic acid coat enables the bacterium to resist some of the killing mechanisms within the phagolysosome.
Some bacteria produce virulence factors that promote infection by exploiting molecules naturally produced by the host. For example, most strains of Staphylococcus aureus produce the exoenzyme coagulase, which exploits the natural mechanism of blood clotting to evade the immune system. Normally, blood clotting is triggered in response to blood vessel damage; platelets begin to plug the clot, and a cascade of reactions occurs in which fibrinogen, a soluble protein made by the liver, is cleaved into fibrin. Fibrin is an insoluble, thread-like protein that binds to blood platelets, cross-links, and contracts to form a mesh of clumped platelets and red blood cells. The resulting clot prevents further loss of blood from the damaged blood vessels. However, if bacteria release coagulase into the bloodstream, the fibrinogen-to-fibrin cascade is triggered in the absence of blood vessel damage. The resulting clot coats the bacteria in fibrin, protecting the bacteria from exposure to phagocytic immune cells circulating in the bloodstream.
Whereas coagulase causes blood to clot, kinases have the opposite effect by triggering the conversion of plasminogen to plasmin, which is involved in the digestion of fibrin clots. By digesting a clot, kinases allow pathogens trapped in the clot to escape and spread, similar to the way that collagenase, hyaluronidase, and DNAse facilitate the spread of infection. Examples of kinases include staphylokinases and streptokinases, produced by Staphylococcus aureusand Streptococcus pyogenes, respectively. It is intriguing that S. aureus can produce both coagulase to promote clotting and staphylokinase to stimulate the digestion of clots. The action of the coagulase provides an important protective barrier from the immune system, but when nutrient supplies are diminished or other conditions signal a need for the pathogen to escape and spread, the production of staphylokinase can initiate this process.
A final mechanism that pathogens can use to protect themselves against the immune system is called antigenic variation, which is the alteration of surface proteins so that a pathogen is no longer recognized by the host’s immune system. For example, the bacterium Borrelia burgdorferi, the causative agent of Lyme disease, contains a surface lipoprotein known as VlsE. Because of genetic recombination during DNA replication and repair, this bacterial protein undergoes antigenic variation. Each time fever occurs, the VlsE protein in B. burgdorferi can differ so much that antibodies against previous VlsE sequences are not effective. It is believed that this variation in the VlsE contributes to the ability B. burgdorferi to cause chronic disease. Another important human bacterial pathogen that uses antigenic variation to avoid the immune system is Neisseria gonorrhoeae, which causes the sexually transmitted disease gonorrhea. This bacterium is well known for its ability to undergo antigenic variation of its type IV pili to avoid immune defenses.
Exercise \(3\)
1. Name at least two ways that a capsule provides protection from the immune system.
2. Besides capsules, name two other virulence factors used by bacteria to evade the immune system.
Clinical Focus: Resolution
Based on Michael’s reported symptoms of stiff neck and hemiparesis, the physician suspects that the infection may have spread to his nervous system. The physician decides to order a spinal tap to look for any bacteria that may have invaded the meninges and cerebrospinal fluid (CSF), which would normally be sterile. To perform the spinal tap, Michael’s lower back is swabbed with an iodine antiseptic and then covered with a sterile sheet. The needle is aseptically removed from the manufacturer’s sealed plastic packaging by the clinician’s gloved hands. The needle is inserted and a small volume of fluid is drawn into an attached sample tube. The tube is removed, capped and a prepared label with Michael’s data is affixed to it. This STAT (urgent or immediate analysis required) specimen is divided into three separate sterile tubes, each with 1 mL of CSF. These tubes are immediately taken to the hospital’s lab, where they are analyzed in the clinical chemistry, hematology, and microbiology departments. The preliminary results from all three departments indicate there is a cerebrospinal infection occurring, with the microbiology department reporting the presence of a gram-positive rod in Michael’s CSF.
These results confirm what his physician had suspected: Michael’s new symptoms are the result of meningitis, acute inflammation of the membranes that protect the brain and spinal cord. Because meningitis can be life threatening and because the first antibiotic therapy was not effective in preventing the spread of infection, Michael is prescribed an aggressive course of two antibiotics, ampicillin and gentamicin, to be delivered intravenously. Michael remains in the hospital for several days for supportive care and for observation. After a week, he is allowed to return home for bed rest and oral antibiotics. After 3 weeks of this treatment, he makes a full recovery.
Viral Virulence
Although viral pathogens are not similar to bacterial pathogens in terms of structure, some of the properties that contribute to their virulence are similar. Viruses use adhesins to facilitate adhesion to host cells, and certain enveloped viruses rely on antigenic variation to avoid the host immune defenses. These virulence factors are discussed in more detail in the following sections.
Viral Adhesins
One of the first steps in any viral infection is adhesion of the virus to specific receptors on the surface of cells. This process is mediated by adhesins that are part of the viral capsid or membrane envelope. The interaction of viral adhesins with specific cell receptors defines the tropism (preferential targeting) of viruses for specific cells, tissues, and organs in the body. The spike protein hemagglutinin found on Influenzavirus is an example of a viral adhesin; it allows the virus to bind to the sialic acid on the membrane of host respiratory and intestinal cells. Another viral adhesin is the glycoprotein gp20, found on HIV. For HIV to infect cells of the immune system, it must interact with two receptors on the surface of cells. The first interaction involves binding between gp120 and the CD4 cellular marker that is found on some essential immune system cells. However, before viral entry into the cell can occur, a second interaction between gp120 and one of two chemokine receptors (CCR5 and CXCR4) must occur. Table \(5\) lists the adhesins for some common viral pathogens and the specific sites to which these adhesins allow viruses to attach.
Table \(5\): Some Viral Adhesins and Their Host Attachment Sites
Pathogen Disease Adhesin Attachment Site
Influenzavirus Influenza Hemagglutinin Sialic acid of respiratory and intestinal cells
Herpes simplex virus I or II Oral herpes, genital herpes Glycoproteins gB, gC, gD Heparan sulfate on mucosal surfaces of the mouth and genitals
Human immunodeficiency virus HIV/AIDS Glycoprotein gp120 CD4 and CCR5 or CXCR4 of immune system cells
Antigenic Variation in Viruses
Antigenic variation also occurs in certain types of enveloped viruses, including influenza viruses, which exhibit two forms of antigenic variation: antigenic drift and antigenic shift (Figure \(9\)). Antigenic drift is the result of point mutations causing slight changes in the spike proteins hemagglutinin (H) and neuraminidase (N). On the other hand, antigenic shift is a major change in spike proteins due to gene reassortment. This reassortment for antigenic shift occurs typically when two different influenza viruses infect the same host.
The rate of antigenic variation in influenza viruses is very high, making it difficult for the immune system to recognize the many different strains of Influenzavirus. Although the body may develop immunity to one strain through natural exposure or vaccination, antigenic variation results in the continual emergence of new strains that the immune system will not recognize. This is the main reason that vaccines against Influenzavirus must be given annually. Each year’s influenza vaccine provides protection against the most prevalent strains for that year, but new or different strains may be more prevalent the following year.
Link to Learning
For another explanation of how antigenic shift and drift occur, watch this video.
Exercise \(4\)
1. Describe the role of adhesins in viral tropism.
2. Explain the difference between antigenic drift and antigenic shift.
Key Concepts and Summary
Virulence factors contribute to a pathogen’s ability to cause disease. Exoenzymes and toxins allow pathogens to invade host tissue and cause tissue damage. Exoenzymes are classified according to the macromolecule they target and exotoxins are classified based on their mechanism of action. Bacterial toxins include endotoxin and exotoxins. Endotoxin is the lipid A component of the LPS of the gram-negative cell envelope. Exotoxins are proteins secreted mainly by gram-positive bacteria, but also are secreted by gram-negative bacteria. Bacterial pathogens may evade the host immune response by producing capsules to avoid phagocytosis, surviving the intracellular environment of phagocytes, degrading antibodies, or through antigenic variation. Viral pathogens use adhesins for initiating infections and antigenic variation to avoid immune defenses. Influenza viruses use both antigenic drift and antigenic shift to avoid being recognized by the immune system.
Footnotes
1. 1 V. Meka. “Panton-Valentine Leukocidin.” http://www.antimicrobe.org/h04c.file...L-S-aureus.asp | textbooks/bio/Microbiology/Microbiology_(OpenStax)/15%3A_Microbial_Mechanisms_of_Pathogenicity/15.03%3A_Virulence_Factors.txt |
Learning Objectives
• Describe virulence factors unique to fungi and parasites
• Compare virulence factors of fungi and bacteria
• Explain the difference between protozoan parasites and helminths
• Describe how helminths evade the host immune system
Although fungi and parasites are important pathogens causing infectious diseases, their pathogenic mechanisms and virulence factors are not as well characterized as those of bacteria. Despite the relative lack of detailed mechanisms, the stages of pathogenesis and general mechanisms of virulence involved in disease production by these pathogens are similar to those of bacteria.
Fungal Virulence
Pathogenic fungi can produce virulence factors that are similar to the bacterial virulence factors that have been discussed earlier in this chapter. In this section, we will look at the virulence factors associated with species of Candida, Cryptococcus, Claviceps, and Aspergillus.
Candida albicans is an opportunistic fungal pathogen and causative agent of oral thrush, vaginal yeast infections, and cutaneous candidiasis. Candida produces adhesins (surface glycoproteins) that bind to the phospholipids of epithelial and endothelial cells. To assist in spread and tissue invasion, Candida produces proteases and phospholipases (i.e., exoenzymes). One of these proteases degrades keratin, a structural protein found on epithelial cells, enhancing the ability of the fungus to invade host tissue. In animal studies, it has been shown that the addition of a protease inhibitor led to attenuation of Candida infection.1 Similarly, the phospholipases can affect the integrity of host cell membranes to facilitate invasion.
The main virulence factor for Cryptococcus, a fungus that causes pneumonia and meningitis, is capsule production. The polysaccharide glucuronoxylomannan is the principal constituent of the Cryptococcus capsule. Similar to encapsulated bacterial cells, encapsulated Cryptococcus cells are more resistant to phagocytosis than nonencapsulated Cryptococcus, which are effectively phagocytosed and, therefore, less virulent.
Like some bacteria, many fungi produce exotoxins. Fungal toxins are called mycotoxins. Claviceps purpurea, a fungus that grows on rye and related grains, produces a mycotoxin called ergot toxin, an alkaloid responsible for the disease known as ergotism. There are two forms of ergotism: gangrenous and convulsive. In gangrenous ergotism, the ergot toxin causes vasoconstriction, resulting in improper blood flow to the extremities, eventually leading to gangrene. A famous outbreak of gangrenous ergotism occurred in Eastern Europe during the 5th century AD due to the consumption of rye contaminated with C. purpurea. In convulsive ergotism, the toxin targets the central nervous system, causing mania and hallucinations.
The mycotoxin aflatoxin is a virulence factor produced by the fungus Aspergillus, an opportunistic pathogen that can enter the body via contaminated food or by inhalation. Inhalation of the fungus can lead to the chronic pulmonary disease aspergillosis, characterized by fever, bloody sputum, and/or asthma. Aflatoxin acts in the host as both a mutagen (a substance that causes mutations in DNA) and a carcinogen (a substance involved in causing cancer), and has been associated with the development of liver cancer. Aflatoxin has also been shown to cross the blood-placental barrier.2 A second mycotoxin produced by Aspergillus is gliotoxin. This toxin promotes virulence by inducing host cells to self-destruct and by evading the host’s immune response by inhibiting the function of phagocytic cells as well as the pro-inflammatory response. Like Candida, Aspergillus also produces several proteases. One is elastase, which breaks down the protein elastin found in the connective tissue of the lung, leading to the development of lung disease. Another is catalase, an enzyme that protects the fungus from hydrogen peroxide produced by the immune system to destroy pathogens.
Exercise \(1\)
1. List virulence factors common to bacteria and fungi.
2. What functions do mycotoxins perform to help fungi survive in the host?
Protozoan Virulence
Protozoan pathogens are unicellular eukaryotic parasites that have virulence factors and pathogenic mechanisms analogous to prokaryotic and viral pathogens, including adhesins, toxins, antigenic variation, and the ability to survive inside phagocytic vesicles.
Protozoans often have unique features for attaching to host cells. The protozoan Giardia lamblia, which causes the intestinal disease giardiasis, uses a large adhesive disc composed of microtubules to attach to the intestinal mucosa. During adhesion, the flagella of G. lamblia move in a manner that draws fluid out from under the disc, resulting in an area of lower pressure that facilitates adhesion to epithelial cells. Giardia does not invade the intestinal cells but rather causes inflammation (possibly through the release of cytopathic substances that cause damage to the cells) and shortens the intestinal villi, inhibiting absorption of nutrients.
Some protozoans are capable of antigenic variation. The obligate intracellular pathogen Plasmodium falciparum (one of the causative agents of malaria) resides inside red blood cells, where it produces an adhesin membrane protein known as PfEMP1. This protein is expressed on the surface of the infected erythrocytes, causing blood cells to stick to each other and to the walls of blood vessels. This process impedes blood flow, sometimes leading to organ failure, anemia, jaundice (yellowing of skin and sclera of the eyes due to buildup of bilirubin from lysed red blood cells), and, subsequently, death. Although PfEMP1 can be recognized by the host’s immune system, antigenic variations in the structure of the protein over time prevent it from being easily recognized and eliminated. This allows malaria to persist as a chronic infection in many individuals.
The virulence factors of Trypanosoma brucei, the causative agent of African sleeping sickness, include the abilities to form capsules and undergo antigenic variation. T. brucei evades phagocytosis by producing a dense glycoprotein coat that resembles a bacterial capsule. Over time, host antibodies are produced that recognize this coat, but T. brucei is able to alter the structure of the glycoprotein to evade recognition.
Exercise \(2\)
Explain how antigenic variation by protozoan pathogens helps them survive in the host.
Helminth Virulence
Helminths, or parasitic worms, are multicellular eukaryotic parasites that depend heavily on virulence factors that allow them to gain entry to host tissues. For example, the aquatic larval form of Schistosoma mansoni, which causes schistosomiasis, penetrates intact skin with the aid of proteases that degrade skin proteins, including elastin.
To survive within the host long enough to perpetuate their often-complex life cycles, helminths need to evade the immune system. Some helminths are so large that the immune system is ineffective against them. Others, such as adult roundworms (which cause trichinosis, ascariasis, and other diseases), are protected by a tough outer cuticle.
Over the course of their life cycles, the surface characteristics of the parasites vary, which may help prevent an effective immune response. Some helminths express polysaccharides called glycans on their external surface; because these glycans resemble molecules produced by host cells, the immune system fails to recognize and attack the helminth as a foreign body. This “glycan gimmickry,” as it has been called, serves as a protective cloak that allows the helminth to escape detection by the immune system.3
In addition to evading host defenses, helminths can actively suppress the immune system. S. mansoni, for example, degrades host antibodies with proteases. Helminths produce many other substances that suppress elements of both innate nonspecific and adaptive specific host defenses. They also release large amounts of material into the host that may locally overwhelm the immune system or cause it to respond inappropriately.
Exercise \(3\)
Describe how helminths avoid being destroyed by the host immune system.
Key Concepts and Summary
• Fungal and parasitic pathogens use pathogenic mechanisms and virulence factors that are similar to those of bacterial pathogens.
• Fungi initiate infections through the interaction of adhesins with receptors on host cells. Some fungi produce toxins and exoenzymes involved in disease production and capsules that provide protection of phagocytosis.
• Protozoa adhere to target cells through complex mechanisms and can cause cellular damage through release of cytopathic substances. Some protozoa avoid the immune system through antigenic variation and production of capsules.
• Helminthic worms are able to avoid the immune system by coating their exteriors with glycan molecules that make them look like host cells or by suppressing the immune system.
Footnotes
1. 1 K. Fallon et al. “Role of Aspartic Proteases in Disseminated Candida albicans Infection in Mice.” Infection and Immunity 65 no. 2 (1997):551–556.
2. 2 C.P. Wild et al. “In-utero exposure to aflatoxin in west Africa.” Lancet 337 no. 8757 (1991):1602.
3. 3 I. van Die, R.D. Cummings. “Glycan Gimmickry by Parasitic Helminths: A Strategy for Modulating the Host Immune Response?” Glycobiology 20 no. 1 (2010):2–12. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/15%3A_Microbial_Mechanisms_of_Pathogenicity/15.04%3A_Aseptic_Techniques.txt |
15.1: Characteristics of Infectious Diseases
In an infection, a microorganism enters a host and begins to multiply. Some infections cause disease, which is any deviation from the normal function or structure of the host. Signs of a disease are objective and are measured. Symptoms of a disease are subjective and are reported by the patient. Diseases can either be noninfectious (due to genetics and environment) or infectious (due to pathogens).
Multiple Choice
Which of the following would be a sign of an infection?
1. muscle aches
2. headache
3. fever
4. nausea
Answer
C
Which of the following is an example of a noncommunicable infectious disease?
1. infection with a respiratory virus
2. food poisoning due to a preformed bacterial toxin in food
3. skin infection acquired from a dog bite
4. infection acquired from the stick of a contaminated needle
Answer
B
During an oral surgery, the surgeon nicked the patient’s gum with a sharp instrument. This allowed Streptococcus, a bacterium normally present in the mouth, to gain access to the blood. As a result, the patient developed bacterial endocarditis (an infection of the heart). Which type of disease is this?
1. iatrogenic
2. nosocomial
3. vectors
4. zoonotic
Answer
A
Which period is the stage of disease during which the patient begins to present general signs and symptoms?
1. convalescence
2. incubation
3. illness
4. prodromal
Answer
D
A communicable disease that can be easily transmitted from person to person is which type of disease?
1. contagious
2. iatrogenic
3. acute
4. nosocomial
Answer
A
Fill in the Blank
A difference between an acute disease and chronic disease is that chronic diseases have an extended period of __________.
Answer
illness
A person steps on a rusty nail and develops tetanus. In this case, the person has acquired a(n) __________ disease.
Answer
noncommunicable
Short Answer
Brian goes to the hospital after not feeling well for a week. He has a fever of 38 °C (100.4 °F) and complains of nausea and a constant migraine. Distinguish between the signs and symptoms of disease in Brian’s case.
Critical Thinking
Two periods of acute disease are the periods of illness and period of decline. (a) In what way are both of these periods similar? (b) In terms of quantity of pathogen, in what way are these periods different? (c) What initiates the period of decline?
In July 2015, a report1 was released indicating the gram-negative bacterium Pseudomonas aeruginosa was found on hospital sinks 10 years after the initial outbreak in a neonatal intensive care unit. P. aeruginosa usually causes localized ear and eye infections but can cause pneumonia or septicemia in vulnerable individuals like newborn babies. Explain how the current discovery of the presence of this reported P. aeruginosa could lead to a recurrence of nosocomial disease.
15.2: How Pathogens Cause Disease
Koch’s postulates are used to determine whether a particular microorganism is a pathogen. Molecular Koch’s postulates are used to determine what genes contribute to a pathogen’s ability to cause disease. Virulence, the degree to which a pathogen can cause disease, can be quantified by calculating either the ID50 or LD50 of a pathogen on a given population. Primary pathogens are capable of causing pathological changes associated with disease in a healthy individual.
Multiple Choice
Which of the following is a pathogen that could not be identified by the original Koch’s postulates?
1. Staphylococcus aureus
2. Pseudomonas aeruginosa
3. Human immunodeficiency virus
4. Salmonella enterica serovar Typhimurium
Answer
C
Pathogen A has an ID50 of 50 particles, pathogen B has an ID50 of 1,000 particles, and pathogen C has an ID50of 1 × 106 particles. Which pathogen is most virulent?
1. pathogen A
2. pathogen B
3. pathogen C
Answer
A
Which of the following choices lists the steps of pathogenesis in the correct order?
1. invasion, infection, adhesion, exposure
2. adhesion, exposure, infection, invasion
3. exposure, adhesion, invasion, infection
4. disease, infection, exposure, invasion
Answer
C
Fill in the Blank
A(n) __________ pathogen causes disease only when conditions are favorable for the microorganism because of transfer to an inappropriate body site or weakened immunity in an individual.
Answer
opportunistic
The concentration of pathogen needed to kill 50% of an infected group of test animals is the __________.
Answer
LD50
A(n) __________ infection is a small region of infection from which a pathogen may move to another part of the body to establish a second infection.
Answer
focal
Cilia, fimbriae, and pili are all examples of structures used by microbes for __________.
Answer
adhesion
Critical Thinking
Diseases that involve biofilm-producing bacteria are of serious concern. They are not as easily treated compared with those involving free-floating (or planktonic) bacteria. Explain three reasons why biofilm formers are more pathogenic.
A microbiologist has identified a new gram-negative pathogen that causes liver disease in rats. She suspects that the bacterium’s fimbriae are a virulence factor. Describe how molecular Koch’s postulates could be used to test this hypothesis.
Acupuncture is a form of alternative medicine that is used for pain relief. Explain how acupuncture could facilitate exposure to pathogens.
15.3: Virulence Factors
Virulence factors contribute to a pathogen’s ability to cause disease. Exoenzymes and toxins allow pathogens to invade host tissue and cause tissue damage. Exoenzymes are classified according to the macromolecule they target and exotoxins are classified based on their mechanism of action. Bacterial toxins include endotoxin and exotoxins. Endotoxin is the lipid A component of the LPS of the gram-negative cell envelope. Exotoxins are proteins secreted mainly by gram-positive bacteria.
Multiple Choice
Which of the following would be a virulence factor of a pathogen?
1. a surface protein allowing the pathogen to bind to host cells
2. a secondary host the pathogen can infect
3. a surface protein the host immune system recognizes
4. the ability to form a provirus
Answer
A
You have recently identified a new toxin. It is produced by a gram-negative bacterium. It is composed mostly of protein, has high toxicity, and is not heat stable. You also discover that it targets liver cells. Based on these characteristics, how would you classify this toxin?
1. superantigen
2. endotoxin
3. exotoxin
4. leukocidin
Answer
C
Which of the following applies to hyaluronidase?
1. It acts as a spreading factor.
2. It promotes blood clotting.
3. It is an example of an adhesin.
4. It is produced by immune cells to target pathogens.
Answer
A
Phospholipases are enzymes that do which of the following?
1. degrade antibodies
2. promote pathogen spread through connective tissue.
3. degrade nucleic acid to promote spread of pathogen
4. degrade cell membranes to allow pathogens to escape phagosomes
Answer
D
Fill in the Blank
The glycoprotein adhesion gp120 on HIV must interact with __________ on some immune cells as the first step in the process of infecting the cell.
Answer
CD4
Adhesins are usually located on __________ of the pathogen and are composed mainly of __________ and __________.
Answer
surface; proteins; sugars
The Shiga and diphtheria toxins target __________ in host cells.
Answer
protein synthesis
Antigenic __________ is the result of reassortment of genes responsible for the production of influenza virus spike proteins between different virus particles while in the same host, whereas antigenic __________ is the result of point mutations in the spike proteins.
Answer
shift; drift
Critical Thinking
Two types of toxins are hemolysins and leukocidins. (a) How are these toxins similar? (b) How do they differ?
Imagine that a mutation in the gene encoding the cholera toxin was made. This mutation affects the A-subunit, preventing it from interacting with any host protein. (a) Would the toxin be able to enter into the intestinal epithelial cell? (b) Would the toxin be able to cause diarrhea?
15.4: Aseptic Techniques
Fungal and parasitic pathogens use pathogenic mechanisms and virulence factors that are similar to those of bacterial pathogens. Fungi initiate infections through the interaction of adhesins with receptors on host cells. Some fungi produce toxins and exoenzymes involved in disease production and capsules that provide protection of phagocytosis. Protozoa adhere to target cells through complex mechanisms and can cause cellular damage through release of cytopathic substances.
Multiple Choice
Which of the following is a major virulence factor for the fungal pathogen Cryptococcus?
1. hemolysin
2. capsule
3. collagenase
4. fimbriae
Answer
B
Which of the following pathogens undergoes antigenic variation to avoid immune defenses?
1. Candida
2. Cryptococcus
3. Plasmodium
4. Giardia
Answer
C
Fill in the Blank
Candida can invade tissue by producing the exoenzymes __________ and __________.
Answer
protease and phospholipase
The larval form of Schistosoma mansoni uses a __________ to help it gain entry through intact skin.
Answer
protease
Short Answer
Describe the virulence factors associated with the fungal pathogen Aspergillus.
Explain how helminths evade the immune system.
Footnotes
1. 1 C. Owens. “P. aeruginosa survives in sinks 10 years after hospital outbreak.” 2015. http://www.healio.com/infectious-disease/nosocomial-infections/news/online/%7B5afba909-56d9-48cc-a9b0-ffe4568161e8%7D/p-aeruginosa-survives-in-sinks-10-years-after-hospital-outbreak | textbooks/bio/Microbiology/Microbiology_(OpenStax)/15%3A_Microbial_Mechanisms_of_Pathogenicity/15.E%3A_Microbial_Mechanisms_of_Pathogenicity_%28Exercises%29.txt |
In the United States and other developed nations, public health is a key function of government. A healthy citizenry is more productive, content, and prosperous; high rates of death and disease, on the other hand, can severely hamper economic productivity and foster social and political instability. The burden of disease makes it difficult for citizens to work consistently, maintain employment, and accumulate wealth to better their lives and support a growing economy.
In this chapter, we will explore the intersections between microbiology and epidemiology, the science that underlies public health. Epidemiology studies how disease originates and spreads throughout a population, with the goal of preventing outbreaks and containing them when they do occur. Over the past two centuries, discoveries in epidemiology have led to public health policies that have transformed life in developed nations, leading to the eradication (or near eradication) of many diseases that were once causes of great human suffering and premature death. However, the work of epidemiologists is far from finished. Numerous diseases continue to plague humanity, and new diseases are always emerging. Moreover, in the developing world, lack of infrastructure continues to pose many challenges to efforts to contain disease.
• 16.1: The Language of Epidemiologists
The field of epidemiology concerns the geographical distribution and timing of infectious disease occurrences and how they are transmitted and maintained in nature, with the goal of recognizing and controlling outbreaks. The science of epidemiology includes etiology (the study of the causes of disease) and investigation of disease transmission (mechanisms by which a disease is spread).
• 16.2: Tracking Infectious Diseases
Some important researchers, such as Florence Nightingale, subscribed to the miasma hypothesis. The transition to acceptance of the germ theory during the 19th century provided a solid mechanistic grounding to the study of disease patterns. The studies of 19th century physicians and researchers such as John Snow, Florence Nightingale, Ignaz Semmelweis, Joseph Lister, Robert Koch, Louis Pasteur, and others sowed the seeds of modern epidemiology.
• 16.3: How Diseases Spread
Pathogens often have elaborate adaptations to exploit host biology, behavior, and ecology to live in and move between hosts. Hosts have evolved defenses against pathogens, but because their rates of evolution are typically slower than their pathogens (because their generation times are longer), hosts are usually at an evolutionary disadvantage. This section will explore where pathogens survive—both inside and outside hosts—and some of the many ways they move from one host to another.
• 16.4: Global Public Health
A large number of international programs and agencies are involved in efforts to promote global public health. Among their goals are developing infrastructure in health care, public sanitation, and public health capacity; monitoring infectious disease occurrences around the world; coordinating communications between national public health agencies in various countries; and coordinating international responses to major health crises.
• 16.E: Disease and Epidemiology (Exercises)
Thumbnail: The biohazard symbol was developed by the Dow Chemical Company in 1966 for their containment products. It is used in the labeling of biological materials that carry a significant health risk. (Public Domain; Silsor).
16: Disease and Epidemiology
Learning Objectives
• Explain the difference between prevalence and incidence of disease
• Distinguish the characteristics of sporadic, endemic, epidemic, and pandemic diseases
• Explain the use of Koch’s postulates and their modifications to determine the etiology of disease
• Explain the relationship between epidemiology and public health
Clinical Focus: Part 1
In late November and early December, a hospital in western Florida started to see a spike in the number of cases of acute gastroenteritis-like symptoms. Patients began arriving at the emergency department complaining of excessive bouts of emesis (vomiting) and diarrhea (with no blood in the stool). They also complained of abdominal pain and cramping, and most were severely dehydrated. Alarmed by the number of cases, hospital staff made some calls and learned that other regional hospitals were also seeing 10 to 20 similar cases per day.
Exercise \(1\)
1. What are some possible causes of this outbreak?
2. In what ways could these cases be linked, and how could any suspected links be confirmed?
The field of epidemiology concerns the geographical distribution and timing of infectious disease occurrences and how they are transmitted and maintained in nature, with the goal of recognizing and controlling outbreaks. The science of epidemiology includes etiology (the study of the causes of disease) and investigation of disease transmission (mechanisms by which a disease is spread).
Analyzing Disease in a Population
Epidemiological analyses are always carried out with reference to a population, which is the group of individuals that are at risk for the disease or condition. The population can be defined geographically, but if only a portion of the individuals in that area are susceptible, additional criteria may be required. Susceptible individuals may be defined by particular behaviors, such as intravenous drug use, owning particular pets, or membership in an institution, such as a college. Being able to define the population is important because most measures of interest in epidemiology are made with reference to the size of the population.
The state of being diseased is called morbidity. Morbidity in a population can be expressed in a few different ways. Morbidity or total morbidity is expressed in numbers of individuals without reference to the size of the population. The morbidity rate can be expressed as the number of diseased individuals out of a standard number of individuals in the population, such as 100,000, or as a percent of the population.
There are two aspects of morbidity that are relevant to an epidemiologist: a disease’s prevalence and its incidence. Prevalence is the number, or proportion, of individuals with a particular illness in a given population at a point in time. For example, the Centers for Disease Control and Prevention (CDC) estimated that in 2012, there were about 1.2 million people 13 years and older with an active human immunodeficiency virus (HIV) infection. Expressed as a proportion, or rate, this is a prevalence of 467 infected persons per 100,000 in the population.1 On the other hand, incidence is the number or proportion of new cases in a period of time. For the same year and population, the CDC estimates that there were 43,165 newly diagnosed cases of HIV infection, which is an incidence of 13.7 new cases per 100,000 in the population.2 The relationship between incidence and prevalence can be seen in Figure \(1\). For a chronic disease like HIV infection, prevalence will generally be higher than incidence because it represents the cumulative number of new cases over many years minus the number of cases that are no longer active (e.g., because the patient died or was cured).
In addition to morbidity rates, the incidence and prevalence of mortality (death) may also be reported. A mortality ratecan be expressed as the percentage of the population that has died from a disease or as the number of deaths per 100,000 persons (or other suitable standard number).
Exercise \(2\)
1. Explain the difference between incidence and prevalence.
2. Describe how morbidity and mortality rates are expressed.
Patterns of Incidence
Diseases that are seen only occasionally, and usually without geographic concentration, are called sporadic diseases. Examples of sporadic diseases include tetanus, rabies, and plague. In the United States, Clostridium tetani, the bacterium that causes tetanus, is ubiquitous in the soil environment, but incidences of infection occur only rarely and in scattered locations because most individuals are vaccinated, clean wounds appropriately, or are only rarely in a situation that would cause infection.3 Likewise in the United States there are a few scattered cases of plague each year, usually contracted from rodents in rural areas in the western states.4
Diseases that are constantly present (often at a low level) in a population within a particular geographic region are called endemic diseases. For example, malaria is endemic to some regions of Brazil, but is not endemic to the United States.
Diseases for which a larger than expected number of cases occurs in a short time within a geographic region are called epidemic diseases. Influenza is a good example of a commonly epidemic disease. Incidence patterns of influenza tend to rise each winter in the northern hemisphere. These seasonal increases are expected, so it would not be accurate to say that influenza is epidemic every winter; however, some winters have an usually large number of seasonal influenza cases in particular regions, and such situations would qualify as epidemics (Figure \(2\) and Figure \(3\)).
An epidemic disease signals the breakdown of an equilibrium in disease frequency, often resulting from some change in environmental conditions or in the population. In the case of influenza, the disruption can be due to antigenic shift or drift (see Virulence Factors of Bacterial and Viral Pathogens), which allows influenza virus strains to circumvent the acquired immunity of their human hosts.
An epidemic that occurs on a worldwide scale is called a pandemic disease. For example, HIV/AIDS is a pandemic disease and novel influenza virus strains often become pandemic.
Exercise \(3\)
1. Explain the difference between sporadic and endemic disease.
2. Explain the difference between endemic and epidemic disease.
Clinical Focus: Part 2
Hospital physicians suspected that some type of food poisoning was to blame for the sudden post-Thanksgiving outbreak of gastroenteritis in western Florida. Over a two-week period, 254 cases were observed, but by the end of the first week of December, the epidemic ceased just as quickly as it had started. Suspecting a link between the cases based on the localized nature of the outbreak, hospitals handed over their medical records to the regional public health office for study.
Laboratory testing of stool samples had indicated that the infections were caused by Salmonella bacteria. Patients ranged from children as young as three to seniors in their late eighties. Cases were nearly evenly split between males and females. Across the region, there had been three confirmed deaths in the outbreak, all due to severe dehydration. In each of the fatal cases, the patients had not sought medical care until their symptoms were severe; also, all of the deceased had preexisting medical conditions such as congestive heart failure, diabetes, or high blood pressure.
After reviewing the medical records, epidemiologists with the public health office decided to conduct interviews with a randomly selected sample of patients.
Exercise \(4\)
1. What conclusions, if any, can be drawn from the medical records?
2. What would epidemiologists hope to learn by interviewing patients? What kinds of questions might they ask?
Etiology
When studying an epidemic, an epidemiologist’s first task is to determinate the cause of the disease, called the etiologic agent or causative agent. Connecting a disease to a specific pathogen can be challenging because of the extra effort typically required to demonstrate direct causation as opposed to a simple association. It is not enough to observe an association between a disease and a suspected pathogen; controlled experiments are needed to eliminate other possible causes. In addition, pathogens are typically difficult to detect when there is no immediate clue as to what is causing the outbreak. Signs and symptoms of disease are also commonly nonspecific, meaning that many different agents can give rise to the same set of signs and symptoms. This complicates diagnosis even when a causative agent is familiar to scientists.
Robert Koch was the first scientist to specifically demonstrate the causative agent of a disease (anthrax) in the late 1800s. Koch developed four criteria, now known as Koch’s postulates, which had to be met in order to positively link a disease with a pathogenic microbe. Without Koch’s postulates, the Golden Age of Microbiology would not have occurred. Between 1876 and 1905, many common diseases were linked with their etiologic agents, including cholera, diphtheria, gonorrhea, meningitis, plague, syphilis, tetanus, and tuberculosis. Today, we use the molecular Koch’s postulates, a variation of Koch’s original postulates that can be used to establish a link between the disease state and virulence traits unique to a pathogenic strain of a microbe. Koch’s original postulates and molecular Koch’s postulates were described in more detail in How Pathogens Cause Disease.
Exercise \(5\)
List some challenges to determining the causative agent of a disease outbreak.
The Role of Public Health Organizations
The main national public health agency in the United States is the Centers for Disease Control and Prevention (CDC), an agency of the Department of Health and Human Services. The CDC is charged with protecting the public from disease and injury. One way that the CDC carries out this mission is by overseeing the National Notifiable Disease Surveillance System (NNDSS) in cooperation with regional, state, and territorial public health departments. The NNDSS monitors diseases considered to be of public health importance on a national scale. Such diseases are called notifiable diseases or reportable diseases because all cases must be reported to the CDC. A physician treating a patient with a notifiable disease is legally required to submit a report on the case. Notifiable diseases include HIV infection, measles, West Nile virus infections, and many others. Some states have their own lists of notifiable diseases that include diseases beyond those on the CDC’s list.
Notifiable diseases are tracked by epidemiological studies and the data is used to inform health-care providers and the public about possible risks. The CDC publishes the Morbidity and Mortality Weekly Report (MMWR), which provides physicians and health-care workers with updates on public health issues and the latest data pertaining to notifiable diseases. Table \(1\) is an example of the kind of data contained in the MMWR.
Table \(1\): Incidence of Four Notifiable Diseases in the United States, Week Ending January 2, 2016
Disease Current Week (Jan 2, 2016) Median of Previous 52 Weeks Maximum of Previous 52 Weeks Cumulative Cases 2015
Campylobacteriosis 406 869 1,385 46,618
Chlamydia trachomatis infection 11,024 28,562 31,089 1,425,303
Giardiasis 115 230 335 11,870
Gonorrhea 3,207 7,155 8,283 369,926
Link to Learning
The current Morbidity and Mortality Weekly Report is available online.
Exercise \(6\)
Describe how health agencies obtain data about the incidence of diseases of public health importance.
Key Concepts and Summary
• Epidemiology is the science underlying public health.
• Morbidity means being in a state of illness, whereas mortality refers to death; both morbidity rates and mortality rates are of interest to epidemiologists.
• Incidence is the number of new cases (morbidity or mortality), usually expressed as a proportion, during a specified time period; prevalence is the total number affected in the population, again usually expressed as a proportion.
• Sporadic diseases only occur rarely and largely without a geographic focus. Endemic diseases occur at a constant (and often low) level within a population. Epidemic diseases and pandemic diseases occur when an outbreak occurs on a significantly larger than expected level, either locally or globally, respectively.
• Koch’s postulates specify the procedure for confirming a particular pathogen as the etiologic agent of a particular disease. Koch’s postulates have limitations in application if the microbe cannot be isolated and cultured or if there is no animal host for the microbe. In this case, molecular Koch’s postulates would be utilized.
• In the United States, the Centers for Disease Control and Prevention monitors notifiable diseases and publishes weekly updates in the Morbidity and Mortality Weekly Report.
Footnotes
1. 1 H. Irene Hall, Qian An, Tian Tang, Ruiguang Song, Mi Chen, Timothy Green, and Jian Kang. “Prevalence of Diagnosed and Undiagnosed HIV Infection—United States, 2008–2012.” Morbidity and Mortality Weekly Report 64, no. 24 (2015): 657–662.
2. 2 Centers for Disease Control and Prevention. “Diagnoses of HIV Infection in the United States and Dependent Areas, 2014.” HIV Surveillance Report 26 (2015).
3. 3 Centers for Disease Control and Prevention. “Tetanus Surveillance—United States, 2001–2008.” Morbidity and Mortality Weekly Report 60, no. 12 (2011): 365.
4. 4 Centers for Disease Control and Prevention. “Plague in the United States.” 2015. http://www.cdc.gov/plague/maps. Accessed June 1, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/16%3A_Disease_and_Epidemiology/16.01%3A_The_Language_of_Epidemiologists.txt |
Learning Objectives
• Explain the research approaches used by the pioneers of epidemiology
• Explain how descriptive, analytical, and experimental epidemiological studies go about determining the cause of morbidity and mortality
Epidemiology has its roots in the work of physicians who looked for patterns in disease occurrence as a way to understand how to prevent it. The idea that disease could be transmitted was an important precursor to making sense of some of the patterns. In 1546, Girolamo Fracastoro first proposed the germ theory of disease in his essay De Contagione et Contagiosis Morbis, but this theory remained in competition with other theories, such as the miasma hypothesis, for many years (see What Our Ancestors Knew). Uncertainty about the cause of disease was not an absolute barrier to obtaining useful knowledge from patterns of disease. Some important researchers, such as Florence Nightingale, subscribed to the miasma hypothesis. The transition to acceptance of the germ theory during the 19th century provided a solid mechanistic grounding to the study of disease patterns. The studies of 19th century physicians and researchers such as John Snow, Florence Nightingale, Ignaz Semmelweis, Joseph Lister, Robert Koch, Louis Pasteur, and others sowed the seeds of modern epidemiology.
Pioneers of Epidemiology
John Snow (Figure \(1\)) was a British physician known as the father of epidemiology for determining the source of the 1854 Broad Street cholera epidemic in London. Based on observations he had made during an earlier cholera outbreak (1848–1849), Snow proposed that cholera was spread through a fecal-oral route of transmission and that a microbe was the infectious agent. He investigated the 1854 cholera epidemic in two ways. First, suspecting that contaminated water was the source of the epidemic, Snow identified the source of water for those infected. He found a high frequency of cholera cases among individuals who obtained their water from the River Thames downstream from London. This water contained the refuse and sewage from London and settlements upstream. He also noted that brewery workers did not contract cholera and on investigation found the owners provided the workers with beer to drink and stated that they likely did not drink water.1 Second, he also painstakingly mapped the incidence of cholera and found a high frequency among those individuals using a particular water pump located on Broad Street. In response to Snow’s advice, local officials removed the pump’s handle,2 resulting in the containment of the Broad Street cholera epidemic.
Snow’s work represents an early epidemiological study and it resulted in the first known public health response to an epidemic. Snow’s meticulous case-tracking methods are now common practice in studying disease outbreaks and in associating new diseases with their causes. His work further shed light on unsanitary sewage practices and the effects of waste dumping in the Thames. Additionally, his work supported the germ theory of disease, which argued disease could be transmitted through contaminated items, including water contaminated with fecal matter.
Snow’s work illustrated what is referred to today as a common source spread of infectious disease, in which there is a single source for all of the individuals infected. In this case, the single source was the contaminated well below the Broad Street pump. Types of common source spread include point source spread, continuous common source spread, and intermittent common source spread. In point source spread of infectious disease, the common source operates for a short time period—less than the incubation period of the pathogen. An example of point source spread is a single contaminated potato salad at a group picnic. In continuous common source spread, the infection occurs for an extended period of time, longer than the incubation period. An example of continuous common source spread would be the source of London water taken downstream of the city, which was continuously contaminated with sewage from upstream. Finally, with intermittent common source spread, infections occur for a period, stop, and then begin again. This might be seen in infections from a well that was contaminated only after large rainfalls and that cleared itself of contamination after a short period.
In contrast to common source spread, propagated spread occurs through direct or indirect person-to-person contact. With propagated spread, there is no single source for infection; each infected individual becomes a source for one or more subsequent infections. With propagated spread, unless the spread is stopped immediately, infections occur for longer than the incubation period. Although point sources often lead to large-scale but localized outbreaks of short duration, propagated spread typically results in longer duration outbreaks that can vary from small to large, depending on the population and the disease (Figure \(2\)). In addition, because of person-to-person transmission, propagated spread cannot be easily stopped at a single source like point source spread.
Florence Nightingale’s work is another example of an early epidemiological study. In 1854, Nightingale was part of a contingent of nurses dispatched by the British military to care for wounded soldiers during the Crimean War. Nightingale kept meticulous records regarding the causes of illness and death during the war. Her recordkeeping was a fundamental task of what would later become the science of epidemiology. Her analysis of the data she collected was published in 1858. In this book, she presented monthly frequency data on causes of death in a wedge chart histogram (Figure \(3\)). This graphical presentation of data, unusual at the time, powerfully illustrated that the vast majority of casualties during the war occurred not due to wounds sustained in action but to what Nightingale deemed preventable infectious diseases. Often these diseases occurred because of poor sanitation and lack of access to hospital facilities. Nightingale’s findings led to many reforms in the British military’s system of medical care.
Joseph Lister provided early epidemiological evidence leading to good public health practices in clinics and hospitals. These settings were notorious in the mid-1800s for fatal infections of surgical wounds at a time when the germ theory of disease was not yet widely accepted (see Foundations of Modern Cell Theory). Most physicians did not wash their hands between patient visits or clean and sterilize their surgical tools. Lister, however, discovered the disinfecting properties of carbolic acid, also known as phenol (see Using Chemicals to Control Microorganisms). He introduced several disinfection protocols that dramatically lowered post-surgical infection rates.3 He demanded that surgeons who worked for him use a 5% carbolic acid solution to clean their surgical tools between patients, and even went so far as to spray the solution onto bandages and over the surgical site during operations (Figure \(4\)). He also took precautions not to introduce sources of infection from his skin or clothing by removing his coat, rolling up his sleeves, and washing his hands in a dilute solution of carbolic acid before and during the surgery.
Link to Learning
John Snow’s own account of his work has additional links and information.
This CDC resource further breaks down the pattern expected from a point-source outbreak.
Learn more about Nightingale’s wedge chart here.
Exercise \(1\)
1. Explain the difference between common source spread and propagated spread of disease.
2. Describe how the observations of John Snow, Florence Nightingale, and Joseph Lister led to improvements in public health.
Types of Epidemiological Studies
Today, epidemiologists make use of study designs, the manner in which data are gathered to test a hypothesis, similar to those of researchers studying other phenomena that occur in populations. These approaches can be divided into observational studies (in which subjects are not manipulated) and experimental studies (in which subjects are manipulated). Collectively, these studies give modern-day epidemiologists multiple tools for exploring the connections between infectious diseases and the populations of susceptible individuals they might infect.
Observational Studies
In an observational study, data are gathered from study participants through measurements (such as physiological variables like white blood cell count), or answers to questions in interviews (such as recent travel or exercise frequency). The subjects in an observational study are typically chosen at random from a population of affected or unaffected individuals. However, the subjects in an observational study are in no way manipulated by the researcher. Observational studies are typically easier to carry out than experimental studies, and in certain situations they may be the only studies possible for ethical reasons.
Observational studies are only able to measure associations between disease occurrence and possible causative agents; they do not necessarily prove a causal relationship. For example, suppose a study finds an association between heavy coffee drinking and lower incidence of skin cancer. This might suggest that coffee prevents skin cancer, but there may be another unmeasured factor involved, such as the amount of sun exposure the participants receive. If it turns out that coffee drinkers work more in offices and spend less time outside in the sun than those who drink less coffee, then it may be possible that the lower rate of skin cancer is due to less sun exposure, not to coffee consumption. The observational study cannot distinguish between these two potential causes.
There are several useful approaches in observational studies. These include methods classified as descriptive epidemiology and analytical epidemiology. Descriptive epidemiology gathers information about a disease outbreak, the affected individuals, and how the disease has spread over time in an exploratory stage of study. This type of study will involve interviews with patients, their contacts, and their family members; examination of samples and medical records; and even histories of food and beverages consumed. Such a study might be conducted while the outbreak is still occurring. Descriptive studies might form the basis for developing a hypothesis of causation that could be tested by more rigorous observational and experimental studies.
Analytical epidemiology employs carefully selected groups of individuals in an attempt to more convincingly evaluate hypotheses about potential causes for a disease outbreak. The selection of cases is generally made at random, so the results are not biased because of some common characteristic of the study participants. Analytical studies may gather their data by going back in time (retrospective studies), or as events unfold forward in time (prospective studies).
Retrospective studies gather data from the past on present-day cases. Data can include things like the medical history, age, gender, or occupational history of the affected individuals. This type of study examines associations between factors chosen or available to the researcher and disease occurrence.
Prospective studies follow individuals and monitor their disease state during the course of the study. Data on the characteristics of the study subjects and their environments are gathered at the beginning and during the study so that subjects who become ill may be compared with those who do not. Again, the researchers can look for associations between the disease state and variables that were measured during the study to shed light on possible causes.
Analytical studies incorporate groups into their designs to assist in teasing out associations with disease. Approaches to group-based analytical studies include cohort studies, case-control studies, and cross-sectional studies. The cohort method examines groups of individuals (called cohorts) who share a particular characteristic. For example, a cohort might consist of individuals born in the same year and the same place; or it might consist of people who practice or avoid a particular behavior, e.g., smokers or nonsmokers. In a cohort study, cohorts can be followed prospectively or studied retrospectively. If only a single cohort is followed, then the affected individuals are compared with the unaffected individuals in the same group. Disease outcomes are recorded and analyzed to try to identify correlations between characteristics of individuals in the cohort and disease incidence. Cohort studies are a useful way to determine the causes of a condition without violating the ethical prohibition of exposing subjects to a risk factor. Cohorts are typically identified and defined based on suspected risk factors to which individuals have already been exposed through their own choices or circumstances.
Case-control studies are typically retrospective and compare a group of individuals with a disease to a similar group of individuals without the disease. Case-control studies are far more efficient than cohort studies because researchers can deliberately select subjects who are already affected with the disease as opposed to waiting to see which subjects from a random sample will develop a disease.
A cross-sectional study analyzes randomly selected individuals in a population and compares individuals affected by a disease or condition to those unaffected at a single point in time. Subjects are compared to look for associations between certain measurable variables and the disease or condition. Cross-sectional studies are also used to determine the prevalence of a condition.
Experimental Studies
Experimental epidemiology uses laboratory or clinical studies in which the investigator manipulates the study subjects to study the connections between diseases and potential causative agents or to assess treatments. Examples of treatments might be the administration of a drug, the inclusion or exclusion of different dietary items, physical exercise, or a particular surgical procedure. Animals or humans are used as test subjects. Because experimental studies involve manipulation of subjects, they are typically more difficult and sometimes impossible for ethical reasons.
Koch’s postulates require experimental interventions to determine the causative agent for a disease. Unlike observational studies, experimental studies can provide strong evidence supporting cause because other factors are typically held constant when the researcher manipulates the subject. The outcomes for one group receiving the treatment are compared to outcomes for a group that does not receive the treatment but is treated the same in every other way. For example, one group might receive a regimen of a drug administered as a pill, while the untreated group receives a placebo (a pill that looks the same but has no active ingredient). Both groups are treated as similarly as possible except for the administration of the drug. Because other variables are held constant in both the treated and the untreated groups, the researcher is more certain that any change in the treated group is a result of the specific manipulation.
Experimental studies provide the strongest evidence for the etiology of disease, but they must also be designed carefully to eliminate subtle effects of bias. Typically, experimental studies with humans are conducted as double-blind studies, meaning neither the subjects nor the researchers know who is a treatment case and who is not. This design removes a well-known cause of bias in research called the placebo effect, in which knowledge of the treatment by either the subject or the researcher can influence the outcomes.
Exercise \(2\)
1. Describe the advantages and disadvantages of observational studies and experimental studies.
2. Explain the ways that groups of subjects can be selected for analytical studies.
Clinical Focus: Part 3
Since laboratory tests had confirmed Salmonella, a common foodborne pathogen, as the etiologic agent, epidemiologists suspected that the outbreak was caused by contamination at a food processing facility serving the region. Interviews with patients focused on food consumption during and after the Thanksgiving holiday, corresponding with the timing of the outbreak. During the interviews, patients were asked to list items consumed at holiday gatherings and describe how widely each item was consumed among family members and relatives. They were also asked about the sources of food items (e.g., brand, location of purchase, date of purchase). By asking such questions, health officials hoped to identify patterns that would lead back to the source of the outbreak.
Analysis of the interview responses eventually linked almost all of the cases to consumption of a holiday dish known as the turducken—a chicken stuffed inside a duck stuffed inside a turkey. Turducken is a dish not generally consumed year-round, which would explain the spike in cases just after the Thanksgiving holiday. Additional analysis revealed that the turduckens consumed by the affected patients were purchased already stuffed and ready to be cooked. Moreover, the pre-stuffed turduckens were all sold at the same regional grocery chain under two different brand names. Upon further investigation, officials traced both brands to a single processing plant that supplied stores throughout the Florida panhandle.
Exercise \(3\)
1. Is this an example of common source spread or propagated spread?
2. What next steps would the public health office likely take after identifying the source of the outbreak?
Key Concepts and Summary
• Early pioneers of epidemiology such as John Snow, Florence Nightingale, and Joseph Lister, studied disease at the population level and used data to disrupt disease transmission.
• Descriptive epidemiology studies rely on case analysis and patient histories to gain information about outbreaks, frequently while they are still occurring.
• Retrospective epidemiology studies use historical data to identify associations with the disease state of present cases. Prospective epidemiology studies gather data and follow cases to find associations with future disease states.
• Analytical epidemiology studies are observational studies that are carefully designed to compare groups and uncover associations between environmental or genetic factors and disease.
• Experimental epidemiology studies generate strong evidence of causation in disease or treatment by manipulating subjects and comparing them with control subjects.
Footnotes
1. John Snow. On the Mode of Communication of Cholera. Second edition, Much Enlarged. John Churchill, 1855.
2. John Snow. “The Cholera near Golden-Wquare, and at Deptford.” Medical Times and Gazette 9 (1854): 321–322. http://www.ph.ucla.edu/epi/snow/chol...densquare.html.
3. O.M. Lidwell. “Joseph Lister and Infection from the Air.” Epidemiology and Infection 99 (1987): 569–578. www.ncbi.nlm.nih.gov/pmc/arti...00006-0004.pdf. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/16%3A_Disease_and_Epidemiology/16.02%3A_Tracking_Infectious_Diseases.txt |
Learning Objectives
• Describe the different types of disease reservoirs
• Compare contact, vector, and vehicle modes of transmission
• Identify important disease vectors
• Explain the prevalence of nosocomial infections
Understanding how infectious pathogens spread is critical to preventing infectious disease. Many pathogens require a living host to survive, while others may be able to persist in a dormant state outside of a living host. But having infected one host, all pathogens must also have a mechanism of transfer from one host to another or they will die when their host dies. Pathogens often have elaborate adaptations to exploit host biology, behavior, and ecology to live in and move between hosts. Hosts have evolved defenses against pathogens, but because their rates of evolution are typically slower than their pathogens (because their generation times are longer), hosts are usually at an evolutionary disadvantage. This section will explore where pathogens survive—both inside and outside hosts—and some of the many ways they move from one host to another.
Reservoirs and Carriers
For pathogens to persist over long periods of time they require reservoirs where they normally reside. Reservoirs can be living organisms or nonliving sites. Nonliving reservoirs can include soil and water in the environment. These may naturally harbor the organism because it may grow in that environment. These environments may also become contaminated with pathogens in human feces, pathogens shed by intermediate hosts, or pathogens contained in the remains of intermediate hosts.
Pathogens may have mechanisms of dormancy or resilience that allow them to survive (but typically not to reproduce) for varying periods of time in nonliving environments. For example, Clostridium tetani survives in the soil and in the presence of oxygen as a resistant endospore. Although many viruses are soon destroyed once in contact with air, water, or other non-physiological conditions, certain types are capable of persisting outside of a living cell for varying amounts of time. For example, a study that looked at the ability of influenza viruses to infect a cell culture after varying amounts of time on a banknote showed survival times from 48 hours to 17 days, depending on how they were deposited on the banknote.1 On the other hand, cold-causing rhinoviruses are somewhat fragile, typically surviving less than a day outside of physiological fluids.
A human acting as a reservoir of a pathogen may or may not be capable of transmitting the pathogen, depending on the stage of infection and the pathogen. To help prevent the spread of disease among school children, the CDC has developed guidelines based on the risk of transmission during the course of the disease. For example, children with chickenpox are considered contagious for five days from the start of the rash, whereas children with most gastrointestinal illnesses should be kept home for 24 hours after the symptoms disappear.
An individual capable of transmitting a pathogen without displaying symptoms is referred to as a carrier. A passive carrier is contaminated with the pathogen and can mechanically transmit it to another host; however, a passive carrier is not infected. For example, a health-care professional who fails to wash his hands after seeing a patient harboring an infectious agent could become a passive carrier, transmitting the pathogen to another patient who becomes infected.
By contrast, an active carrier is an infected individual who can transmit the disease to others. An active carrier may or may not exhibit signs or symptoms of infection. For example, active carriers may transmit the disease during the incubation period (before they show signs and symptoms) or the period of convalescence (after symptoms have subsided). Active carriers who do not present signs or symptoms of disease despite infection are called asymptomatic carriers. Pathogens such as hepatitis B virus, herpes simplex virus, and HIV are frequently transmitted by asymptomatic carriers. Mary Mallon, better known as Typhoid Mary, is a famous historical example of an asymptomatic carrier. An Irish immigrant, Mallon worked as a cook for households in and around New York City between 1900 and 1915. In each household, the residents developed typhoid fever (caused by Salmonella typhi) a few weeks after Mallon started working. Later investigations determined that Mallon was responsible for at least 122 cases of typhoid fever, five of which were fatal.2 See Eye on Ethics: Typhoid Mary for more about the Mallon case.
A pathogen may have more than one living reservoir. In zoonotic diseases, animals act as reservoirs of human disease and transmit the infectious agent to humans through direct or indirect contact. In some cases, the disease also affects the animal, but in other cases the animal is asymptomatic.
In parasitic infections, the parasite’s preferred host is called the definitive host. In parasites with complex life cycles, the definitive host is the host in which the parasite reaches sexual maturity. Some parasites may also infect one or more intermediate hosts in which the parasite goes through several immature life cycle stages or reproduces asexually.
Link to Learning
George Soper, the sanitary engineer who traced the typhoid outbreak to Mary Mallon, gives an account of his investigation, an example of descriptive epidemiology, in “The Curious Career of Typhoid Mary.”
Exercise \(1\)
1. List some nonliving reservoirs for pathogens.
2. Explain the difference between a passive carrier and an active carrier.
Transmission
Regardless of the reservoir, transmission must occur for an infection to spread. First, transmission from the reservoir to the individual must occur. Then, the individual must transmit the infectious agent to other susceptible individuals, either directly or indirectly. Pathogenic microorganisms employ diverse transmission mechanisms.
Contact Transmission
Contact transmission includes direct contact or indirect contact. Person-to-person transmission is a form of direct contact transmission. Here the agent is transmitted by physical contact between two individuals (Figure \(1\)) through actions such as touching, kissing, sexual intercourse, or droplet sprays. Direct contact can be categorized as vertical, horizontal, or droplet transmission. Vertical direct contact transmission occurs when pathogens are transmitted from mother to child during pregnancy, birth, or breastfeeding. Other kinds of direct contact transmission are called horizontal direct contact transmission. Often, contact between mucous membranes is required for entry of the pathogen into the new host, although skin-to-skin contact can lead to mucous membrane contact if the new host subsequently touches a mucous membrane. Contact transmission may also be site-specific; for example, some diseases can be transmitted by sexual contact but not by other forms of contact.
When an individual coughs or sneezes, small droplets of mucus that may contain pathogens are ejected. This leads to direct droplet transmission, which refers to droplet transmission of a pathogen to a new host over distances of one meter or less. A wide variety of diseases are transmitted by droplets, including influenza and many forms of pneumonia. Transmission over distances greater than one meter is called airborne transmission.
Indirect contact transmission involves inanimate objects called fomites that become contaminated by pathogens from an infected individual or reservoir (Figure \(2\)). For example, an individual with the common cold may sneeze, causing droplets to land on a fomite such as a tablecloth or carpet, or the individual may wipe her nose and then transfer mucus to a fomite such as a doorknob or towel. Transmission occurs indirectly when a new susceptible host later touches the fomite and transfers the contaminated material to a susceptible portal of entry. Fomites can also include objects used in clinical settings that are not properly sterilized, such as syringes, needles, catheters, and surgical equipment. Pathogens transmitted indirectly via such fomites are a major cause of healthcare-associated infections (see Controlling Microbial Growth).
Vehicle Transmission
The term vehicle transmission refers to the transmission of pathogens through vehicles such as water, food, and air. Water contamination through poor sanitation methods leads to waterborne transmission of disease. Waterborne disease remains a serious problem in many regions throughout the world. The World Health Organization (WHO) estimates that contaminated drinking water is responsible for more than 500,000 deaths each year.3 Similarly, food contaminated through poor handling or storage can lead to foodborne transmission of disease (Figure \(3\)).
Dust and fine particles known as aerosols, which can float in the air, can carry pathogens and facilitate the airborne transmission of disease. For example, dust particles are the dominant mode of transmission of hantavirus to humans. Hantavirus is found in mouse feces, urine, and saliva, but when these substances dry, they can disintegrate into fine particles that can become airborne when disturbed; inhalation of these particles can lead to a serious and sometimes fatal respiratory infection.
Although droplet transmission over short distances is considered contact transmission as discussed above, longer distance transmission of droplets through the air is considered vehicle transmission. Unlike larger particles that drop quickly out of the air column, fine mucus droplets produced by coughs or sneezes can remain suspended for long periods of time, traveling considerable distances. In certain conditions, droplets desiccate quickly to produce a droplet nucleus that is capable of transmitting pathogens; air temperature and humidity can have an impact on effectiveness of airborne transmission.
Tuberculosis is often transmitted via airborne transmission when the causative agent, Mycobacterium tuberculosis, is released in small particles with coughs. Because tuberculosis requires as few as 10 microbes to initiate a new infection, patients with tuberculosis must be treated in rooms equipped with special ventilation, and anyone entering the room should wear a mask.
Clinical Focus: Resolution
After identifying the source of the contaminated turduckens, the Florida public health office notified the CDC, which requested an expedited inspection of the facility by state inspectors. Inspectors found that a machine used to process the chicken was contaminated with Salmonella as a result of substandard cleaning protocols. Inspectors also found that the process of stuffing and packaging the turduckens prior to refrigeration allowed the meat to remain at temperatures conducive to bacterial growth for too long. The contamination and the delayed refrigeration led to vehicle (food) transmission of the bacteria in turduckens.
Based on these findings, the plant was shut down for a full and thorough decontamination. All turduckens produced in the plant were recalled and pulled from store shelves ahead of the December holiday season, preventing further outbreaks.
Vector Transmission
Diseases can also be transmitted by a mechanical or biological vector, an animal (typically an arthropod) that carries the disease from one host to another. Mechanical transmission is facilitated by a mechanical vector, an animal that carries a pathogen from one host to another without being infected itself. For example, a fly may land on fecal matter and later transmit bacteria from the feces to food that it lands on; a human eating the food may then become infected by the bacteria, resulting in a case of diarrhea or dysentery (Figure \(4\)).
Biological transmission occurs when the pathogen reproduces within a biological vector that transmits the pathogen from one host to another (Figure \(4\)). Arthropods are the main vectors responsible for biological transmission (Figure \(5\)). Most arthropod vectors transmit the pathogen by biting the host, creating a wound that serves as a portal of entry. The pathogen may go through part of its reproductive cycle in the gut or salivary glands of the arthropod to facilitate its transmission through the bite. For example, hemipterans (called “kissing bugs” or “assassin bugs”) transmit Chagas disease to humans by defecating when they bite, after which the human scratches or rubs the infected feces into a mucous membrane or break in the skin.
Biological insect vectors include mosquitoes, which transmit malaria and other diseases, and lice, which transmit typhus. Other arthropod vectors can include arachnids, primarily ticks, which transmit Lyme disease and other diseases, and mites, which transmit scrub typhus and rickettsial pox. Biological transmission, because it involves survival and reproduction within a parasitized vector, complicates the biology of the pathogen and its transmission. There are also important non-arthropod vectors of disease, including mammals and birds. Various species of mammals can transmit rabies to humans, usually by means of a bite that transmits the rabies virus. Chickens and other domestic poultry can transmit avian influenza to humans through direct or indirect contact with avian influenza virus A shed in the birds’ saliva, mucous, and feces.
Exercise \(2\)
1. Describe how diseases can be transmitted through the air.
2. Explain the difference between a mechanical vector and a biological vector.
Using GMOs to Stop the Spread of Zika
In 2016, an epidemic of the Zika virus was linked to a high incidence of birth defects in South America and Central America. As winter turned to spring in the northern hemisphere, health officials correctly predicted the virus would spread to North America, coinciding with the breeding season of its major vector, the Aedes aegypti mosquito.
The range of the A. aegypti mosquito extends well into the southern United States (Figure \(6\)). Because these same mosquitoes serve as vectors for other problematic diseases (dengue fever, yellow fever, and others), various methods of mosquito control have been proposed as solutions. Chemical pesticides have been used effectively in the past, and are likely to be used again; but because chemical pesticides can have negative impacts on the environment, some scientists have proposed an alternative that involves genetically engineering A. aegypti so that it cannot reproduce. This method, however, has been the subject of some controversy.
One method that has worked in the past to control pests, with little apparent downside, has been sterile male introductions. This method controlled the screw-worm fly pest in the southwest United States and fruit fly pests of fruit crops. In this method, males of the target species are reared in the lab, sterilized with radiation, and released into the environment where they mate with wild females, who subsequently bear no live offspring. Repeated releases shrink the pest population.
A similar method, taking advantage of recombinant DNA technology,4 introduces a dominant lethal allele into male mosquitoes that is suppressed in the presence of tetracycline (an antibiotic) during laboratory rearing. The males are released into the environment and mate with female mosquitoes. Unlike the sterile male method, these matings produce offspring, but they die as larvae from the lethal gene in the absence of tetracycline in the environment. As of 2016, this method has yet to be implemented in the United States, but a UK company tested the method in Piracicaba, Brazil, and found an 82% reduction in wild A. aegypti larvae and a 91% reduction in dengue cases in the treated area.5 In August 2016, amid news of Zika infections in several Florida communities, the FDA gave the UK company permission to test this same mosquito control method in Key West, Florida, pending compliance with local and state regulations and a referendum in the affected communities.
The use of genetically modified organisms (GMOs) to control a disease vector has its advocates as well as its opponents. In theory, the system could be used to drive the A. aegypti mosquito extinct—a noble goal according to some, given the damage they do to human populations.6 But opponents of the idea are concerned that the gene could escape the species boundary of A. aegypti and cause problems in other species, leading to unforeseen ecological consequences. Opponents are also wary of the program because it is being administered by a for-profit corporation, creating the potential for conflicts of interest that would have to be tightly regulated; and it is not clear how any unintended consequences of the program could be reversed.
There are other epidemiological considerations as well. Aedes aegypti is apparently not the only vector for the Zika virus. Aedes albopictus, the Asian tiger mosquito, is also a vector for the Zika virus.7 A. albopictus is now widespread around the planet including much of the United States (Figure \(6\)). Many other mosquitoes have been found to harbor Zika virus, though their capacity to act as vectors is unknown.8 Genetically modified strains of A. aegypti will not control the other species of vectors. Finally, the Zika virus can apparently be transmitted sexually between human hosts, from mother to child, and possibly through blood transfusion. All of these factors must be considered in any approach to controlling the spread of the virus.
Clearly there are risks and unknowns involved in conducting an open-environment experiment of an as-yet poorly understood technology. But allowing the Zika virus to spread unchecked is also risky. Does the threat of a Zika epidemic justify the ecological risk of genetically engineering mosquitos? Are current methods of mosquito control sufficiently ineffective or harmful that we need to try untested alternatives? These are the questions being put to public health officials now.
Quarantining
Individuals suspected or known to have been exposed to certain contagious pathogens may be quarantined, or isolated to prevent transmission of the disease to others. Hospitals and other health-care facilities generally set up special wards to isolate patients with particularly hazardous diseases such as tuberculosis or Ebola (Figure \(7\)). Depending on the setting, these wards may be equipped with special air-handling methods, and personnel may implement special protocols to limit the risk of transmission, such as personal protective equipment or the use of chemical disinfectant sprays upon entry and exit of medical personnel.
The duration of the quarantine depends on factors such as the incubation period of the disease and the evidence suggestive of an infection. The patient may be released if signs and symptoms fail to materialize when expected or if preventive treatment can be administered in order to limit the risk of transmission. If the infection is confirmed, the patient may be compelled to remain in isolation until the disease is no longer considered contagious.
In the United States, public health authorities may only quarantine patients for certain diseases, such as cholera, diphtheria, infectious tuberculosis, and strains of influenza capable of causing a pandemic. Individuals entering the United States or moving between states may be quarantined by the CDC if they are suspected of having been exposed to one of these diseases. Although the CDC routinely monitors entry points to the United States for crew or passengers displaying illness, quarantine is rarely implemented.
Healthcare-Associated (Nosocomial) Infections
Hospitals, retirement homes, and prisons attract the attention of epidemiologists because these settings are associated with increased incidence of certain diseases. Higher rates of transmission may be caused by characteristics of the environment itself, characteristics of the population, or both. Consequently, special efforts must be taken to limit the risks of infection in these settings.
Infections acquired in health-care facilities, including hospitals, are called nosocomial infections or healthcare-associated infections (HAI). HAIs are often connected with surgery or other invasive procedures that provide the pathogen with access to the portal of infection. For an infection to be classified as an HAI, the patient must have been admitted to the health-care facility for a reason other than the infection. In these settings, patients suffering from primary disease are often afflicted with compromised immunity and are more susceptible to secondary infection and opportunistic pathogens.
In 2011, more than 720,000 HAIs occurred in hospitals in the United States, according to the CDC. About 22% of these HAIs occurred at a surgical site, and cases of pneumonia accounted for another 22%; urinary tract infections accounted for an additional 13%, and primary bloodstream infections 10%.9 Such HAIs often occur when pathogens are introduced to patients’ bodies through contaminated surgical or medical equipment, such as catheters and respiratory ventilators. Health-care facilities seek to limit nosocomial infections through training and hygiene protocols such as those described in Control of Microbial Growth.
Exercise \(3\)
Give some reasons why HAIs occur.
Key Concepts and Summary
• Reservoirs of human disease can include the human and animal populations, soil, water, and inanimate objects or materials.
• Contact transmission can be direct or indirect through physical contact with either an infected host (direct) or contact with a fomite that an infected host has made contact with previously (indirect).
• Vector transmission occurs when a living organism carries an infectious agent on its body (mechanical) or as an infection host itself (biological), to a new host.
• Vehicle transmission occurs when a substance, such as soil, water, or air, carries an infectious agent to a new host.
• Healthcare-associated infections (HAI), or nosocomial infections, are acquired in a clinical setting. Transmission is facilitated by medical interventions and the high concentration of susceptible, immunocompromised individuals in clinical settings.
Footnotes
1. 1 Yves Thomas, Guido Vogel, Werner Wunderli, Patricia Suter, Mark Witschi, Daniel Koch, Caroline Tapparel, and Laurent Kaiser. “Survival of Influenza Virus on Banknotes.” Applied and Environmental Microbiology 74, no. 10 (2008): 3002–3007.
2. 2 Filio Marineli, Gregory Tsoucalas, Marianna Karamanou, and George Androutsos. “Mary Mallon (1869–1938) and the History of Typhoid Fever.” Annals of Gastroenterology 26 (2013): 132–134. www.ncbi.nlm.nih.gov/pmc/arti...rol-26-132.pdf.
3. 3 World Health Organization. Fact sheet No. 391—Drinking Water. June 2005. www.who.int/mediacentre/factsheets/fs391/en.
4. 4 Blandine Massonnet-Bruneel, Nicole Corre-Catelin, Renaud Lacroix, Rosemary S. Lees, Kim Phuc Hoang, Derric Nimmo, Luke Alphey, and Paul Reiter. “Fitness of Transgenic Mosquito Aedes aegypti Males Carrying a Dominant Lethal Genetic System.” PLOS ONE 8, no. 5 (2013): e62711.
5. 5 Richard Levine. “Cases of Dengue Drop 91 Percent Due to Genetically Modified Mosquitoes.” Entomology Today. entomologytoday.org/2016/07/...ied-mosquitoes.
6. 6 Olivia Judson. “A Bug’s Death.” The New York Times, September 25, 2003. www.nytimes.com/2003/09/25/op...g-s-death.html.
7. 7 Gilda Grard, Mélanie Caron, Illich Manfred Mombo, Dieudonné Nkoghe, Statiana Mboui Ondo, Davy Jiolle, Didier Fontenille, Christophe Paupy, and Eric Maurice Leroy. “Zika Virus in Gabon (Central Africa)–2007: A New Threat from Aedes albopictus?” PLOS Neglected Tropical Diseases 8, no. 2 (2014): e2681.
8. 8 Constância F.J. Ayres. “Identification of Zika Virus Vectors and Implications for Control.” The Lancet Infectious Diseases 16, no. 3 (2016): 278–279.
9. 9 Centers for Disease Control and Prevention. “HAI Data and Statistics.” 2016. http://www.cdc.gov/hai/surveillance. Accessed Jan 2, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/16%3A_Disease_and_Epidemiology/16.03%3A_How_Diseases_Spread.txt |
Learning Objectives
• Describe the entities involved in international public health and their activities
• Identify and differentiate between emerging and reemerging infectious diseases
A large number of international programs and agencies are involved in efforts to promote global public health. Among their goals are developing infrastructure in health care, public sanitation, and public health capacity; monitoring infectious disease occurrences around the world; coordinating communications between national public health agencies in various countries; and coordinating international responses to major health crises. In large part, these international efforts are necessary because disease-causing microorganisms know no national boundaries.
The World Health Organization (WHO)
International public health issues are coordinated by the World Health Organization (WHO), an agency of the United Nations. Of its roughly \$4 billion budget for 2015–161, about \$1 billion was funded by member states and the remaining \$3 billion by voluntary contributions. In addition to monitoring and reporting on infectious disease, WHO also develops and implements strategies for their control and prevention. WHO has had a number of successful international public health campaigns. For example, its vaccination program against smallpox, begun in the mid-1960s, resulted in the global eradication of the disease by 1980. WHO continues to be involved in infectious disease control, primarily in the developing world, with programs targeting malaria, HIV/AIDS, and tuberculosis, among others. It also runs programs to reduce illness and mortality that occur as a result of violence, accidents, lifestyle-associated illnesses such as diabetes, and poor health-care infrastructure.
WHO maintains a global alert and response system that coordinates information from member nations. In the event of a public health emergency or epidemic, it provides logistical support and coordinates international response to the emergency. The United States contributes to this effort through the CDC. The CDC carries out international monitoring and public health efforts, mainly in the service of protecting US public health in an increasingly connected world. Similarly, the European Union maintains a Health Security Committee that monitors disease outbreaks within its member countries and internationally, coordinating with WHO.
Exercise \(1\)
Name the organizations that participate in international public health monitoring.
Emerging and Reemerging Infectious Diseases
Both WHO and some national public health agencies such as the CDC monitor and prepare for emerging infectious diseases. An emerging infectious disease is either new to the human population or has shown an increase in prevalence in the previous twenty years. Whether the disease is new or conditions have changed to cause an increase in frequency, its status as emerging implies the need to apply resources to understand and control its growing impact.
Emerging diseases may change their frequency gradually over time, or they may experience sudden epidemic growth. The importance of vigilance was made clear during the Ebola hemorrhagic fever epidemic in western Africa through 2014–2015. Although health experts had been aware of the Ebola virus since the 1970s, an outbreak on such a large scale had never happened before (Figure \(1\)). Previous human epidemics had been small, isolated, and contained. Indeed, the gorilla and chimpanzee populations of western Africa had suffered far worse from Ebola than the human population. The pattern of small isolated human epidemics changed in 2014. Its high transmission rate, coupled with cultural practices for treatment of the dead and perhaps its emergence in an urban setting, caused the disease to spread rapidly, and thousands of people died. The international public health community responded with a large emergency effort to treat patients and contain the epidemic.
Emerging diseases are found in all countries, both developed and developing (Table \(1\)). Some nations are better equipped to deal with them. National and international public health agencies watch for epidemics like the Ebola outbreak in developing countries because those countries rarely have the health-care infrastructure and expertise to deal with large outbreaks effectively. Even with the support of international agencies, the systems in western Africa struggled to identify and care for the sick and control spread. In addition to the altruistic goal of saving lives and assisting nations lacking in resources, the global nature of transportation means that an outbreak anywhere can spread quickly to every corner of the planet. Managing an epidemic in one location—its source—is far easier than fighting it on many fronts.
Ebola is not the only disease that needs to be monitored in the global environment. In 2015, WHO set priorities on several emerging diseases that had a high probability of causing epidemics and that were poorly understood (and thus urgently required research and development efforts).
A reemerging infectious disease is a disease that is increasing in frequency after a previous period of decline. Its reemergence may be a result of changing conditions or old prevention regimes that are no longer working. Examples of such diseases are drug-resistant forms of tuberculosis, bacterial pneumonia, and malaria. Drug-resistant strains of the bacteria causing gonorrhea and syphilis are also becoming more widespread, raising concerns of untreatable infections.
Table \(1\): Some Emerging and Reemerging Infectious Diseases
Disease Pathogen Year Discovered Affected Regions Transmission
AIDS HIV 1981 Worldwide Contact with infected body fluids
Chikungunya fever Chikungunya virus 1952 Africa, Asia, India; spreading to Europe and the Americas Mosquito-borne
Ebola virus disease Ebola virus 1976 Central and Western Africa Contact with infected body fluids
H1N1 Influenza (swine flu) H1N1 virus 2009 Worldwide Droplet transmission
Lyme disease Borrelia burgdorferi bacterium 1981 Northern hemisphere From mammal reservoirs to humans by tick vectors
West Nile virus disease West Nile virus 1937 Africa, Australia, Canada to Venezuela, Europe, Middle East, Western Asia Mosquito-borne
Exercise \(2\)
1. Explain why it is important to monitor emerging infectious diseases.
2. Explain how a bacterial disease could reemerge, even if it had previously been successfully treated and controlled.
SARS Outbreak and Identification
On November 16, 2002, the first case of a SARS outbreak was reported in Guangdong Province, China. The patient exhibited influenza-like symptoms such as fever, cough, myalgia, sore throat, and shortness of breath. As the number of cases grew, the Chinese government was reluctant to openly communicate information about the epidemic with the World Health Organization (WHO) and the international community. The slow reaction of Chinese public health officials to this new disease contributed to the spread of the epidemic within and later outside China. In April 2003, the Chinese government finally responded with a huge public health effort involving quarantines, medical checkpoints, and massive cleaning projects. Over 18,000 people were quarantined in Beijing alone. Large funding initiatives were created to improve health-care facilities, and dedicated outbreak teams were created to coordinate the response. By August 16, 2003, the last SARS patients were released from a hospital in Beijing nine months after the first case was reported in China.
In the meantime, SARS spread to other countries on its way to becoming a global pandemic. Though the infectious agent had yet to be identified, it was thought to be an influenza virus. The disease was named SARS, an acronym for severe acute respiratory syndrome, until the etiologic agent could be identified. Travel restrictions to Southeast Asia were enforced by many countries. By the end of the outbreak, there were 8,098 cases and 774 deaths worldwide. China and Hong Kong were hit hardest by the epidemic, but Taiwan, Singapore, and Toronto, Canada, also saw significant numbers of cases (Figure \(2\)).
Fortunately, timely public health responses in many countries effectively suppressed the outbreak and led to its eventual containment. For example, the disease was introduced to Canada in February 2003 by an infected traveler from Hong Kong, who died shortly after being hospitalized. By the end of March, hospital isolation and home quarantine procedures were in place in the Toronto area, stringent anti-infection protocols were introduced in hospitals, and the media were actively reporting on the disease. Public health officials tracked down contacts of infected individuals and quarantined them. A total of 25,000 individuals were quarantined in the city. Thanks to the vigorous response of the Canadian public health community, SARS was brought under control in Toronto by June, a mere four months after it was introduced.
In 2003, WHO established a collaborative effort to identify the causative agent of SARS, which has now been identified as a coronavirus that was associated with horseshoe bats. The genome of the SARS virus was sequenced and published by researchers at the CDC and in Canada in May 2003, and in the same month researchers in the Netherlands confirmed the etiology of the disease by fulfilling Koch’s postulates for the SARS coronavirus. The last known case of SARS worldwide was reported in 2004.
Link to Learning
This database of reports chronicles outbreaks of infectious disease around the world. It was on this system that the first information about the SARS outbreak in China emerged.
The CDC publishes Emerging Infectious Diseases, a monthly journal available online.
Key Concepts and Summary
• The World Health Organization (WHO) is an agency of the United Nations that collects and analyzes data on disease occurrence from member nations. WHO also coordinates public health programs and responses to international health emergencies.
• Emerging diseases are those that are new to human populations or that have been increasing in the past two decades. Reemerging diseases are those that are making a resurgence in susceptible populations after previously having been controlled in some geographic areas.
Footnotes
1. 1 World Health Organization. “Programme Budget 2014–2015.” www.who.int/about/finances-ac...lity/budget/en. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/16%3A_Disease_and_Epidemiology/16.04%3A_Global_Public_Health.txt |
16.1: The Language of Epidemiologists
The field of epidemiology concerns the geographical distribution and timing of infectious disease occurrences and how they are transmitted and maintained in nature, with the goal of recognizing and controlling outbreaks. The science of epidemiology includes etiology (the study of the causes of disease) and investigation of disease transmission (mechanisms by which a disease is spread).
Matching
Match each term with its description.
___sporadic disease A. the number of disease cases per 100,000 individuals
___endemic disease B. a disease in higher than expected numbers around the world
___pandemic disease C. the number of deaths from a disease for every 10,000 individuals
___morbidity rate D. a disease found occasionally in a region with cases occurring mainly in isolation from each other
___mortality rate E. a disease found regularly in a region
Answer
D, E, B, A, C
Fill in the Blank
The ________ collects data and conducts epidemiologic studies in the United States.
Answer
Centers for Disease Control and Prevention, or CDC
Short Answer
During an epidemic, why might the prevalence of a disease at a particular time not be equal to the sum of the incidences of the disease?
In what publication would you find data on emerging/reemerging diseases in the United States?
Critical Thinking
Why might an epidemiological population in a state not be the same size as the number of people in a state? Use an example.
16.2: Tracking Infectious Diseases
Some important researchers, such as Florence Nightingale, subscribed to the miasma hypothesis. The transition to acceptance of the germ theory during the 19th century provided a solid mechanistic grounding to the study of disease patterns. The studies of 19th century physicians and researchers such as John Snow, Florence Nightingale, Ignaz Semmelweis, Joseph Lister, Robert Koch, Louis Pasteur, and others sowed the seeds of modern epidemiology.
Matching
Match each type of epidemiology study with its description.
___experimental A. examination of past case histories and medical test results conducted on patients in an outbreak
___analytical B. examination of current case histories, interviews with patients and their contacts, interpretation of medical test results; frequently conducted while outbreak is still in progress
___prospective C. use of a set of test subjects (human or animal) and control subjects that are treated the same as the test subjects except for the specific treatment being studied
___descriptive D. observing groups of individuals to look for associations with disease
___retrospective E. a comparison of a cohort of individuals through the course of the study
Answer
C, D, E, B, A
Match each pioneer of epidemiology with his or her contribution.
___Florence Nightingale A. determined the source of a cholera outbreak in London
___Robert Koch B. showed that surgical wound infection rates could be dramatically reduced by using carbolic acid to disinfect surgical tools, bandages, and surgical sites
___Joseph Lister C. compiled data on causes of mortality in soldiers, leading to innovations in military medical care
___John Snow D. developed a methodology for conclusively determining the etiology of disease
Answer
C, D, B, A
Fill in the Blank
________occurs when an infected individual passes the infection on to other individuals, who pass it on to still others, increasing the penetration of the infection into the susceptible population.
Answer
Propagated spread
A batch of food contaminated with botulism exotoxin, consumed at a family reunion by most of the members of a family, would be an example of a ________ outbreak.
Answer
point source
Short Answer
What activity did John Snow conduct, other than mapping, that contemporary epidemiologists also use when trying to understand how to control a disease?
16.3: How Diseases Spread
Pathogens often have elaborate adaptations to exploit host biology, behavior, and ecology to live in and move between hosts. Hosts have evolved defenses against pathogens, but because their rates of evolution are typically slower than their pathogens (because their generation times are longer), hosts are usually at an evolutionary disadvantage. This section will explore where pathogens survive—both inside and outside hosts—and some of the many ways they move from one host to another.
Multiple Choice
Which is the most common type of biological vector of human disease?
1. viruses
2. bacteria
3. mammals
4. arthropods
Answer
D
A mosquito bites a person who subsequently develops a fever and abdominal rash. What type of transmission would this be?
1. mechanical vector transmission
2. biological vector transmission
3. direct contact transmission
4. vehicle transmission
Answer
B
Cattle are allowed to pasture in a field that contains the farmhouse well, and the farmer’s family becomes ill with a gastrointestinal pathogen after drinking the water. What type of transmission of infectious agents would this be?
1. biological vector transmission
2. direct contact transmission
3. indirect contact transmission
4. vehicle transmission
Answer
D
A blanket from a child with chickenpox is likely to be contaminated with the virus that causes chickenpox (Varicella-zoster virus). What is the blanket called?
1. fomite
2. host
3. pathogen
4. vector
Answer
A
Fill in the Blank
A patient in the hospital with a urinary catheter develops a bladder infection. This is an example of a(n) ________ infection.
Answer
nosocomial or healthcare-associated
A ________ is an animal that can transfer infectious pathogens from one host to another.
Answer
vector
Short Answer
Differentiate between droplet vehicle transmission and airborne transmission.
Critical Thinking
Many people find that they become ill with a cold after traveling by airplane. The air circulation systems of commercial aircraft use HEPA filters that should remove any infectious agents that pass through them. What are the possible reasons for increased incidence of colds after flights?
16.4: Global Public Health
A large number of international programs and agencies are involved in efforts to promote global public health. Among their goals are developing infrastructure in health care, public sanitation, and public health capacity; monitoring infectious disease occurrences around the world; coordinating communications between national public health agencies in various countries; and coordinating international responses to major health crises.
Multiple Choice
Which of the following would NOT be considered an emerging disease?
1. Ebola hemorrhagic fever
2. West Nile virus fever/encephalitis
3. Zika virus disease
4. Tuberculosis
Answer
D
Which of the following would NOT be considered a reemerging disease?
1. Drug-resistant tuberculosis
2. Drug-resistant gonorrhea
3. Malaria
4. West Nile virus fever/encephalitis
Answer
D
Which of the following factors can lead to reemergence of a disease?
1. A mutation that allows it to infect humans
2. A period of decline in vaccination rates
3. A change in disease reporting procedures
4. Better education on the signs and symptoms of the disease
Answer
B
Why are emerging diseases with very few cases the focus of intense scrutiny?
1. They tend to be more deadly
2. They are increasing and therefore not controlled
3. They naturally have higher transmission rates
4. They occur more in developed countries
Answer
B
Fill in the Blank
The ________ collects data and conducts epidemiologic studies at the global level.
Answer
WHO (World Health Organization)
Critical Thinking
An Atlantic crossing by boat from England to New England took 60–80 days in the 18th century. In the late 19th century the voyage took less than a week. How do you think these time differences for travel might have impacted the spread of infectious diseases from Europe to the Americas, or vice versa? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/16%3A_Disease_and_Epidemiology/16.E%3A_Disease_and_Epidemiology_%28Exercises%29.txt |
Despite relatively constant exposure to pathogenic microbes in the environment, humans do not generally suffer from constant infection or disease. Under most circumstances, the body is able to defend itself from the threat of infection thanks to a complex immune system designed to repel, kill, and expel disease-causing invaders. Immunity as a whole can be described as two interrelated parts: nonspecific innate immunity, which is the subject of this chapter, and specific adaptive host defenses, which are discussed in the next chapter.
The nonspecific innate immune response provides a first line of defense that can often prevent infections from gaining a solid foothold in the body. These defenses are described as nonspecific because they do not target any specific pathogen; rather, they defend against a wide range of potential pathogens. They are called innate because they are built-in mechanisms of the human organism. Unlike the specific adaptive defenses, they are not acquired over time and they have no “memory” (they do not improve after repeated exposures to specific pathogens).
Broadly speaking, nonspecific innate defenses provide an immediate (or very rapid) response against potential pathogens. However, these responses are neither perfect nor impenetrable. They can be circumvented by pathogens on occasion, and sometimes they can even cause damage to the body, contributing to the signs and symptoms of infection (Figure \(1\)).
• 17.1: Physical Defenses
Nonspecific innate immunity provides a first line of defense against infection by nonspecifically blocking entry of microbes and targeting them for destruction or removal from the body. The physical defenses of innate immunity include physical barriers, mechanical actions that remove microbes and debris, and the microbiome, which competes with and inhibits the growth of pathogens. The skin, mucous membranes, and endothelia throughout the body serve as physical barriers.
• 17.2: Chemical Defenses
Numerous chemical mediators produced endogenously and exogenously exhibit nonspecific antimicrobial functions. Many chemical mediators are found in body fluids such as sebum, saliva, mucus, gastric and intestinal fluids, urine, tears, cerumen, and vaginal secretions. Antimicrobial peptides (AMPs) found on the skin and in other areas of the body are largely produced in response to the presence of pathogens. These include dermcidin, cathelicidin, defensins, histatins, and bacteriocins.
• 17.3: Cellular Defenses
The formed elements of the blood include red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Of these, leukocytes are primarily involved in the immune response. All formed elements originate in the bone marrow as stem cells (HSCs) that differentiate through hematopoiesis. Granulocytes are leukocytes characterized by a lobed nucleus and granules in the cytoplasm. These include neutrophils (PMNs), eosinophils, and basophils.
• 17.4: Pathogen Recognition and Phagocytosis
Phagocytes are cells that recognize pathogens and destroy them through phagocytosis. Recognition often takes place by the use of phagocyte receptors that bind molecules commonly found on pathogens, known as pathogen-associated molecular patterns (PAMPs). The receptors that bind PAMPs are called pattern recognition receptors, or PRRs. Toll-like receptors (TLRs) are one type of PRR found on phagocytes.
• 17.5: Inflammation and Fever
Inflammation results from the collective response of chemical mediators and cellular defenses to an injury or infection. Acute inflammation is short lived and localized to the site of injury or infection. Chronic inflammation occurs when the inflammatory response is unsuccessful, and may result in the formation of granulomas (e.g., with tuberculosis) and scarring (e.g., with hepatitis C viral infections and liver cirrhosis).
• 17.E: Innate Nonspecific Host Defenses (Exercises)
Thumbnail: Scanning electron micrograph of a phagocyte (yellow, right) phagocytosing anthrax bacilli (orange, left). (CC BY 2.5; Volker Brinkmann via PLOS).
17: Innate Nonspecific Host Defenses
Learning Objectives
• Describe the various physical barriers and mechanical defenses that protect the human body against infection and disease
• Describe the role of microbiota as a first-line defense against infection and disease
Clinical Focus: Part 1
Angela, a 25-year-old female patient in the emergency department, is having some trouble communicating verbally because of shortness of breath. A nurse observes constriction and swelling of the airway and labored breathing. The nurse asks Angela if she has a history of asthma or allergies. Angela shakes her head no, but there is fear in her eyes. With some difficulty, she explains that her father died suddenly at age 27, when she was just a little girl, of a similar respiratory attack. The underlying cause had never been identified.
Exercise \(1\)
1. What are some possible causes of constriction and swelling of the airway?
2. What causes swelling of body tissues in general?
Nonspecific innate immunity can be characterized as a multifaceted system of defenses that targets invading pathogens in a nonspecific manner. In this chapter, we have divided the numerous defenses that make up this system into three categories: physical defenses, chemical defenses, and cellular defenses. However, it is important to keep in mind that these defenses do not function independently, and the categories often overlap. Table \(1\) provides an overview of the nonspecific defenses discussed in this chapter.
Table \(1\): Overview of Nonspecific Innate Immune Defenses
Overview of Nonspecific Innate Immune Defenses
Physical defenses Physical barriers
Mechanical defenses
Microbiome
Chemical defenses Chemicals and enzymes in body fluids
Antimicrobial peptides
Plasma protein mediators
Cytokines
Inflammation-eliciting mediators
Cellular defenses Granulocytes
Agranulocytes
Physical defenses provide the body’s most basic form of nonspecific defense. They include physical barriers to microbes, such as the skin and mucous membranes, as well as mechanical defenses that physically remove microbes and debris from areas of the body where they might cause harm or infection. In addition, the microbiome provides a measure of physical protection against disease, as microbes of the normal microbiota compete with pathogens for nutrients and cellular binding sites necessary to cause infection.
Physical Barriers
Physical barriers play an important role in preventing microbes from reaching tissues that are susceptible to infection. At the cellular level, barriers consist of cells that are tightly joined to prevent invaders from crossing through to deeper tissue. For example, the endothelial cells that line blood vessels have very tight cell-to-cell junctions, blocking microbes from gaining access to the bloodstream. Cell junctions are generally composed of cell membrane proteins that may connect with the extracellular matrix or with complementary proteins from neighboring cells. Tissues in various parts of the body have different types of cell junctions. These include tight junctions, desmosomes, and gap junctions, as illustrated in Figure \(1\). Invading microorganisms may attempt to break down these substances chemically, using enzymes such as proteases that can cause structural damage to create a point of entry for pathogens.
The Skin Barrier
One of the body’s most important physical barriers is the skin barrier, which is composed of three layers of closely packed cells. The thin upper layer is called the epidermis. A second, thicker layer, called the dermis, contains hair follicles, sweat glands, nerves, and blood vessels. A layer of fatty tissue called the hypodermis lies beneath the dermis and contains blood and lymph vessels (Figure \(2\)).
The topmost layer of skin, the epidermis, consists of cells that are packed with keratin. These dead cells remain as a tightly connected, dense layer of protein-filled cell husks on the surface of the skin. The keratin makes the skin’s surface mechanically tough and resistant to degradation by bacterial enzymes. Fatty acids on the skin’s surface create a dry, salty, and acidic environment that inhibits the growth of some microbes and is highly resistant to breakdown by bacterial enzymes. In addition, the dead cells of the epidermis are frequently shed, along with any microbes that may be clinging to them. Shed skin cells are continually replaced with new cells from below, providing a new barrier that will soon be shed in the same way.
Infections can occur when the skin barrier is compromised or broken. A wound can serve as a point of entry for opportunistic pathogens, which can infect the skin tissue surrounding the wound and possibly spread to deeper tissues.
Every Rose Has its Thorn
Mike, a gardener from southern California, recently noticed a small red bump on his left forearm. Initially, he did not think much of it, but soon it grew larger and then ulcerated (opened up), becoming a painful lesion that extended across a large part of his forearm (Figure \(3\)). He went to an urgent care facility, where a physician asked about his occupation. When he said he was a landscaper, the physician immediately suspected a case of sporotrichosis, a type of fungal infection known as rose gardener’s disease because it often afflicts landscapers and gardening enthusiasts.
Under most conditions, fungi cannot produce skin infections in healthy individuals. Fungi grow filaments known as hyphae, which are not particularly invasive and can be easily kept at bay by the physical barriers of the skin and mucous membranes. However, small wounds in the skin, such as those caused by thorns, can provide an opening for opportunistic pathogens like Sporothrix schenkii, a soil-dwelling fungus and the causative agent of rose gardener’s disease. Once it breaches the skin barrier, S. schenkii can infect the skin and underlying tissues, producing ulcerated lesions like Mike’s. Compounding matters, other pathogens may enter the infected tissue, causing secondary bacterial infections.
Luckily, rose gardener’s disease is treatable. Mike’s physician wrote him a prescription for some antifungal drugs as well as a course of antibiotics to combat secondary bacterial infections. His lesions eventually healed, and Mike returned to work with a new appreciation for gloves and protective clothing.
Mucous Membranes
The mucous membranes lining the nose, mouth, lungs, and urinary and digestive tracts provide another nonspecific barrier against potential pathogens. Mucous membranes consist of a layer of epithelial cells bound by tight junctions. The epithelial cells secrete a moist, sticky substance called mucus, which covers and protects the more fragile cell layers beneath it and traps debris and particulate matter, including microbes. Mucus secretions also contain antimicrobial peptides.
In many regions of the body, mechanical actions serve to flush mucus (along with trapped or dead microbes) out of the body or away from potential sites of infection. For example, in the respiratory system, inhalation can bring microbes, dust, mold spores, and other small airborne debris into the body. This debris becomes trapped in the mucus lining the respiratory tract, a layer known as the mucociliary blanket. The epithelial cells lining the upper parts of the respiratory tract are called ciliated epithelial cells because they have hair-like appendages known as cilia. Movement of the cilia propels debris-laden mucus out and away from the lungs. The expelled mucus is then swallowed and destroyed in the stomach, or coughed up, or sneezed out (Figure \(4\)). This system of removal is often called the mucociliary escalator.
The mucociliary escalator is such an effective barrier to microbes that the lungs, the lowermost (and most sensitive) portion of the respiratory tract, were long considered to be a sterile environment in healthy individuals. Only recently has research suggested that healthy lungs may have a small normal microbiota. Disruption of the mucociliary escalator by the damaging effects of smoking or diseases such as cystic fibrosis can lead to increased colonization of bacteria in the lower respiratory tract and frequent infections, which highlights the importance of this physical barrier to host defenses.
Like the respiratory tract, the digestive tract is a portal of entry through which microbes enter the body, and the mucous membranes lining the digestive tract provide a nonspecific physical barrier against ingested microbes. The intestinal tract is lined with epithelial cells, interspersed with mucus-secreting goblet cells (Figure \(5\)). This mucus mixes with material received from the stomach, trapping foodborne microbes and debris. The mechanical action of peristalsis, a series of muscular contractions in the digestive tract, moves the sloughed mucus and other material through the intestines, rectum, and anus, excreting the material in feces.
Endothelia
The epithelial cells lining the urogenital tract, blood vessels, lymphatic vessels, and certain other tissues are known as endothelia. These tightly packed cells provide a particularly effective frontline barrier against invaders. The endothelia of the blood-brain barrier, for example, protect the central nervous system (CNS), which consists of the brain and the spinal cord. The CNS is one of the most sensitive and important areas of the body, as microbial infection of the CNS can quickly lead to serious and often fatal inflammation. The cell junctions in the blood vessels traveling through the CNS are some of the tightest and toughest in the body, preventing any transient microbes in the bloodstream from entering the CNS. This keeps the cerebrospinal fluid that surrounds and bathes the brain and spinal cord sterile under normal conditions.
Exercise \(2\)
1. Describe how the mucociliary escalator functions.
2. Name two places you would find endothelia.
Mechanical Defenses
In addition to physical barriers that keep microbes out, the body has a number of mechanical defenses that physically remove pathogens from the body, preventing them from taking up residence. We have already discussed several examples of mechanical defenses, including the shedding of skin cells, the expulsion of mucus via the mucociliary escalator, and the excretion of feces through intestinal peristalsis. Other important examples of mechanical defenses include the flushing action of urine and tears, which both serve to carry microbes away from the body. The flushing action of urine is largely responsible for the normally sterile environment of the urinary tract, which includes the kidneys, ureters, and urinary bladder. Urine passing out of the body washes out transient microorganisms, preventing them from taking up residence. The eyes also have physical barriers and mechanical mechanisms for preventing infections. The eyelashes and eyelids prevent dust and airborne microorganisms from reaching the surface of the eye. Any microbes or debris that make it past these physical barriers may be flushed out by the mechanical action of blinking, which bathes the eye in tears, washing debris away (Figure \(6\)).
Exercise \(3\)
Name two mechanical defenses that protect the eyes.
Microbiome
In various regions of the body, resident microbiota serve as an important first-line defense against invading pathogens. Through their occupation of cellular binding sites and competition for available nutrients, the resident microbiota prevent the critical early steps of pathogen attachment and proliferation required for the establishment of an infection. For example, in the vagina, members of the resident microbiota compete with opportunistic pathogens like the yeast Candida. This competition prevents infections by limiting the availability of nutrients, thus inhibiting the growth of Candida, keeping its population in check. Similar competitions occur between the microbiota and potential pathogens on the skin, in the upper respiratory tract, and in the gastrointestinal tract. As will be discussed later in this chapter, the resident microbiota also contribute to the chemical defenses of the innate nonspecific host defenses.
The importance of the normal microbiota in host defenses is highlighted by the increased susceptibility to infectious diseases when the microbiota is disrupted or eliminated. Treatment with antibiotics can significantly deplete the normal microbiota of the gastrointestinal tract, providing an advantage for pathogenic bacteria to colonize and cause diarrheal infection. In the case of diarrhea caused by Clostridium difficile, the infection can be severe and potentially lethal. One strategy for treating C. difficile infections is fecal transplantation, which involves the transfer of fecal material from a donor (screened for potential pathogens) into the intestines of the recipient patient as a method of restoring the normal microbiota and combating C. difficile infections.
Table \(2\) provides a summary of the physical defenses discussed in this section.
Table \(2\): Physical Defenses of Nonspecific Innate Immunity
Defense Examples Function
Cellular barriers Skin, mucous membranes, endothelial cells Deny entry to pathogens
Mechanical defenses Shedding of skin cells, mucociliary sweeping, peristalsis, flushing action of urine and tears Remove pathogens from potential sites of infection
Microbiome Resident bacteria of the skin, upper respiratory tract, gastrointestinal tract, and genitourinary tract Compete with pathogens for cellular binding sites and nutrients
Exercise \(4\)
List two ways resident microbiota defend against pathogens.
Key Concepts and Summary
• Nonspecific innate immunity provides a first line of defense against infection by nonspecifically blocking entry of microbes and targeting them for destruction or removal from the body.
• The physical defenses of innate immunity include physical barriers, mechanical actions that remove microbes and debris, and the microbiome, which competes with and inhibits the growth of pathogens.
• The skin, mucous membranes, and endothelia throughout the body serve as physical barriers that prevent microbes from reaching potential sites of infection. Tight cell junctions in these tissues prevent microbes from passing through.
• Microbes trapped in dead skin cells or mucus are removed from the body by mechanical actions such as shedding of skin cells, mucociliary sweeping, coughing, peristalsis, and flushing of bodily fluids (e.g., urination, tears)
• The resident microbiota provide a physical defense by occupying available cellular binding sites and competing with pathogens for available nutrients. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/17%3A_Innate_Nonspecific_Host_Defenses/17.01%3A_Physical_Defenses.txt |
Learning Objectives
• Describe how enzymes in body fluids provide protection against infection or disease
• List and describe the function of antimicrobial peptides, complement components, cytokines, and acute-phase proteins
• Describe similarities and differences among classic, alternate, and lectin complement pathways
In addition to physical defenses, the innate nonspecific immune system uses a number of chemical mediators that inhibit microbial invaders. The term “chemical mediators” encompasses a wide array of substances found in various body fluids and tissues throughout the body. Chemical mediators may work alone or in conjunction with each other to inhibit microbial colonization and infection.
Some chemical mediators are endogenously produced, meaning they are produced by human body cells; others are produced exogenously, meaning that they are produced by certain microbes that are part of the microbiome. Some mediators are produced continually, bathing the area in the antimicrobial substance; others are produced or activated primarily in response to some stimulus, such as the presence of microbes.
Chemical and Enzymatic Mediators Found in Body Fluids
Fluids produced by the skin include examples of both endogenous and exogenous mediators. Sebaceous glands in the dermis secrete an oil called sebum that is released onto the skin surface through hair follicles. This sebum is an endogenous mediator, providing an additional layer of defense by helping seal off the pore of the hair follicle, preventing bacteria on the skin’s surface from invading sweat glands and surrounding tissue (Figure \(1\)). Certain members of the microbiome, such as the bacterium Propionibacterium acnes and the fungus Malassezia, among others, can use lipase enzymes to degrade sebum, using it as a food source. This produces oleic acid, which creates a mildly acidic environment on the surface of the skin that is inhospitable to many pathogenic microbes. Oleic acid is an example of an exogenously produced mediator because it is produced by resident microbes and not directly by body cells.
Environmental factors that affect the microbiota of the skin can have a direct impact on the production of chemical mediators. Low humidity or decreased sebum production, for example, could make the skin less habitable for microbes that produce oleic acid, thus making the skin more susceptible to pathogens normally inhibited by the skin’s low pH. Many skin moisturizers are formulated to counter such effects by restoring moisture and essential oils to the skin.
The digestive tract also produces a large number of chemical mediators that inhibit or kill microbes. In the oral cavity, saliva contains mediators such as lactoperoxidase enzymes, and mucus secreted by the esophagus contains the antibacterial enzyme lysozyme. In the stomach, highly acidic gastric fluid kills most microbes. In the lower digestive tract, the intestines have pancreatic and intestinal enzymes, antibacterial peptides (cryptins), bile produced from the liver, and specialized Paneth cells that produce lysozyme. Together, these mediators are able to eliminate most pathogens that manage to survive the acidic environment of the stomach.
In the urinary tract, urine flushes microbes out of the body during urination. Furthermore, the slight acidity of urine (the average pH is about 6) inhibits the growth of many microbes and potential pathogens in the urinary tract.
The female reproductive system employs lactate, an exogenously produced chemical mediator, to inhibit microbial growth. The cells and tissue layers composing the vagina produce glycogen, a branched and more complex polymer of glucose. Lactobacilli in the area ferment glycogen to produce lactate, lowering the pH in the vagina and inhibiting transient microbiota, opportunistic pathogens like Candida (a yeast associated with vaginal infections), and other pathogens responsible for sexually transmitted diseases.
In the eyes, tears contain the chemical mediators lysozyme and lactoferrin, both of which are capable of eliminating microbes that have found their way to the surface of the eyes. Lysozyme cleaves the bond between NAG and NAM in peptidoglycan, a component of the cell wall in bacteria. It is more effective against gram-positive bacteria, which lack the protective outer membrane associated with gram-negative bacteria. Lactoferrin inhibits microbial growth by chemically binding and sequestering iron. This effectually starves many microbes that require iron for growth.
In the ears, cerumen (earwax) exhibits antimicrobial properties due to the presence of fatty acids, which lower the pH to between 3 and 5.
The respiratory tract uses various chemical mediators in the nasal passages, trachea, and lungs. The mucus produced in the nasal passages contains a mix of antimicrobial molecules similar to those found in tears and saliva (e.g., lysozyme, lactoferrin, lactoperoxidase). Secretions in the trachea and lungs also contain lysozyme and lactoferrin, as well as a diverse group of additional chemical mediators, such as the lipoprotein complex called surfactant, which has antibacterial properties.
Exercise \(1\)
1. Explain the difference between endogenous and exogenous mediators
2. Describe how pH affects antimicrobial defenses
Antimicrobial Peptides
The antimicrobial peptides (AMPs) are a special class of nonspecific cell-derived mediators with broad-spectrum antimicrobial properties. Some AMPs are produced routinely by the body, whereas others are primarily produced (or produced in greater quantities) in response to the presence of an invading pathogen. Research has begun exploring how AMPs can be used in the diagnosis and treatment of disease.
AMPs may induce cell damage in microorganisms in a variety of ways, including by inflicting damage to membranes, destroying DNA and RNA, or interfering with cell-wall synthesis. Depending on the specific antimicrobial mechanism, a particular AMP may inhibit only certain groups of microbes (e.g., gram-positive or gram-negative bacteria) or it may be more broadly effective against bacteria, fungi, protozoa, and viruses. Many AMPs are found on the skin, but they can also be found in other regions of the body.
A family of AMPs called defensins can be produced by epithelial cells throughout the body as well as by cellular defenses such as macrophages and neutrophils (see Cellular Defenses). Defensins may be secreted or act inside host cells; they combat microorganisms by damaging their plasma membranes. AMPs called bacteriocins are produced exogenously by certain members of the resident microbiota within the gastrointestinal tract. The genes coding for these types of AMPs are often carried on plasmids and can be passed between different species within the resident microbiota through lateral or horizontal gene transfer.
There are numerous other AMPs throughout the body. The characteristics of a few of the more significant AMPs are summarized in Table \(1\).
Table \(1\): Characteristics of Selected Antimicrobial Peptides (AMPs)
AMP Secreted by Body site Pathogens inhibited Mode of action
Bacteriocins Resident microbiota Gastrointestinal tract Bacteria Disrupt membrane
Cathelicidin Epithelial cells, macrophages, and other cell types Skin Bacteria and fungi Disrupts membrane
Defensins Epithelial cells, macrophages, neutrophils Throughout the body Fungi, bacteria, and many viruses Disrupt membrane
Dermicidin Sweat glands Skin Bacteria and fungi Disrupts membrane integrity and ion channels
Histatins Salivary glands Oral cavity Fungi Disrupt intracellular function
Exercise \(2\)
Why are antimicrobial peptides (AMPs) considered nonspecific defenses?
Plasma Protein Mediators
Many nonspecific innate immune factors are found in plasma, the fluid portion of blood. Plasma contains electrolytes, sugars, lipids, and proteins, each of which helps to maintain homeostasis (i.e., stable internal body functioning), and contains the proteins involved in the clotting of blood. Additional proteins found in blood plasma, such as acute-phase proteins, complement proteins, and cytokines, are involved in the nonspecific innate immune response.
Plasma versus Serum
There are two terms for the fluid portion of blood: plasma and serum. How do they differ if they are both fluid and lack cells? The fluid portion of blood left over after coagulation (blood cell clotting) has taken place is serum. Although molecules such as many vitamins, electrolytes, certain sugars, complement proteins, and antibodies are still present in serum, clotting factors are largely depleted. Plasma, conversely, still contains all the clotting elements. To obtain plasma from blood, an anticoagulant must be used to prevent clotting. Examples of anticoagulants include heparin and ethylene diamine tetraacetic acid (EDTA). Because clotting is inhibited, once obtained, the sample must be gently spun down in a centrifuge. The heavier, denser blood cells form a pellet at the bottom of a centrifuge tube, while the fluid plasma portion, which is lighter and less dense, remains above the cell pellet.
Acute-Phase Proteins
The acute-phase proteins are another class of antimicrobial mediators. Acute-phase proteins are primarily produced in the liver and secreted into the blood in response to inflammatory molecules from the immune system. Examples of acute-phase proteins include C-reactive protein, serum amyloid A, ferritin, transferrin, fibrinogen, and mannose-binding lectin. Each of these proteins has a different chemical structure and inhibits or destroys microbes in some way (Table \(1\)).
Table \(2\): Some Acute-Phase Proteins and Their Functions
Some Acute-Phase Proteins and Their Functions
C-reactive protein Coats bacteria (opsonization), preparing them for ingestion by phagocytes
Serum amyloid A
Ferritin Bind and sequester iron, thereby inhibiting the growth of pathogens
Transferrin
Fibrinogen Involved in formation of blood clots that trap bacterial pathogens
Mannose-binding lectin Activates complement cascade
The Complement System
The complement system is a group of plasma protein mediators that can act as an innate nonspecific defense while also serving to connect innate and adaptive immunity (discussed in the next chapter). The complement system is composed of more than 30 proteins (including C1 through C9) that normally circulate as precursor proteins in blood. These precursor proteins become activated when stimulated or triggered by a variety of factors, including the presence of microorganisms. Complement proteins are considered part of innate nonspecific immunity because they are always present in the blood and tissue fluids, allowing them to be activated quickly. Also, when activated through the alternative pathway (described later in this section), complement proteins target pathogens in a nonspecific manner.
The process by which circulating complement precursors become functional is called complement activation. This process is a cascade that can be triggered by one of three different mechanisms, known as the alternative, classical, and lectin pathways.
The alternative pathway is initiated by the spontaneous activation of the complement protein C3. The hydrolysis of C3 produces two products, C3a and C3b. When no invader microbes are present, C3b is very quickly degraded in a hydrolysis reaction using the water in the blood. However, if invading microbes are present, C3b attaches to the surface of these microbes. Once attached, C3b will recruit other complement proteins in a cascade (Figure \(2\)).
The classical pathway provides a more efficient mechanism of activating the complement cascade, but it depends upon the production of antibodies by the specific adaptive immune defenses. To initiate the classical pathway, a specific antibody must first bind to the pathogen to form an antibody-antigen complex. This activates the first protein in the complement cascade, the C1 complex. The C1 complex is a multipart protein complex, and each component participates in the full activation of the overall complex. Following recruitment and activation of the C1 complex, the remaining classical pathway complement proteins are recruited and activated in a cascading sequence (Figure \(2\)).
The lectin activation pathway is similar to the classical pathway, but it is triggered by the binding of mannose-binding lectin, an acute-phase protein, to carbohydrates on the microbial surface. Like other acute-phase proteins, lectins are produced by liver cells and are commonly upregulated in response to inflammatory signals received by the body during an infection (Figure \(2\)).
Although each complement activation pathway is initiated in a different way, they all provide the same protective outcomes: opsonization, inflammation, chemotaxis, and cytolysis. The term opsonization refers to the coating of a pathogen by a chemical substance (called an opsonin) that allows phagocytic cells to recognize, engulf, and destroy it more easily. Opsonins from the complement cascade include C1q, C3b, and C4b. Additional important opsonins include mannose-binding proteins and antibodies. The complement fragments C3a and C5a are well-characterized anaphylatoxins with potent proinflammatory functions. Anaphylatoxins activate mast cells, causing degranulation and the release of inflammatory chemical signals, including mediators that cause vasodilation and increased vascular permeability. C5a is also one of the most potent chemoattractants for neutrophils and other white blood cells, cellular defenses that will be discussed in the next section.
The complement proteins C6, C7, C8, and C9 assemble into a membrane attack complex (MAC), which allows C9 to polymerize into pores in the membranes of gram-negative bacteria. These pores allow water, ions, and other molecules to move freely in and out of the targeted cells, eventually leading to cell lysis and death of the pathogen (Figure \(2\)). However, the MAC is only effective against gram-negative bacteria; it cannot penetrate the thick layer of peptidoglycan associated with cell walls of gram-positive bacteria. Since the MAC does not pose a lethal threat to gram-positive bacterial pathogens, complement-mediated opsonization is more important for their clearance.
Cytokines
Cytokines are soluble proteins that act as communication signals between cells. In a nonspecific innate immune response, various cytokines may be released to stimulate production of chemical mediators or other cell functions, such as cell proliferation, cell differentiation, inhibition of cell division, apoptosis, and chemotaxis.
When a cytokine binds to its target receptor, the effect can vary widely depending on the type of cytokine and the type of cell or receptor to which it has bound. The function of a particular cytokine can be described as autocrine, paracrine, or endocrine (Figure \(3\)). In autocrine function, the same cell that releases the cytokine is the recipient of the signal; in other words, autocrine function is a form of self-stimulation by a cell. In contrast, paracrine function involves the release of cytokines from one cell to other nearby cells, stimulating some response from the recipient cells. Last, endocrine function occurs when cells release cytokines into the bloodstream to be carried to target cells much farther away.
Three important classes of cytokines are the interleukins, chemokines, and interferons. The interleukins were originally thought to be produced only by leukocytes (white blood cells) and to only stimulate leukocytes, thus the reasons for their name. Although interleukins are involved in modulating almost every function of the immune system, their role in the body is not restricted to immunity. Interleukins are also produced by and stimulate a variety of cells unrelated to immune defenses.
The chemokines are chemotactic factors that recruit leukocytes to sites of infection, tissue damage, and inflammation. In contrast to more general chemotactic factors, like complement factor C5a, chemokines are very specific in the subsets of leukocytes they recruit.
Interferons are a diverse group of immune signaling molecules and are especially important in our defense against viruses. Type I interferons (interferon-α and interferon-β) are produced and released by cells infected with virus. These interferons stimulate nearby cells to stop production of mRNA, destroy RNA already produced, and reduce protein synthesis. These cellular changes inhibit viral replication and production of mature virus, slowing the spread of the virus. Type I interferons also stimulate various immune cells involved in viral clearance to more aggressively attack virus-infected cells. Type II interferon (interferon-γ) is an important activator of immune cells (Figure \(4\)).
Inflammation-Eliciting Mediators
Many of the chemical mediators discussed in this section contribute in some way to inflammation and fever, which are nonspecific immune responses discussed in more detail in Inflammation and Fever. Cytokines stimulate the production of acute-phase proteins such as C-reactive protein and mannose-binding lectin in the liver. These acute-phase proteins act as opsonins, activating complement cascades through the lectin pathway.
Some cytokines also bind mast cells and basophils, inducing them to release histamine, a proinflammatory compound. Histamine receptors are found on a variety of cells and mediate proinflammatory events, such as bronchoconstriction (tightening of the airways) and smooth muscle contraction.
In addition to histamine, mast cells may release other chemical mediators, such as leukotrienes. Leukotrienes are lipid-based proinflammatory mediators that are produced from the metabolism of arachidonic acid in the cell membrane of leukocytes and tissue cells. Compared with the proinflammatory effects of histamine, those of leukotrienes are more potent and longer lasting. Together, these chemical mediators can induce coughing, vomiting, and diarrhea, which serve to expel pathogens from the body.
Certain cytokines also stimulate the production of prostaglandins, chemical mediators that promote the inflammatory effects of kinins and histamines. Prostaglandins can also help to set the body temperature higher, leading to fever, which promotes the activities of white blood cells and slightly inhibits the growth of pathogenic microbes (see Inflammation and Fever).
Another inflammatory mediator, bradykinin, contributes to edema, which occurs when fluids and leukocytes leak out of the bloodstream and into tissues. It binds to receptors on cells in the capillary walls, causing the capillaries to dilate and become more permeable to fluids.
Exercise \(3\)
1. What do the three complement activation pathways have in common?
2. Explain autocrine, paracrine, and endocrine signals.
3. Name two important inflammation-eliciting mediators.
Clinical Focus: Part 2
To relieve the constriction of her airways, Angela is immediately treated with antihistamines and administered corticosteroids through an inhaler, and then monitored for a period of time. Though her condition does not worsen, the drugs do not seem to be alleviating her condition. She is admitted to the hospital for further observation, testing, and treatment.
Following admission, a clinician conducts allergy testing to try to determine if something in her environment might be triggering an allergic inflammatory response. A doctor orders blood analysis to check for levels of particular cytokines. A sputum sample is also taken and sent to the lab for microbial staining, culturing, and identification of pathogens that could be causing an infection.
Exercise \(4\)
1. Which aspects of the innate immune system could be contributing to Angela’s airway constriction?
2. Why was Angela treated with antihistamines?
3. Why would the doctor be interested in levels of cytokines in Angela’s blood?
Table \(3\) provides a summary of the chemical defenses discussed in this section.
Table \(3\): Chemical Defenses of Nonspecific Innate Immunity
Defense Examples Function
Chemicals and enzymes in body fluids Sebum from sebaceous glands Provides oil barrier protecting hair follicle pores from pathogens
Oleic acid from sebum and skin microbiota Lowers pH to inhibit pathogens
Lysozyme in secretions Kills bacteria by attacking cell wall
Acid in stomach, urine, and vagina Inhibits or kills bacteria
Digestive enzymes and bile Kill bacteria
Lactoferrin and transferrin Bind and sequester iron, inhibiting bacterial growth
Surfactant in lungs Kills bacteria
Antimicrobial peptides Defensins, bacteriocins, dermicidin, cathelicidin, histatins, Kill bacteria by attacking membranes or interfering with cell functions
Plasma protein mediators Acute-phase proteins (C-reactive protein, serum amyloid A, ferritin, fibrinogen, transferrin, and mannose-binding lectin) Inhibit the growth of bacteria and assist in the trapping and killing of bacteria
Complements C3b and C4b Opsonization of pathogens to aid phagocytosis
Complement C5a Chemoattractant for phagocytes
Complements C3a and C5a Proinflammatory anaphylatoxins
Cytokines Interleukins Stimulate and modulate most functions of immune system
Chemokines Recruit white blood cells to infected area
Interferons Alert cells to viral infection, induce apoptosis of virus-infected cells, induce antiviral defenses in infected and nearby uninfected cells, stimulate immune cells to attack virus-infected cells
Inflammation-eliciting mediators Histamine Promotes vasodilation, bronchoconstriction, smooth muscle contraction, increased secretion and mucus production
Leukotrienes Promote inflammation; stronger and longer lasting than histamine
Prostaglandins Promote inflammation and fever
Bradykinin Increases vasodilation and vascular permeability, leading to edema
Key Concepts and Summary
• Numerous chemical mediators produced endogenously and exogenously exhibit nonspecific antimicrobial functions.
• Many chemical mediators are found in body fluids such as sebum, saliva, mucus, gastric and intestinal fluids, urine, tears, cerumen, and vaginal secretions.
• Antimicrobial peptides (AMPs) found on the skin and in other areas of the body are largely produced in response to the presence of pathogens. These include dermcidin, cathelicidin, defensins, histatins, and bacteriocins.
• Plasma contains various proteins that serve as chemical mediators, including acute-phase proteins, complement proteins, and cytokines.
• The complement system involves numerous precursor proteins that circulate in plasma. These proteins become activated in a cascading sequence in the presence of microbes, resulting in the opsonization of pathogens, chemoattraction of leukocytes, induction of inflammation, and cytolysis through the formation of a membrane attack complex (MAC).
• Cytokines are proteins that facilitate various nonspecific responses by innate immune cells, including production of other chemical mediators, cell proliferation, cell death, and differentiation.
• Cytokines play a key role in the inflammatory response, triggering production of inflammation-eliciting mediators such as acute-phase proteins, histamine, leukotrienes, prostaglandins, and bradykinin. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/17%3A_Innate_Nonspecific_Host_Defenses/17.02%3A_Chemical_Defenses.txt |
Learning Objectives
• Identify and describe the components of blood
• Explain the process by which the formed elements of blood are formed (hematopoiesis)
• Describe the characteristics of formed elements found in peripheral blood, as well as their respective functions within the innate immune system
In the previous section, we discussed some of the chemical mediators found in plasma, the fluid portion of blood. The nonfluid portion of blood consists of various types of formed elements, so called because they are all formed from the same stem cells found in bone marrow. The three major categories of formed elements are: red blood cells (RBCs), also called erythrocytes; platelets, also called thrombocytes; and white blood cells (WBCs), also called leukocytes.
Red blood cells are primarily responsible for carrying oxygen to tissues. Platelets are cellular fragments that participate in blood clot formation and tissue repair. Several different types of WBCs participate in various nonspecific mechanisms of innate and adaptive immunity. In this section, we will focus primarily on the innate mechanisms of various types of WBCs.
Hematopoiesis
All of the formed elements of blood are derived from pluripotent hematopoietic stem cells (HSCs) in the bone marrow. As the HSCs make copies of themselves in the bone marrow, individual cells receive different cues from the body that control how they develop and mature. As a result, the HSCs differentiate into different types of blood cells that, once mature, circulate in peripheral blood. This process of differentiation, called hematopoiesis, is shown in more detail in Figure \(1\).
In terms of sheer numbers, the vast majority of HSCs become erythrocytes. Much smaller numbers become leukocytes and platelets. Leukocytes can be further subdivided into granulocytes, which are characterized by numerous granules visible in the cytoplasm, and agranulocytes, which lack granules. Figure \(2\) provides an overview of the various types of formed elements, including their relative numbers, primary function, and lifespans.
Granulocytes
The various types of granulocytes can be distinguished from one another in a blood smear by the appearance of their nuclei and the contents of their granules, which confer different traits, functions, and staining properties. The neutrophils, also called polymorphonuclear neutrophils (PMNs), have a nucleus with three to five lobes and small, numerous, lilac-colored granules. Each lobe of the nucleus is connected by a thin strand of material to the other lobes. The eosinophils have fewer lobes in the nucleus (typically 2–3) and larger granules that stain reddish-orange. The basophils have a two-lobed nucleus and large granules that stain dark blue or purple (Figure \(3\)).
Neutrophils (PMNs)
Neutrophils (PMNs) are frequently involved in the elimination and destruction of extracellular bacteria. They are capable of migrating through the walls of blood vessels to areas of bacterial infection and tissue damage, where they seek out and kill infectious bacteria. PMN granules contain a variety of defensins and hydrolytic enzymes that help them destroy bacteria through phagocytosis (described in more detail in Pathogen Recognition and Phagocytosis) In addition, when many neutrophils are brought into an infected area, they can be stimulated to release toxic molecules into the surrounding tissue to better clear infectious agents. This is called degranulation.
Another mechanism used by neutrophils is neutrophil extracellular traps (NETs), which are extruded meshes of chromatin that are closely associated with antimicrobial granule proteins and components. Chromatin is DNA with associated proteins (usually histone proteins, around which DNA wraps for organization and packing within a cell). By creating and releasing a mesh or lattice-like structure of chromatin that is coupled with antimicrobial proteins, the neutrophils can mount a highly concentrated and efficient attack against nearby pathogens. Proteins frequently associated with NETs include lactoferrin, gelatinase, cathepsin G, and myeloperoxidase. Each has a different means of promoting antimicrobial activity, helping neutrophils eliminate pathogens. The toxic proteins in NETs may kill some of the body’s own cells along with invading pathogens. However, this collateral damage can be repaired after the danger of the infection has been eliminated.
As neutrophils fight an infection, a visible accumulation of leukocytes, cellular debris, and bacteria at the site of infection can be observed. This buildup is what we call pus (also known as purulent or suppurative discharge or drainage). The presence of pus is a sign that the immune defenses have been activated against an infection; historically, some physicians believed that inducing pus formation could actually promote the healing of wounds. The practice of promoting “laudable pus” (by, for instance, wrapping a wound in greasy wool soaked in wine) dates back to the ancient physician Galen in the 2nd century AD, and was practiced in variant forms until the 17th century (though it was not universally accepted). Today, this method is no longer practiced because we now know that it is not effective. Although a small amount of pus formation can indicate a strong immune response, artificially inducing pus formation does not promote recovery.
Eosinophils
Eosinophils are granulocytes that protect against protozoa and helminths; they also play a role in allergic reactions. The granules of eosinophils, which readily absorb the acidic reddish dye eosin, contain histamine, degradative enzymes, and a compound known as major basic protein (MBP) (Figure \(3\)). MBP binds to the surface carbohydrates of parasites, and this binding is associated with disruption of the cell membrane and membrane permeability.
Basophils
Basophils have cytoplasmic granules of varied size and are named for their granules’ ability to absorb the basic dye methylene blue (Figure \(3\)). Their stimulation and degranulation can result from multiple triggering events. Activated complement fragments C3a and C5a, produced in the activation cascades of complement proteins, act as anaphylatoxins by inducing degranulation of basophils and inflammatory responses. This cell type is important in allergic reactions and other responses that involve inflammation. One of the most abundant components of basophil granules is histamine, which is released along with other chemical factors when the basophil is stimulated. These chemicals can be chemotactic and can help to open the gaps between cells in the blood vessels. Other mechanisms for basophil triggering require the assistance of antibodies, as discussed in B Lymphocytes and Humoral Immunity.
Mast Cells
Hematopoiesis also gives rise to mast cells, which appear to be derived from the same common myeloid progenitor cell as neutrophils, eosinophils, and basophils. Functionally, mast cells are very similar to basophils, containing many of the same components in their granules (e.g., histamine) and playing a similar role in allergic responses and other inflammatory reactions. However, unlike basophils, mast cells leave the circulating blood and are most frequently found residing in tissues. They are often associated with blood vessels and nerves or found close to surfaces that interface with the external environment, such as the skin and mucous membranes in various regions of the body (Figure \(4\)).
Exercise \(1\)
1. Describe the granules and nuclei of neutrophils, eosinophils, basophils, and mast cells.
2. Name three antimicrobial mechanisms of neutrophils
Clinical Focus: Part 3
Angela’s tests come back negative for all common allergens, and her sputum samples contain no abnormal presence of pathogenic microbes or elevated levels of members of the normal respiratory microbiota. She does, however, have elevated levels of inflammatory cytokines in her blood.
The swelling of her airway has still not responded to treatment with antihistamines or corticosteroids. Additional blood work shows that Angela has a mildly elevated white blood cell count but normal antibody levels. Also, she has a lower-than-normal level of the complement protein C4.
Exercise \(2\)
1. What does this new information reveal about the cause of Angela’s constricted airways?
2. What are some possible conditions that could lead to low levels of complement proteins?
Agranulocytes
As their name suggests, agranulocytes lack visible granules in the cytoplasm. Agranulocytes can be categorized as lymphocytes or monocytes (Figure \(2\)). Among the lymphocytes are natural killer cells, which play an important role in nonspecific innate immune defenses. Lymphocytes also include the B cells and T cells, which are discussed in the next chapter because they are central players in the specific adaptive immune defenses. The monocytes differentiate into macrophages and dendritic cells, which are collectively referred to as the mononuclear phagocyte system.
Natural Killer Cells
Most lymphocytes are primarily involved in the specific adaptive immune response, and thus will be discussed in the following chapter. An exception is the natural killer cells (NK cells); these mononuclear lymphocytes use nonspecific mechanisms to recognize and destroy cells that are abnormal in some way. Cancer cells and cells infected with viruses are two examples of cellular abnormalities that are targeted by NK cells. Recognition of such cells involves a complex process of identifying inhibitory and activating molecular markers on the surface of the target cell. Molecular markers that make up the major histocompatibility complex (MHC) are expressed by healthy cells as an indication of “self.” This will be covered in more detail in next chapter. NK cells are able to recognize normal MHC markers on the surface of healthy cells, and these MHC markers serve as an inhibitory signal preventing NK cell activation. However, cancer cells and virus-infected cells actively diminish or eliminate expression of MHC markers on their surface. When these MHC markers are diminished or absent, the NK cell interprets this as an abnormality and a cell in distress. This is one part of the NK cell activation process (Figure \(5\)). NK cells are also activated by binding to activating molecular molecules on the target cell. These activating molecular molecules include “altered self” or “nonself” molecules. When a NK cell recognizes a decrease in inhibitory normal MHC molecules and an increase in activating molecules on the surface of a cell, the NK cell will be activated to eliminate the cell in distress.
Once a cell has been recognized as a target, the NK cell can use several different mechanisms to kill its target. For example, it may express cytotoxic membrane proteins and cytokines that stimulate the target cell to undergo apoptosis, or controlled cell suicide. NK cells may also use perforin-mediated cytotoxicity to induce apoptosis in target cells. This mechanism relies on two toxins released from granules in the cytoplasm of the NK cell: perforin, a protein that creates pores in the target cell, and granzymes, proteases that enter through the pores into the target cell’s cytoplasm, where they trigger a cascade of protein activation that leads to apoptosis. The NK cell binds to the abnormal target cell, releases its destructive payload, and detaches from the target cell. While the target cell undergoes apoptosis, the NK cell synthesizes more perforin and proteases to use on its next target.
NK cells contain these toxic compounds in granules in their cytoplasm. When stained, the granules are azurophilic and can be visualized under a light microscope (Figure \(6\)). Even though they have granules, NK cells are not considered granulocytes because their granules are far less numerous than those found in true granulocytes. Furthermore, NK cells have a different lineage than granulocytes, arising from lymphoid rather than myeloid stem cells (Figure \(1\)).
Monocytes
The largest of the white blood cells, monocytes have a nucleus that lacks lobes, and they also lack granules in the cytoplasm (Figure \(7\)). Nevertheless, they are effective phagocytes, engulfing pathogens and apoptotic cells to help fight infection.
When monocytes leave the bloodstream and enter a specific body tissue, they differentiate into tissue-specific phagocytes called macrophages and dendritic cells. They are particularly important residents of lymphoid tissue, as well as nonlymphoid sites and organs. Macrophages and dendritic cells can reside in body tissues for significant lengths of time. Macrophages in specific body tissues develop characteristics suited to the particular tissue. Not only do they provide immune protection for the tissue in which they reside but they also support normal function of their neighboring tissue cells through the production of cytokines. Macrophages are given tissue-specific names, and a few examples of tissue-specific macrophages are listed in Table \(1\). Dendritic cells are important sentinels residing in the skin and mucous membranes, which are portals of entry for many pathogens. Monocytes, macrophages, and dendritic cells are all highly phagocytic and important promoters of the immune response through their production and release of cytokines. These cells provide an essential bridge between innate and adaptive immune responses, as discussed in the next section as well as the next chapter.
Table \(1\): Macrophages Found in Various Body Tissues
Tissue Macrophage
Brain and central nervous system Microglial cells
Liver Kupffer cells
Lungs Alveolar macrophages (dust cells)
Peritoneal cavity Peritoneal macrophages
Exercise \(3\)
1. Describe the signals that activate natural killer cells.
2. What is the difference between monocytes and macrophages?
Key Concepts and Summary
• The formed elements of the blood include red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Of these, leukocytes are primarily involved in the immune response.
• All formed elements originate in the bone marrow as stem cells (HSCs) that differentiate through hematopoiesis.
• Granulocytes are leukocytes characterized by a lobed nucleus and granules in the cytoplasm. These include neutrophils (PMNs), eosinophils, and basophils.
• Neutrophils are the leukocytes found in the largest numbers in the bloodstream and they primarily fight bacterial infections.
• Eosinophils target parasitic infections. Eosinophils and basophils are involved in allergic reactions. Both release histamine and other proinflammatory compounds from their granules upon stimulation.
• Mast cells function similarly to basophils but can be found in tissues outside the bloodstream.
• Natural killer (NK) cells are lymphocytes that recognize and kill abnormal or infected cells by releasing proteins that trigger apoptosis.
• Monocytes are large, mononuclear leukocytes that circulate in the bloodstream. They may leave the bloodstream and take up residence in body tissues, where they differentiate and become tissue-specific macrophages and dendritic cells. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/17%3A_Innate_Nonspecific_Host_Defenses/17.03%3A_Cellular_Defenses.txt |
Learning Objectives
• Explain how leukocytes migrate from peripheral blood into infected tissues
• Explain the mechanisms by which leukocytes recognize pathogens
• Explain the process of phagocytosis and the mechanisms by which phagocytes destroy and degrade pathogens
Several of the cell types discussed in the previous section can be described as phagocytes—cells whose main function is to seek, ingest, and kill pathogens. This process, called phagocytosis, was first observed in starfish in the 1880s by Nobel Prize-winning zoologist Ilya Metchnikoff (1845–1916), who made the connection to white blood cells (WBCs) in humans and other animals. At the time, Pasteur and other scientists believed that WBCs were spreading pathogens rather than killing them (which is true for some diseases, such as tuberculosis). But in most cases, phagocytes provide a strong, swift, and effective defense against a broad range of microbes, making them a critical component of innate nonspecific immunity. This section will focus on the mechanisms by which phagocytes are able to seek, recognize, and destroy pathogens.
Extravasation (Diapedesis) of Leukocytes
Some phagocytes are leukocytes (WBCs) that normally circulate in the bloodstream. To reach pathogens located in infected tissue, leukocytes must pass through the walls of small capillary blood vessels within tissues. This process, called extravasation, or diapedesis, is initiated by complement factor C5a, as well as cytokines released into the immediate vicinity by resident macrophages and tissue cells responding to the presence of the infectious agent (Figure \(1\)). Similar to C5a, many of these cytokines are proinflammatory and chemotactic, and they bind to cells of small capillary blood vessels, initiating a response in the endothelial cells lining the inside of the blood vessel walls. This response involves the upregulation and expression of various cellular adhesion molecules and receptors. Leukocytes passing through will stick slightly to the adhesion molecules, slowing down and rolling along the blood vessel walls near the infected area. When they reach a cellular junction, they will bind to even more of these adhesion molecules, flattening out and squeezing through the cellular junction in a process known as transendothelial migration. This mechanism of “rolling adhesion” allows leukocytes to exit the bloodstream and enter the infected areas, where they can begin phagocytosing the invading pathogens.
Note that extravasation does not occur in arteries or veins. These blood vessels are surrounded by thicker, multilayer protective walls, in contrast to the thin single-cell-layer walls of capillaries. Furthermore, the blood flow in arteries is too turbulent to allow for rolling adhesion. Also, some leukocytes tend to respond to an infection more quickly than others. The first to arrive typically are neutrophils, often within hours of a bacterial infection. By contract, monocytes may take several days to leave the bloodstream and differentiate into macrophages.
Link to Learning
Watch the following videos on leukocyte extravasation and leukocyte rolling to learn more.
Exercise \(1\)
Explain the role of adhesion molecules in the process of extravasation.
Pathogen Recognition
As described in the previous section, opsonization of pathogens by antibody; complement factors C1q, C3b, and C4b; and lectins can assist phagocytic cells in recognition of pathogens and attachment to initiate phagocytosis. However, not all pathogen recognition is opsonin dependent. Phagocytes can also recognize molecular structures that are common to many groups of pathogenic microbes. Such structures are called pathogen-associated molecular patterns (PAMPs). Common PAMPs include the following:
• peptidoglycan, found in bacterial cell walls;
• flagellin, a protein found in bacterial flagella;
• lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria;
• lipopeptides, molecules expressed by most bacteria; and
• nucleic acids such as viral DNA or RNA.
Like numerous other PAMPs, these substances are integral to the structure of broad classes of microbes.
The structures that allow phagocytic cells to detect PAMPs are called pattern recognition receptors (PRRs). One group of PRRs is the toll-like receptors (TLRs), which bind to various PAMPs and communicate with the nucleus of the phagocyte to elicit a response. Many TLRs (and other PRRs) are located on the surface of a phagocyte, but some can also be found embedded in the membranes of interior compartments and organelles (Figure \(2\)). These interior PRRs can be useful for the binding and recognition of intracellular pathogens that may have gained access to the inside of the cell before phagocytosis could take place. Viral nucleic acids, for example, might encounter an interior PRR, triggering production of the antiviral cytokine interferon.
In addition to providing the first step of pathogen recognition, the interaction between PAMPs and PRRs on macrophages provides an intracellular signal that activates the phagocyte, causing it to transition from a dormant state of readiness and slow proliferation to a state of hyperactivity, proliferation, production/secretion of cytokines, and enhanced intracellular killing. PRRs on macrophages also respond to chemical distress signals from damaged or stressed cells. This allows macrophages to extend their responses beyond protection from infectious diseases to a broader role in the inflammatory response initiated from injuries or other diseases.
Exercise \(2\)
1. Name four pathogen-associated molecular patterns (PAMPs).
2. Describe the process of phagocyte activation.
Pathogen Degradation
Once pathogen recognition and attachment occurs, the pathogen is engulfed in a vesicle and brought into the internal compartment of the phagocyte in a process called phagocytosis (Figure \(3\)). PRRs can aid in phagocytosis by first binding to the pathogen’s surface, but phagocytes are also capable of engulfing nearby items even if they are not bound to specific receptors. To engulf the pathogen, the phagocyte forms a pseudopod that wraps around the pathogen and then pinches it off into a membrane vesicle called a phagosome. Acidification of the phagosome (pH decreases to the range of 4–5) provides an important early antibacterial mechanism. The phagosome containing the pathogen fuses with one or more lysosomes, forming a phagolysosome. Formation of the phagolysosome enhances the acidification, which is essential for activation of pH-dependent digestive lysosomal enzymes and production of hydrogen peroxide and toxic reactive oxygen species. Lysosomal enzymes such as lysozyme, phospholipase, and proteases digest the pathogen. Other enzymes are involved a respiratory burst. During the respiratory burst, phagocytes will increase their uptake and consumption of oxygen, but not for energy production. The increased oxygen consumption is focused on the production of superoxide anion, hydrogen peroxide, hydroxyl radicals, and other reactive oxygen species that are antibacterial.
In addition to the reactive oxygen species produced by the respiratory burst, reactive nitrogen compounds with cytotoxic (cell-killing) potential can also form. For example, nitric oxide can react with superoxide to form peroxynitrite, a highly reactive nitrogen compound with degrading capabilities similar to those of the reactive oxygen species. Some phagocytes even contain an internal storehouse of microbicidal defensin proteins (e.g., neutrophil granules). These destructive forces can be released into the area around the cell to degrade microbes externally. Neutrophils, especially, can be quite efficient at this secondary antimicrobial mechanism.
Once degradation is complete, leftover waste products are excreted from the cell in an exocytic vesicle. However, it is important to note that not all remains of the pathogen are excreted as waste. Macrophages and dendritic cells are also antigen-presenting cells involved in the specific adaptive immune response. These cells further process the remains of the degraded pathogen and present key antigens (specific pathogen proteins) on their cellular surface. This is an important step for stimulation of some adaptive immune responses, as will be discussed in more detail in the next chapter.
Link to Learning
Visit this link to view a phagocyte chasing and engulfing a pathogen.
Exercise \(3\)
What is the difference between a phagosome and a lysosome?
When Phagocytosis Fails
Although phagocytosis successfully destroys many pathogens, some are able to survive and even exploit this defense mechanism to multiply in the body and cause widespread infection. Protozoans of the genus Leishmania are one example. These obligate intracellular parasites are flagellates transmitted to humans by the bite of a sand fly. Infections cause serious and sometimes disfiguring sores and ulcers in the skin and other tissues (Figure \(4\)). Worldwide, an estimated 1.3 million people are newly infected with leishmaniasis annually.1
Salivary peptides from the sand fly activate host macrophages at the site of their bite. The classic or alternate pathway for complement activation ensues with C3b opsonization of the parasite. Leishmania cells are phagocytosed, lose their flagella, and multiply in a form known as an amastigote (Leishman-Donovan body) within the phagolysosome. Although many other pathogens are destroyed in the phagolysosome, survival of the Leishmania amastigotes is maintained by the presence of surface lipophosphoglycan and acid phosphatase. These substances inhibit the macrophage respiratory burst and lysosomal enzymes. The parasite then multiplies inside the cell and lyses the infected macrophage, releasing the amastigotes to infect other macrophages within the same host. Should another sand fly bite an infected person, it might ingest amastigotes and then transmit them to another individual through another bite.
There are several different forms of leishmaniasis. The most common is a localized cutaneous form of the illness caused by L. tropica, which typically resolves spontaneously over time but with some significant lymphocyte infiltration and permanent scarring. A mucocutaneous form of the disease, caused by L. viannia brasilienfsis, produces lesions in the tissue of the nose and mouth and can be life threatening. A visceral form of the illness can be caused by several of the different Leishmania species. It affects various organ systems and causes abnormal enlargement of the liver and spleen. Irregular fevers, anemia, liver dysfunction, and weight loss are all signs and symptoms of visceral leishmaniasis. If left untreated, it is typically fatal.
Key Concepts and Summary
• Phagocytes are cells that recognize pathogens and destroy them through phagocytosis.
• Recognition often takes place by the use of phagocyte receptors that bind molecules commonly found on pathogens, known as pathogen-associated molecular patterns (PAMPs).
• The receptors that bind PAMPs are called pattern recognition receptors, or PRRs. Toll-like receptors (TLRs) are one type of PRR found on phagocytes.
• Extravasation of white blood cells from the bloodstream into infected tissue occurs through the process of transendothelial migration.
• Phagocytes degrade pathogens through phagocytosis, which involves engulfing the pathogen, killing and digesting it within a phagolysosome, and then excreting undigested matter.
Footnotes
1. 1 World Health Organization. “Leishmaniasis.” 2016. http://www.who.int/mediacentre/factsheets/fs375/en/. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/17%3A_Innate_Nonspecific_Host_Defenses/17.04%3A_Pathogen_Recognition_and_Phagocytosis.txt |
Learning Objectives
• Identify the signs of inflammation and fever and explain why they occur
• Explain the advantages and risks posed by inflammatory responses
The inflammatory response, or inflammation, is triggered by a cascade of chemical mediators and cellular responses that may occur when cells are damaged and stressed or when pathogens successfully breach the physical barriers of the innate immune system. Although inflammation is typically associated with negative consequences of injury or disease, it is a necessary process insofar as it allows for recruitment of the cellular defenses needed to eliminate pathogens, remove damaged and dead cells, and initiate repair mechanisms. Excessive inflammation, however, can result in local tissue damage and, in severe cases, may even become deadly.
Acute Inflammation
An early, if not immediate, response to tissue injury is acute inflammation. Immediately following an injury, vasoconstriction of blood vessels will occur to minimize blood loss. The amount of vasoconstriction is related to the amount of vascular injury, but it is usually brief. Vasoconstriction is followed by vasodilation and increased vascular permeability, as a direct result of the release of histamine from resident mast cells. Increased blood flow and vascular permeability can dilute toxins and bacterial products at the site of injury or infection. They also contribute to the five observable signs associated with the inflammatory response: erythema (redness), edema (swelling), heat, pain, and altered function. Vasodilation and increased vascular permeability are also associated with an influx of phagocytes at the site of injury and/or infection. This can enhance the inflammatory response because phagocytes may release proinflammatory chemicals when they are activated by cellular distress signals released from damaged cells, by PAMPs, or by opsonins on the surface of pathogens. Activation of the complement system can further enhance the inflammatory response through the production of the anaphylatoxin C5a. Figure \(1\) illustrates a typical case of acute inflammation at the site of a skin wound.
During the period of inflammation, the release of bradykinin causes capillaries to remain dilated, flooding tissues with fluids and leading to edema. Increasing numbers of neutrophils are recruited to the area to fight pathogens. As the fight rages on, pus forms from the accumulation of neutrophils, dead cells, tissue fluids, and lymph. Typically, after a few days, macrophages will help to clear out this pus. Eventually, tissue repair can begin in the wounded area.
Chronic Inflammation
When acute inflammation is unable to clear an infectious pathogen, chronic inflammation may occur. This often results in an ongoing (and sometimes futile) lower-level battle between the host organism and the pathogen. The wounded area may heal at a superficial level, but pathogens may still be present in deeper tissues, stimulating ongoing inflammation. Additionally, chronic inflammation may be involved in the progression of degenerative neurological diseases such as Alzheimer’s and Parkinson’s, heart disease, and metastatic cancer.
Chronic inflammation may lead to the formation of granulomas, pockets of infected tissue walled off and surrounded by WBCs. Macrophages and other phagocytes wage an unsuccessful battle to eliminate the pathogens and dead cellular materials within a granuloma. One example of a disease that produces chronic inflammation is tuberculosis, which results in the formation of granulomas in lung tissues. A tubercular granuloma is called a tubercle (Figure \(2\)). Tuberculosis will be covered in more detail in Bacterial Infections of the Respiratory Tract.
Chronic inflammation is not just associated with bacterial infections. Chronic inflammation can be an important cause of tissue damage from viral infections. The extensive scarring observed with hepatitis C infections and liver cirrhosis is the result of chronic inflammation.
Exercise \(1\)
1. Name the five signs of inflammation.
2. Is a granuloma an acute or chronic form of inflammation? Explain.
Chronic Edema
In addition to granulomas, chronic inflammation can also result in long-term edema. A condition known as lymphatic filariasis (also known as elephantiasis) provides an extreme example. Lymphatic filariasis is caused by microscopic nematodes (parasitic worms) whose larvae are transmitted between human hosts by mosquitoes. Adult worms live in the lymphatic vessels, where their presence stimulates infiltration by lymphocytes, plasma cells, eosinophils, and thrombocytes (a condition known as lymphangitis). Because of the chronic nature of the illness, granulomas, fibrosis, and blocking of the lymphatic system may eventually occur. Over time, these blockages may worsen with repeated infections over decades, leading to skin thickened with edema and fibrosis. Lymph (extracellular tissue fluid) may spill out of the lymphatic areas and back into tissues, causing extreme swelling (Figure \(3\)). Secondary bacterial infections commonly follow. Because it is a disease caused by a parasite, eosinophilia (a dramatic rise in the number of eosinophils in the blood) is characteristic of acute infection. However, this increase in antiparasite granulocytes is not sufficient to clear the infection in many cases.
Lymphatic filariasis affects an estimated 120 million people worldwide, mostly concentrated in Africa and Asia.1 Improved sanitation and mosquito control can reduce transmission rates.
Fever
A fever is an inflammatory response that extends beyond the site of infection and affects the entire body, resulting in an overall increase in body temperature. Body temperature is normally regulated and maintained by the hypothalamus, an anatomical section of the brain that functions to maintain homeostasis in the body. However, certain bacterial or viral infections can result in the production of pyrogens, chemicals that effectively alter the “thermostat setting” of the hypothalamus to elevate body temperature and cause fever. Pyrogens may be exogenous or endogenous. For example, the endotoxin lipopolysaccharide (LPS), produced by gram-negative bacteria, is an exogenous pyrogen that may induce the leukocytes to release endogenous pyrogens such as interleukin-1 (IL-1), IL-6, interferon-γ (IFN-γ), and tumor necrosis factor (TNF). In a cascading effect, these molecules can then lead to the release of prostaglandin E2 (PGE2) from other cells, resetting the hypothalamus to initiate fever (Figure \(4\)).
Like other forms of inflammation, a fever enhances the innate immune defenses by stimulating leukocytes to kill pathogens. The rise in body temperature also may inhibit the growth of many pathogens since human pathogens are mesophiles with optimum growth occurring around 35 °C (95 °F). In addition, some studies suggest that fever may also stimulate release of iron-sequestering compounds from the liver, thereby starving out microbes that rely on iron for growth.2
During fever, the skin may appear pale due to vasoconstriction of the blood vessels in the skin, which is mediated by the hypothalamus to divert blood flow away from extremities, minimizing the loss of heat and raising the core temperature. The hypothalamus will also stimulate shivering of muscles, another effective mechanism of generating heat and raising the core temperature.
The crisis phase occurs when the fever breaks. The hypothalamus stimulates vasodilation, resulting in a return of blood flow to the skin and a subsequent release of heat from the body. The hypothalamus also stimulates sweating, which cools the skin as the sweat evaporates.
Although a low-level fever may help an individual overcome an illness, in some instances, this immune response can be too strong, causing tissue and organ damage and, in severe cases, even death. The inflammatory response to bacterial superantigens is one scenario in which a life-threatening fever may develop. Superantigens are bacterial or viral proteins that can cause an excessive activation of T cells from the specific adaptive immune defense, as well as an excessive release of cytokines that overstimulates the inflammatory response. For example, Staphylococcus aureus and Streptococcus pyogenes are capable of producing superantigens that cause toxic shock syndrome and scarlet fever, respectively. Both of these conditions can be associated with very high, life-threatening fevers in excess of 42 °C (108 °F).
Exercise \(2\)
1. Explain the difference between exogenous and endogenous pyrogens.
2. How does a fever inhibit pathogens?
Clinical Focus: Resolution
Given her father’s premature death, Angela’s doctor suspects that she has hereditary angioedema, a genetic disorder that compromises the function of C1 inhibitor protein. Patients with this genetic abnormality may have occasional episodes of swelling in various parts of the body. In Angela’s case, the swelling has occurred in the respiratory tract, leading to difficulty breathing. Swelling may also occur in the gastrointestinal tract, causing abdominal cramping, diarrhea, and vomiting, or in the muscles of the face or limbs. This swelling may be nonresponsive to steroid treatment and is often misdiagnosed as an allergy.
Because there are three types of hereditary angioedema, the doctor orders a more specific blood test to look for levels of C1-INH, as well as a functional assay of Angela’s C1 inhibitors. The results suggest that Angela has type I hereditary angioedema, which accounts for 80%–85% of all cases. This form of the disorder is caused by a deficiency in C1 esterase inhibitors, the proteins that normally help suppress activation of the complement system. When these proteins are deficient or nonfunctional, overstimulation of the system can lead to production of inflammatory anaphylatoxins, which results in swelling and fluid buildup in tissues.
There is no cure for hereditary angioedema, but timely treatment with purified and concentrated C1-INH from blood donors can be effective, preventing tragic outcomes like the one suffered by Angela’s father. A number of therapeutic drugs, either currently approved or in late-stage human trials, may also be considered as options for treatment in the near future. These drugs work by inhibiting inflammatory molecules or the receptors for inflammatory molecules.
Thankfully, Angela’s condition was quickly diagnosed and treated. Although she may experience additional episodes in the future, her prognosis is good and she can expect to live a relatively normal life provided she seeks treatment at the onset of symptoms.
Key Concepts and Summary
• Inflammation results from the collective response of chemical mediators and cellular defenses to an injury or infection.
• Acute inflammation is short lived and localized to the site of injury or infection. Chronic inflammation occurs when the inflammatory response is unsuccessful, and may result in the formation of granulomas (e.g., with tuberculosis) and scarring (e.g., with hepatitis C viral infections and liver cirrhosis).
• The five cardinal signs of inflammation are erythema, edema, heat, pain, and altered function. These largely result from innate responses that draw increased blood flow to the injured or infected tissue.
• Fever is a system-wide sign of inflammation that raises the body temperature and stimulates the immune response.
• Both inflammation and fever can be harmful if the inflammatory response is too severe.
Footnotes
1. 1 Centers for Disease Control and Prevention. “Parasites–Lymphatic Filiariasis.” 2016. http://www.cdc.gov/parasites/lymphat...info/faqs.html.
2. 2 N. Parrow et al. “Sequestration and Scavenging of Iron in Infection.” Infection and Immunity 81 no. 10 (2013):3503–3514 | textbooks/bio/Microbiology/Microbiology_(OpenStax)/17%3A_Innate_Nonspecific_Host_Defenses/17.05%3A_Inflammation_and_Fever.txt |
17.1: Physical Defenses
Nonspecific innate immunity provides a first line of defense against infection by nonspecifically blocking entry of microbes and targeting them for destruction or removal from the body. The physical defenses of innate immunity include physical barriers, mechanical actions that remove microbes and debris, and the microbiome, which competes with and inhibits the growth of pathogens. The skin, mucous membranes, and endothelia throughout the body serve as physical barriers.
Multiple Choice
Which of the following best describes the innate nonspecific immune system?
1. a targeted and highly specific response to a single pathogen or molecule
2. a generalized and nonspecific set of defenses against a class or group of pathogens
3. a set of barrier mechanisms that adapts to specific pathogens after repeated exposure
4. the production of antibody molecules against pathogens
Answer
B
Which of the following constantly sheds dead cells along with any microbes that may be attached to those cells?
1. epidermis
2. dermis
3. hypodermis
4. mucous membrane
Answer
A
Which of the following uses a particularly dense suite of tight junctions to prevent microbes from entering the underlying tissue?
1. the mucociliary escalator
2. the epidermis
3. the blood-brain barrier
4. the urethra
Answer
C
Fill in the Blank
The muscular contraction of the intestines that results in movement of material through the digestive tract is called ________.
Answer
peristalsis
______ are the hair-like appendages of cells lining parts of the respiratory tract that sweep debris away from the lungs.
Answer
cilia
Secretions that bathe and moisten the interior of the intestines are produced by _______ cells.
Answer
goblet
Short Answer
Differentiate a physical barrier from a mechanical removal mechanism and give an example of each.
Identify some ways that pathogens can breach the physical barriers of the innate immune system.
17.2: Chemical Defenses
Numerous chemical mediators produced endogenously and exogenously exhibit nonspecific antimicrobial functions. Many chemical mediators are found in body fluids such as sebum, saliva, mucus, gastric and intestinal fluids, urine, tears, cerumen, and vaginal secretions. Antimicrobial peptides (AMPs) found on the skin and in other areas of the body are largely produced in response to the presence of pathogens. These include dermcidin, cathelicidin, defensins, histatins, and bacteriocins.
Multiple Choice
Which of the following serve as chemical signals between cells and stimulate a wide range of nonspecific defenses?
1. cytokines
2. antimicrobial peptides
3. complement proteins
4. antibodies
Answer
A
Bacteriocins and defensins are types of which of the following?
1. leukotrienes
2. cytokines
3. inflammation-eliciting mediators
4. antimicrobial peptides
Answer
D
Which of the following chemical mediators is secreted onto the surface of the skin?
1. cerumen
2. sebum
3. gastric acid
4. prostaglandin
Answer
B
Identify the complement activation pathway that is triggered by the binding of an acute-phase protein to a pathogen.
1. classical
2. alternate
3. lectin
4. cathelicidin
Answer
C
Histamine, leukotrienes, prostaglandins, and bradykinin are examples of which of the following?
1. chemical mediators primarily found in the digestive system
2. chemical mediators that promote inflammation
3. antimicrobial peptides found on the skin
4. complement proteins that form MACs
Answer
B
Fill in the Blank
________ are antimicrobial peptides produced by members of the normal microbiota.
Answer
bacteriocins
________ is the fluid portion of a blood sample that has been drawn in the presence of an anticoagulant compound.
Answer
plasma
The process by which cells are drawn or attracted to an area by a microbe invader is known as ________.
Answer
chemotaxis
Short Answer
Differentiate the main activation methods of the classic, alternative, and lectin complement cascades.
What are the four protective outcomes of complement activation?
17.3: Cellular Defenses
The formed elements of the blood include red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Of these, leukocytes are primarily involved in the immune response. All formed elements originate in the bone marrow as stem cells (HSCs) that differentiate through hematopoiesis. Granulocytes are leukocytes characterized by a lobed nucleus and granules in the cytoplasm. These include neutrophils (PMNs), eosinophils, and basophils.
Multiple Choice
White blood cells are also referred to as which of the following?
1. platelets
2. erythrocytes
3. leukocytes
4. megakaryocytes
Answer
C
Hematopoiesis occurs inwhich of the following?
1. liver
2. bone marrow
3. kidneys
4. central nervous system
Answer
B
Granulocytes are which type of cell?
1. lymphocyte
2. erythrocyte
3. megakaryocyte
4. leukocyte
Answer
D
Matching
Match each cell type with its description.
___natural killer cell A. stains with basic dye methylene blue, has large amounts of histamine in granules, and facilitates allergic responses and inflammation
___basophil B. stains with acidic dye eosin, has histamine and major basic protein in granules, and facilitates responses to protozoa and helminths
___macrophage C. recognizes abnormal cells, binds to them, and releases perforin and granzyme molecules, which induce apoptosis
___eosinophil D. large agranular phagocyte that resides in tissues such as the brain and lungs
Answer
C, A, D, B
Match each cellular defense with the infection it would most likely target.
___natural killer cell A. virus-infected cell
___neutrophil B. tapeworm in the intestines
___eosinophil C. bacteria in a skin lesion
Answer
A, C, B
Fill in the Blank
Platelets are also called ________.
Answer
thrombocytes
The cell in the bone marrow that gives rise to all other blood cell types is the ________.
Answer
pluripotent hematopoietic stem cell (HSC)
PMNs are another name for ________.
Answer
neutrophils
Kupffer cells residing in the liver are a type of ________.
Answer
macrophage
_____________ are similar to basophils, but reside in tissues rather than circulating in the blood.
Answer
mast cells
Short Answer
Explain the difference between plasma and the formed elements of the blood.
List three ways that a neutrophil can destroy an infectious bacterium.
Critical Thinking
Neutrophils can sometimes kill human cells along with pathogens when they release the toxic contents of their granules into the surrounding tissue. Likewise, natural killer cells target human cells for destruction. Explain why it is advantageous for the immune system to have cells that can kill human cells as well as pathogens.
Refer to Figure 17.3.2. In a blood smear taken from a healthy patient, which type of leukocyte would you expect to observe in the highest numbers?
17.4: Pathogen Recognition and Phagocytosis
Phagocytes are cells that recognize pathogens and destroy them through phagocytosis. Recognition often takes place by the use of phagocyte receptors that bind molecules commonly found on pathogens, known as pathogen-associated molecular patterns (PAMPs). The receptors that bind PAMPs are called pattern recognition receptors, or PRRs. Toll-like receptors (TLRs) are one type of PRR found on phagocytes.
Multiple Choice
PAMPs would be found on the surface of which of the following?
1. pathogen
2. phagocyte
3. skin cell
4. blood vessel wall
Answer
A
________ on phagocytes bind to PAMPs on bacteria, which triggers the uptake and destruction of the bacterial pathogens?
1. PRRs
2. AMPs
3. PAMPs
4. PMNs
Answer
A
Which of the following best characterizes the mode of pathogen recognition for opsonin-dependent phagocytosis?
1. Opsonins produced by a pathogen attract phagocytes through chemotaxis.
2. A PAMP on the pathogen’s surface is recognized by a phagocyte’s toll-like receptors.
3. A pathogen is first coated with a molecule such as a complement protein, which allows it to be recognized by phagocytes.
4. A pathogen is coated with a molecule such as a complement protein that immediately lyses the cell.
Answer
C
Fill in the Blank
________, also known as diapedesis, refers to the exit from the bloodstream of neutrophils and other circulating leukocytes.
Answer
extravasation
Toll-like receptors are examples of ________.
Answer
pattern-recognition receptors (PRRs)
Short Answer
Briefly summarize the events leading up to and including the process of transendothelial migration.
17.5: Inflammation and Fever
Inflammation results from the collective response of chemical mediators and cellular defenses to an injury or infection. Acute inflammation is short lived and localized to the site of injury or infection. Chronic inflammation occurs when the inflammatory response is unsuccessful, and may result in the formation of granulomas (e.g., with tuberculosis) and scarring (e.g., with hepatitis C viral infections and liver cirrhosis).
Multiple Choice
Which refers to swelling as a result of inflammation?
1. erythema
2. edema
3. granuloma
4. vasodilation
Answer
B
Which type of inflammation occurs at the site of an injury or infection?
1. acute
2. chronic
3. endogenous
4. exogenous
Answer
A
Fill in the Blank
A(n) ________ is a walled-off area of infected tissue that exhibits chronic inflammation.
Answer
granuloma
The ________ is the part of the body responsible for regulating body temperature.
Answer
hypothalamus
Heat and redness, or ________, occur when the small blood vessels in an inflamed area dilate (open up), bringing more blood much closer to the surface of the skin.
Answer
erythema
Short Answer
Differentiate exogenous and endogenous pyrogens, and provide an example of each.
Critical Thinking
If a gram-negative bacterial infection reaches the bloodstream, large quantities of LPS can be released into the blood, resulting in a syndrome called septic shock. Death due to septic shock is a real danger. The overwhelming immune and inflammatory responses that occur with septic shock can cause a perilous drop in blood pressure; intravascular blood clotting; development of thrombi and emboli that block blood vessels, leading to tissue death; failure of multiple organs; and death of the patient. Identify and characterize two to three therapies that might be useful in stopping the dangerous events and outcomes of septic shock once it has begun, given what you have learned about inflammation and innate immunity in this chapter.
In Lubeck, Germany, in 1930, a group of 251 infants was accidentally administered a tainted vaccine for tuberculosis that contained live Mycobacterium tuberculosis. This vaccine was administered orally, directly exposing the infants to the deadly bacterium. Many of these infants contracted tuberculosis, and some died. However, 44 of the infants never contracted tuberculosis. Based on your knowledge of the innate immune system, what innate defenses might have inhibited M. tuberculosis enough to prevent these infants from contracting the disease? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/17%3A_Innate_Nonspecific_Host_Defenses/17.E%3A_Innate_Nonspecific_Host_Defenses_%28Exercises%29.txt |
People living in developed nations and born in the 1960s or later may have difficulty understanding the once heavy burden of devastating infectious diseases. For example, smallpox, a deadly viral disease, once destroyed entire civilizations but has since been eradicated. Thanks to the vaccination efforts by multiple groups, including the World Health Organization, Rotary International, and the United Nations Children’s Fund (UNICEF), smallpox has not been diagnosed in a patient since 1977. Polio is another excellent example. This crippling viral disease paralyzed patients, who were often kept alive in “iron lung wards” as recently as the 1950s (Figure \(1\)). Today, vaccination against polio has nearly eradicated the disease. Vaccines have also reduced the prevalence of once-common infectious diseases such as chickenpox, German measles, measles, mumps, and whooping cough. The success of these and other vaccines is due to the very specific and adaptive host defenses that are the focus of this chapter.
Innate Nonspecific Host Defenses described innate immunity against microbial pathogens. Higher animals, such as humans, also possess an adaptive immune defense, which is highly specific for individual microbial pathogens. This specific adaptive immunity is acquired through active infection or vaccination and serves as an important defense against pathogens that evade the defenses of innate immunity.
• 18.1: Architecture of the Immune System
Adaptive immunity is defined by two important characteristics: specificity and memory. Specificity refers to the adaptive immune system’s ability to target specific pathogens, and memory refers to its ability to quickly respond to pathogens to which it has previously been exposed. For example, when an individual recovers from chickenpox, the body develops a memory of the infection that will specifically protect it from the causative agent if it is exposed to the virus again later.
• 18.2: Antigens, Antigen Presenting Cells, and Major Histocompatibility Complexes
Major histocompatibility complex (MHC) molecules are expressed on the surface of healthy cells, identifying them as normal and “self” to natural killer (NK) cells. MHC molecules also play an important role in the presentation of foreign antigens, which is a critical step in the activation of T cells and thus an important mechanism of the adaptive immune system.
• 18.3: T Lymphocytes
The antibodies involved in humoral immunity often bind pathogens and toxins before they can attach to and invade host cells. Thus, humoral immunity is primarily concerned with fighting pathogens in extracellular spaces. However, pathogens that have already gained entry to host cells are largely protected from the humoral antibody-mediated defenses. Cellular immunity, on the other hand, targets and eliminates intracellular pathogens through the actions of T lymphocytes, or T cells.
• 18.4: B Lymphocytes and Antibodies
Humoral immunity refers to mechanisms of the adaptive immune defenses that are mediated by antibodies secreted by B lymphocytes, or B cells. This section will focus on B cells and discuss their production and maturation, receptors, and mechanisms of activation.
• 18.5: Vaccines
By artificially stimulating the adaptive immune defenses, a vaccine triggers memory cell production similar to that which would occur during a primary response. In so doing, the patient is able to mount a strong secondary response upon exposure to the pathogen—but without having to first suffer through an initial infection. In this section, we will explore several different kinds of artificial immunity along with various types of vaccines and their mechanisms for inducing artificial immunity.
• 18.E: Specific Adaptive Host Defenses (Exercises)
Thumbnail: From left to right: erythrocyte, platelet and lymphocyte. (Public Domain; The National Cancer Institute at Frederick).
18: Specific Adaptive Host Defenses
Learning Objectives
• Define memory, primary response, secondary response, and specificity
• Distinguish between humoral and cellular immunity
• Differentiate between antigens, epitopes, and haptens
• Describe the structure and function of antibodies and distinguish between the different classes of antibodies
Clinical Focus: Part 1
Olivia, a one-year old infant, is brought to the emergency room by her parents, who report her symptoms: excessive crying, irritability, sensitivity to light, unusual lethargy, and vomiting. A physician feels swollen lymph nodes in Olivia’s throat and armpits. In addition, the area of the abdomen over the spleen is swollen and tender.
Exercise \(1\)
1. What do these symptoms suggest?
2. What tests might be ordered to try to diagnose the problem?
Adaptive immunity is defined by two important characteristics: specificity and memory. Specificity refers to the adaptive immune system’s ability to target specific pathogens, and memory refers to its ability to quickly respond to pathogens to which it has previously been exposed. For example, when an individual recovers from chickenpox, the body develops a memory of the infection that will specifically protect it from the causative agent, the varicella-zoster virus, if it is exposed to the virus again later.
Specificity and memory are achieved by essentially programming certain cells involved in the immune response to respond rapidly to subsequent exposures of the pathogen. This programming occurs as a result of the first exposure to a pathogen or vaccine, which triggers a primary response. Subsequent exposures result in a secondary response that is faster and stronger as a result of the body’s memory of the first exposure (Figure \(1\)). This secondary response, however, is specific to the pathogen in question. For example, exposure to one virus (e.g., varicella-zoster virus) will not provide protection against other viral diseases (e.g., measles, mumps, or polio).
Adaptive specific immunity involves the actions of two distinct cell types: B lymphocytes (B cells) and T lymphocytes (T cells). Although B cells and T cells arise from a common hematopoietic stem cell differentiation pathway, their sites of maturation and their roles in adaptive immunity are very different.
B cells mature in the bone marrow and are responsible for the production of glycoproteins called antibodies, or immunoglobulins. Antibodies are involved in the body’s defense against pathogens and toxins in the extracellular environment. Mechanisms of adaptive specific immunity that involve B cells and antibody production are referred to as humoral immunity. The maturation of T cells occurs in the thymus. T cells function as the central orchestrator of both innate and adaptive immune responses. They are also responsible for destruction of cells infected with intracellular pathogens. The targeting and destruction of intracellular pathogens by T cells is called cell-mediated immunity, or cellular immunity.
Exercise \(2\)
1. List the two defining characteristics of adaptive immunity.
2. Explain the difference between a primary and secondary immune response.
3. How do humoral and cellular immunity differ?
Antigens
Activation of the adaptive immune defenses is triggered by pathogen-specific molecular structures called antigens. Antigens are similar to the pathogen-associated molecular patterns (PAMPs) discussed in Pathogen Recognition and Phagocytosis; however, whereas PAMPs are molecular structures found on numerous pathogens, antigens are unique to a specific pathogen. The antigens that stimulate adaptive immunity to chickenpox, for example, are unique to the varicella-zoster virus but significantly different from the antigens associated with other viral pathogens.
The term antigen was initially used to describe molecules that stimulate the production of antibodies; in fact, the term comes from a combination of the words antibody and generator, and a molecule that stimulates antibody production is said to be antigenic. However, the role of antigens is not limited to humoral immunity and the production of antibodies; antigens also play an essential role in stimulating cellular immunity, and for this reason antigens are sometimes more accurately referred to as immunogens. In this text, however, we will typically refer to them as antigens.
Pathogens possess a variety of structures that may contain antigens. For example, antigens from bacterial cells may be associated with their capsules, cell walls, fimbriae, flagella, or pili. Bacterial antigens may also be associated with extracellular toxins and enzymes that they secrete. Viruses possess a variety of antigens associated with their capsids, envelopes, and the spike structures they use for attachment to cells.
Antigens may belong to any number of molecular classes, including carbohydrates, lipids, nucleic acids, proteins, and combinations of these molecules. Antigens of different classes vary in their ability to stimulate adaptive immune defenses as well as in the type of response they stimulate (humoral or cellular). The structural complexity of an antigenic molecule is an important factor in its antigenic potential. In general, more complex molecules are more effective as antigens. For example, the three-dimensional complex structure of proteins make them the most effective and potent antigens, capable of stimulating both humoral and cellular immunity. In comparison, carbohydrates are less complex in structure and therefore less effective as antigens; they can only stimulate humoral immune defenses. Lipids and nucleic acids are the least antigenic molecules, and in some cases may only become antigenic when combined with proteins or carbohydrates to form glycolipids, lipoproteins, or nucleoproteins.
One reason the three-dimensional complexity of antigens is so important is that antibodies and T cells do not recognize and interact with an entire antigen but with smaller exposed regions on the surface of antigens called epitopes. A single antigen may possess several different epitopes (Figure \(2\)), and different antibodies may bind to different epitopes on the same antigen (Figure \(3\)). For example, the bacterial flagellum is a large, complex protein structure that can possess hundreds or even thousands of epitopes with unique three-dimensional structures. Moreover, flagella from different bacterial species (or even strains of the same species) contain unique epitopes that can only be bound by specific antibodies.
An antigen’s size is another important factor in its antigenic potential. Whereas large antigenic structures like flagella possess multiple epitopes, some molecules are too small to be antigenic by themselves. Such molecules, called haptens, are essentially free epitopes that are not part of the complex three-dimensional structure of a larger antigen. For a hapten to become antigenic, it must first attach to a larger carrier molecule (usually a protein) to produce a conjugate antigen. The hapten-specific antibodies produced in response to the conjugate antigen are then able to interact with unconjugated free hapten molecules. Haptens are not known to be associated with any specific pathogens, but they are responsible for some allergic responses. For example, the hapten urushiol, a molecule found in the oil of plants that cause poison ivy, causes an immune response that can result in a severe rash (called contact dermatitis). Similarly, the hapten penicillin can cause allergic reactions to drugs in the penicillin class.
Exercise \(3\)
1. What is the difference between an antigen and an epitope?
2. What factors affect an antigen’s antigenic potential?
3. Why are haptens typically not antigenic, and how do they become antigenic?
Antibodies
Antibodies (also called immunoglobulins) are glycoproteins that are present in both the blood and tissue fluids. The basic structure of an antibody monomer consists of four protein chains held together by disulfide bonds (Figure \(4\)). A disulfide bond is a covalent bond between the sulfhydryl R groups found on two cysteine amino acids. The two largest chains are identical to each other and are called the heavy chains. The two smaller chains are also identical to each other and are called the light chains. Joined together, the heavy and light chains form a basic Y-shaped structure.
The two ‘arms’ of the Y-shaped antibody molecule are known as the Fab region, for “fragment of antigen binding.” The far end of the Fab region is the variable region, which serves as the site of antigen binding. The amino acid sequence in the variable region dictates the three-dimensional structure, and thus the specific three-dimensional epitope to which the Fab region is capable of binding. Although the epitope specificity of the Fab regions is identical for each arm of a single antibody molecule, this region displays a high degree of variability between antibodies with different epitope specificities. Binding to the Fab region is necessary for neutralization of pathogens, agglutination or aggregation of pathogens, and antibody-dependent cell-mediated cytotoxicity.
The constant region of the antibody molecule includes the trunk of the Y and lower portion of each arm of the Y. The trunk of the Y is also called the Fc region, for “fragment of crystallization,” and is the site of complement factor binding and binding to phagocytic cells during antibody-mediated opsonization.
Exercise \(4\)
Describe the different functions of the Fab region and the Fc region.
Antibody Classes
The constant region of an antibody molecule determines its class, or isotype. The five classes of antibodies are IgG, IgM, IgA, IgD, and IgE. Each class possesses unique heavy chains designated by Greek letters γ, μ, α, δ, and ε, respectively. Antibody classes also exhibit important differences in abundance in serum, arrangement, body sites of action, functional roles, and size (Figure \(5\)).
IgG is a monomer that is by far the most abundant antibody in human blood, accounting for about 80% of total serum antibody. IgG penetrates efficiently into tissue spaces, and is the only antibody class with the ability to cross the placental barrier, providing passive immunity to the developing fetus during pregnancy. IgG is also the most versatile antibody class in terms of its role in the body’s defense against pathogens.
IgM is initially produced in a monomeric membrane-bound form that serves as an antigen-binding receptor on B cells. The secreted form of IgM assembles into a pentamer with five monomers of IgM bound together by a protein structure called the J chain. Although the location of the J chain relative to the Fc regions of the five monomers prevents IgM from performing some of the functions of IgG, the ten available Fab sites associated with a pentameric IgM make it an important antibody in the body’s arsenal of defenses. IgM is the first antibody produced and secreted by B cells during the primary and secondary immune responses, making pathogen-specific IgM a valuable diagnostic marker during active or recent infections.
IgA accounts for about 13% of total serum antibody, and secretory IgA is the most common and abundant antibody class found in the mucus secretions that protect the mucous membranes. IgA can also be found in other secretions such as breast milk, tears, and saliva. Secretory IgA is assembled into a dimeric form with two monomers joined by a protein structure called the secretory component. One of the important functions of secretory IgA is to trap pathogens in mucus so that they can later be eliminated from the body.
Similar to IgM, IgD is a membrane-bound monomer found on the surface of B cells, where it serves as an antigen-binding receptor. However, IgD is not secreted by B cells, and only trace amounts are detected in serum. These trace amounts most likely come from the degradation of old B cells and the release of IgD molecules from their cytoplasmic membranes.
IgE is the least abundant antibody class in serum. Like IgG, it is secreted as a monomer, but its role in adaptive immunity is restricted to anti-parasitic defenses. The Fc region of IgE binds to basophils and mast cells. The Fab region of the bound IgE then interacts with specific antigen epitopes, causing the cells to release potent pro-inflammatory mediators. The inflammatory reaction resulting from the activation of mast cells and basophils aids in the defense against parasites, but this reaction is also central to allergic reactions (see Diseases of the Immune System.)
Exercise \(5\)
1. What part of an antibody molecule determines its class?
2. What class of antibody is involved in protection against parasites?
3. Describe the difference in structure between IgM and IgG.
Antigen-Antibody Interactions
Different classes of antibody play important roles in the body’s defense against pathogens. These functions include neutralization of pathogens, opsonization for phagocytosis, agglutination, complement activation, and antibody-dependent cell-mediated cytotoxicity. For most of these functions, antibodies also provide an important link between adaptive specific immunity and innate nonspecific immunity.
Neutralization involves the binding of certain antibodies (IgG, IgM, or IgA) to epitopes on the surface of pathogens or toxins, preventing their attachment to cells. For example, Secretory IgA can bind to specific pathogens and block initial attachment to intestinal mucosal cells. Similarly, specific antibodies can bind to certain toxins, blocking them from attaching to target cells and thus neutralizing their toxic effects. Viruses can be neutralized and prevented from infecting a cell by the same mechanism (Figure \(6\)).
As described in Chemical Defenses, opsonization is the coating of a pathogen with molecules, such as complementfactors, C-reactive protein, and serum amyloid A, to assist in phagocyte binding to facilitate phagocytosis. IgG antibodies also serve as excellent opsonins, binding their Fab sites to specific epitopes on the surface of pathogens. Phagocytic cells such as macrophages, dendritic cells, and neutrophils have receptors on their surfaces that recognize and bind to the Fc portion of the IgG molecules; thus, IgG helps such phagocytes attach to and engulf the pathogens they have bound (Figure \(7\)).
Agglutination or aggregation involves the cross-linking of pathogens by antibodies to create large aggregates (Figure \(8\)). IgG has two Fab antigen-binding sites, which can bind to two separate pathogen cells, clumping them together. When multiple IgG antibodies are involved, large aggregates can develop; these aggregates are easier for the kidneys and spleen to filter from the blood and easier for phagocytes to ingest for destruction. The pentameric structure of IgMprovides ten Fab binding sites per molecule, making it the most efficient antibody for agglutination.
Another important function of antibodies is activation of the complement cascade. As discussed in the previous chapter, the complement system is an important component of the innate defenses, promoting the inflammatory response, recruiting phagocytes to site of infection, enhancing phagocytosis by opsonization, and killing gram-negative bacterial pathogens with the membrane attack complex (MAC). Complement activation can occur through three different pathways (see Figure 17.2.2), but the most efficient is the classical pathway, which requires the initial binding of IgG or IgM antibodies to the surface of a pathogen cell, allowing for recruitment and activation of the C1 complex.
Yet another important function of antibodies is antibody-dependent cell-mediated cytotoxicity (ADCC), which enhances killing of pathogens that are too large to be phagocytosed. This process is best characterized for natural killer cells (NK cells), as shown in Figure \(9\), but it can also involve macrophages and eosinophils. ADCC occurs when the Fab region of an IgG antibody binds to a large pathogen; Fc receptors on effector cells (e.g., NK cells) then bind to the Fc region of the antibody, bringing them into close proximity with the target pathogen. The effector cell then secretes powerful cytotoxins (e.g., perforin and granzymes) that kill the pathogen.
Exercise \(6\)
1. Where is IgA normally found?
2. Which class of antibody crosses the placenta, providing protection to the fetus?
3. Compare the mechanisms of opsonization and antibody-dependent cell-mediated cytotoxicity.
Key Concepts and Summary
• Adaptive immunity is an acquired defense against foreign pathogens that is characterized by specificity and memory. The first exposure to an antigen stimulates a primary response, and subsequent exposures stimulate a faster and strong secondary response.
• Adaptive immunity is a dual system involving humoral immunity (antibodies produced by B cells) and cellular immunity (T cells directed against intracellular pathogens).
• Antigens, also called immunogens, are molecules that activate adaptive immunity. A single antigen possesses smaller epitopes, each capable of inducing a specific adaptive immune response.
• An antigen’s ability to stimulate an immune response depends on several factors, including its molecular class, molecular complexity, and size.
• Antibodies (immunoglobulins) are Y-shaped glycoproteins with two Fab sites for binding antigens and an Fc portion involved in complement activation and opsonization.
• The five classes of antibody are IgM, IgG, IgA, IgE, and IgD, each differing in size, arrangement, location within the body, and function. The five primary functions of antibodies are neutralization, opsonization, agglutination, complement activation, and antibody-dependent cell-mediated cytotoxicity (ADCC). | textbooks/bio/Microbiology/Microbiology_(OpenStax)/18%3A_Specific_Adaptive_Host_Defenses/18.01%3A_Architecture_of_the_Immune_System.txt |
Learning Objectives
• Identify cells that express MHC I and/or MHC II molecules and describe the structures and cellular location of MHC I and MHC II molecules
• Identify the cells that are antigen-presenting cells
• Describe the process of antigen processing and presentation with MHC I and MHC II
As discussed in Cellular Defenses, major histocompatibility complex (MHC) molecules are expressed on the surface of healthy cells, identifying them as normal and “self” to natural killer (NK) cells. MHC molecules also play an important role in the presentation of foreign antigens, which is a critical step in the activation of T cells and thus an important mechanism of the adaptive immune system.
Major Histocompatibility Complex Molecules
The major histocompatibility complex (MHC) is a collection of genes coding for MHC molecules found on the surface of all nucleated cells of the body. In humans, the MHC genes are also referred to as human leukocyte antigen (HLA) genes. Mature red blood cells, which lack a nucleus, are the only cells that do not express MHC molecules on their surface.
There are two classes of MHC molecules involved in adaptive immunity, MHC I and MHC II (Figure \(1\)). MHC I molecules are found on all nucleated cells; they present normal self-antigens as well as abnormal or nonself pathogens to the effector T cells involved in cellular immunity. In contrast, MHC II molecules are only found on macrophages, dendritic cells, and B cells; they present abnormal or nonself pathogen antigens for the initial activation of T cells.
Both types of MHC molecules are transmembrane glycoproteins that assemble as dimers in the cytoplasmic membrane of cells, but their structures are quite different. MHC I molecules are composed of a longer α protein chain coupled with a smaller β2 microglobulin protein, and only the α chain spans the cytoplasmic membrane. The α chain of the MHC I molecule folds into three separate domains: α1, α2 and α3. MHC II molecules are composed of two protein chains (an α and a β chain) that are approximately similar in length. Both chains of the MHC II molecule possess portions that span the plasma membrane, and each chain folds into two separate domains: α1 and α2, and β1, and β2. In order to present abnormal or non-self-antigens to T cells, MHC molecules have a cleft that serves as the antigen-binding site near the “top” (or outermost) portion of the MHC-I or MHC-II dimer. For MHC I, the antigen-binding cleft is formed by the α1 and α2 domains, whereas for MHC II, the cleft is formed by the α1 and β1 domains (Figure \(1\)).
Exercise \(1\)
Compare the structures of the MHC I and MHC II molecules.
Antigen-Presenting Cells (APCs)
All nucleated cells in the body have mechanisms for processing and presenting antigens in association with MHC molecules. This signals the immune system, indicating whether the cell is normal and healthy or infected with an intracellular pathogen. However, only macrophages, dendritic cells, and B cells have the ability to present antigens specifically for the purpose of activating T cells; for this reason, these types of cells are sometimes referred to as antigen-presenting cells (APCs).
While all APCs play a similar role in adaptive immunity, there are some important differences to consider. Macrophages and dendritic cells are phagocytes that ingest and kill pathogens that penetrate the first-line barriers (i.e., skin and mucous membranes). B cells, on the other hand, do not function as phagocytes but play a primary role in the production and secretion of antibodies. In addition, whereas macrophages and dendritic cells recognize pathogens through nonspecific receptor interactions (e.g., PAMPs, toll-like receptors, and receptors for opsonizing complement or antibody), B cells interact with foreign pathogens or their free antigens using antigen-specific immunoglobulin as receptors (monomeric IgD and IgM). When the immunoglobulin receptors bind to an antigen, the B cell internalizes the antigen by endocytosis before processing and presentting the antigen to T cells.
Antigen Presentation with MHC II Molecules
MHC II molecules are only found on the surface of APCs. Macrophages and dendritic cells use similar mechanisms for processing and presentation of antigens and their epitopes in association with MHC II; B cells use somewhat different mechanisms that will be described further in B Lymphocytes and Humoral Immunity. For now, we will focus on the steps of the process as they pertain to dendritic cells.
After a dendritic cell recognizes and attaches to a pathogen cell, the pathogen is internalized by phagocytosis and is initially contained within a phagosome. Lysosomes containing antimicrobial enzymes and chemicals fuse with the phagosome to create a phagolysosome, where degradation of the pathogen for antigen processing begins. Proteases (protein-degrading) are especially important in antigen processing because only protein antigen epitopes are presented to T cells by MHC II (Figure \(2\)).
APCs do not present all possible epitopes to T cells; only a selection of the most antigenic or immunodominantepitopes are presented. The mechanism by which epitopes are selected for processing and presentation by an APC is complicated and not well understood; however, once the most antigenic, immunodominant epitopes have been processed, they associate within the antigen-binding cleft of MHC II molecules and are translocated to the cell surface of the dendritic cell for presentation to T cells.
Exercise \(2\)
1. What are the three kinds of APCs?
2. What role to MHC II molecules play in antigen presentation?
3. What is the role of antigen presentation in adaptive immunity?
Antigen Presentation with MHC I Molecules
MHC I molecules, found on all normal, healthy, nucleated cells, signal to the immune system that the cell is a normal “self” cell. In a healthy cell, proteins normally found in the cytoplasm are degraded by proteasomes (enzyme complexes responsible for degradation and processing of proteins) and processed into self-antigen epitopes; these self-antigen epitopes bind within the MHC I antigen-binding cleft and are then presented on the cell surface. Immune cells, such as NK cells, recognize these self-antigens and do not target the cell for destruction. However, if a cell becomes infected with an intracellular pathogen (e.g., a virus), protein antigens specific to the pathogen are processed in the proteasomes and bind with MHC I molecules for presentation on the cell surface. This presentation of pathogen-specific antigens with MHC I signals that the infected cell must be targeted for destruction along with the pathogen.
Before elimination of infected cells can begin, APCs must first activate the T cells involved in cellular immunity. If an intracellular pathogen directly infects the cytoplasm of an APC, then the processing and presentation of antigens can occur as described (in proteasomes and on the cell surface with MHC I). However, if the intracellular pathogen does not directly infect APCs, an alternative strategy called cross-presentation is utilized. In cross-presentation, antigens are brought into the APC by mechanisms normally leading to presentation with MHC II (i.e., through phagocytosis), but the antigen is presented on an MHC I molecule for CD8 T cells. The exact mechanisms by which cross-presentation occur are not yet well understood, but it appears that cross-presentation is primarily a function of dendritic cells and not macrophages or B cells.
Exercise \(3\)
1. Compare and contrast antigen processing and presentation associated with MHC I and MHC II molecules.
2. What is cross-presentation, and when is it likely to occur?
Key Concepts and Summary
• Major histocompatibility complex (MHC) is a collection of genes coding for glycoprotein molecules expressed on the surface of all nucleated cells.
• MHC I molecules are expressed on all nucleated cells and are essential for presentation of normal “self” antigens. Cells that become infected by intracellular pathogens can present foreign antigens on MHC I as well, marking the infected cell for destruction.
• MHC II molecules are expressed only on the surface of antigen-presenting cells (macrophages, dendritic cells, and B cells). Antigen presentation with MHC II is essential for the activation of T cells.
• Antigen-presenting cells (APCs) primarily ingest pathogens by phagocytosis, destroy them in the phagolysosomes, process the protein antigens, and select the most antigenic/immunodominant epitopes with MHC II for presentation to T cells.
• Cross-presentation is a mechanism of antigen presentation and T-cell activation used by dendritic cells not directly infected by the pathogen; it involves phagocytosis of the pathogen but presentation on MHC I rather than MHC II. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/18%3A_Specific_Adaptive_Host_Defenses/18.02%3A_Antigens_Antigen_Presenting_Cells_and_Major_Histocompatibility_Complexes.txt |
Learning Objectives
• Describe the process of T-cell maturation and thymic selection
• Explain the genetic events that lead to diversity of T-cell receptors
• Compare and contrast the various classes and subtypes of T cells in terms of activation and function
• Explain the mechanism by which superantigens effect unregulated T-cell activation
As explained in Overview of Specific Adaptive Immunity, the antibodies involved in humoral immunity often bind pathogens and toxins before they can attach to and invade host cells. Thus, humoral immunity is primarily concerned with fighting pathogens in extracellular spaces. However, pathogens that have already gained entry to host cells are largely protected from the humoral antibody-mediated defenses. Cellular immunity, on the other hand, targets and eliminates intracellular pathogens through the actions of T lymphocytes, or T cells (Figure \(1\)). T cells also play a more central role in orchestrating the overall adaptive immune response (humoral as well as cellular) along with the cellular defenses of innate immunity.
T Cell Production and Maturation
T cells, like all other white blood cells involved in innate and adaptive immunity, are formed from multipotent hematopoietic stem cells (HSCs) in the bone marrow (see Figure 17.3.1). However, unlike the white blood cells of innate immunity, eventual T cells differentiate first into lymphoid stem cells that then become small, immature lymphocytes, sometimes called lymphoblasts. The first steps of differentiation occur in the red marrow of bones (Figure \(2\)), after which immature T lymphocytes enter the bloodstream and travel to the thymus for the final steps of maturation (Figure \(3\)). Once in the thymus, the immature T lymphocytes are referred to as thymocytes.
The maturation of thymocytes within the thymus can be divided into tree critical steps of positive and negative selection, collectively referred to as thymic selection. The first step of thymic selection occurs in the cortex of the thymus and involves the development of a functional T-cell receptor (TCR) that is required for activation by APCs. Thymocytes with defective TCRs are removed by negative selection through the induction of apoptosis (programmed controlled cell death). The second step of thymic selection also occurs in the cortex and involves the positive selection of thymocytes that will interact appropriately with MHC molecules. Thymocytes that can interact appropriately with MHC molecules receive a positive stimulation that moves them further through the process of maturation, whereas thymocytes that do not interact appropriately are not stimulated and are eliminated by apoptosis. The third and final step of thymic selection occurs in both the cortex and medulla and involves negative selection to remove self-reacting thymocytes, those that react to self-antigens, by apoptosis. This final step is sometimes referred to as central tolerance because it prevents self-reacting T cells from reaching the bloodstream and potentially causing autoimmune disease, which occurs when the immune system attacks healthy “self” cells.
Despite central tolerance, some self-reactive T cells generally escape the thymus and enter the peripheral bloodstream. Therefore, a second line of defense called peripheral tolerance is needed to protect against autoimmune disease. Peripheral tolerance involves mechanisms of anergy and inhibition of self-reactive T cells by regulatory T cells. Anergy refers to a state of nonresponsiveness to antigen stimulation. In the case of self-reactive T cells that escape the thymus, lack of an essential co-stimulatory signal required for activation causes anergy and prevents autoimmune activation. Regulatory T cells participate in peripheral tolerance by inhibiting the activation and function of self-reactive T cells and by secreting anti-inflammatory cytokines.
It is not completely understood what events specifically direct maturation of thymocytes into regulatory T cells. Current theories suggest the critical events may occur during the third step of thymic selection, when most self-reactive T cells are eliminated. Regulatory T cells may receive a unique signal that is below the threshold required to target them for negative selection and apoptosis. Consequently, these cells continue to mature and then exit the thymus, armed to inhibit the activation of self-reactive T cells.
It has been estimated that the three steps of thymic selection eliminate 98% of thymocytes. The remaining 2% that exit the thymus migrate through the bloodstream and lymphatic system to sites of secondary lymphoid organs/tissues, such as the lymph nodes, spleen, and tonsils (Figure \(3\)), where they await activation through the presentation of specific antigens by APCs. Until they are activated, they are known as mature naïve T cells.
Exercise \(1\)
1. What anatomical sites are involved in T cell production and maturation?
2. What are the three steps involved in thymic selection?
3. Why are central tolerance and peripheral tolerance important? What do they prevent?
Classes of T Cells
T cells can be categorized into three distinct classes: helper T cells, regulatory T cells, and cytotoxic T cells. These classes are differentiated based on their expression of certain surface molecules, their mode of activation, and their functional roles in adaptive immunity (Table \(1\)).
All T cells produce cluster of differentiation (CD) molecules, cell surface glycoproteins that can be used to identify and distinguish between the various types of white blood cells. Although T cells can produce a variety of CD molecules, CD4and CD8 are the two most important used for differentiation of the classes. Helper T cells and regulatory T cells are characterized by the expression of CD4 on their surface, whereas cytotoxic T cells are characterized by the expression of CD8.
Classes of T cells can also be distinguished by the specific MHC molecules and APCs with which they interact for activation. Helper T cells and regulatory T cells can only be activated by APCs presenting antigens associated with MHC II. In contrast, cytotoxic T cells recognize antigens presented in association with MHC I, either by APCs or by nucleated cells infected with an intracellular pathogen.
The different classes of T cells also play different functional roles in the immune system. Helper T cells serve as the central orchestrators that help activate and direct functions of humoral and cellular immunity. In addition, helper T cells enhance the pathogen-killing functions of macrophages and NK cells of innate immunity. In contrast, the primary role of regulatory T cells is to prevent undesirable and potentially damaging immune responses. Their role in peripheral tolerance, for example, protects against autoimmune disorders, as discussed earlier. Finally, cytotoxic T cells are the primary effector cells for cellular immunity. They recognize and target cells that have been infected by intracellular pathogens, destroying infected cells along with the pathogens inside.
Table \(1\): Classes of T Cells
Class Surface CD Molecules Activation Functions
Helper T cells CD4 APCs presenting antigens associated with MHC II Orchestrate humoral and cellular immunity
Involved in the activation of macrophages and NK cells
Regulatory T cells CD4 APCs presenting antigens associated with MHC II Involved in peripheral tolerance and prevention of autoimmune responses
Cytotoxic T cells CD8 APCs or infected nucleated cells presenting antigens associated with MHC I Destroy cells infected with intracellular pathogens
Exercise \(2\)
1. What are the unique functions of the three classes of T cells?
2. Which T cells can be activated by antigens presented by cells other than APCs?
T-Cell Receptors
For both helper T cells and cytotoxic T cells, activation is a complex process that requires the interactions of multiple molecules and exposure to cytokines. The T-cell receptor (TCR) is involved in the first step of pathogen epitope recognition during the activation process.
The TCR comes from the same receptor family as the antibodies IgD and IgM, the antigen receptors on the B cell membrane surface, and thus shares common structural elements. Similar to antibodies, the TCR has a variable regionand a constant region, and the variable region provides the antigen-binding site (Figure \(4\)). However, the structure of TCR is smaller and less complex than the immunoglobulin molecules (Figure 18.1.4). Whereas immunoglobulins have four peptide chains and Y-shaped structures, the TCR consists of just two peptide chains (α and β chains), both of which span the cytoplasmic membrane of the T cell.
TCRs are epitope-specific, and it has been estimated that 25 million T cells with unique epitope-binding TCRs are required to protect an individual against a wide range of microbial pathogens. Because the human genome only contains about 25,000 genes, we know that each specific TCR cannot be encoded by its own set of genes. This raises the question of how such a vast population of T cells with millions of specific TCRs can be achieved. The answer is a process called genetic rearrangement, which occurs in the thymus during the first step of thymic selection.
The genes that code for the variable regions of the TCR are divided into distinct gene segments called variable (V), diversity (D), and joining (J) segments. The genes segments associated with the α chain of the TCR consist 70 or more different Vα segments and 61 different Jα segments. The gene segments associated with the β chain of the TCR consist of 52 different Vβ segments, two different Dβ segments, and 13 different Jβ segments. During the development of the functional TCR in the thymus, genetic rearrangement in a T cell brings together one Vα segment and one Jα segment to code for the variable region of the α chain. Similarly, genetic rearrangement brings one of the Vβ segments together with one of the Dβ segments and one of thetJβ segments to code for the variable region of the β chain. All the possible combinations of rearrangements between different segments of V, D, and J provide the genetic diversity required to produce millions of TCRs with unique epitope-specific variable regions.
Exercise \(3\)
1. What are the similarities and differences between TCRs and immunoglobulins?
2. What process is used to provide millions of unique TCR binding sites?
Activation and Differentiation of Helper T Cells
Helper T cells can only be activated by APCs presenting processed foreign epitopes in association with MHC II. The first step in the activation process is TCR recognition of the specific foreign epitope presented within the MHC II antigen-binding cleft. The second step involves the interaction of CD4 on the helper T cell with a region of the MHC II molecule separate from the antigen-binding cleft. This second interaction anchors the MHC II-TCR complex and ensures that the helper T cell is recognizing both the foreign (“nonself”) epitope and “self” antigen of the APC; both recognitions are required for activation of the cell. In the third step, the APC and T cell secrete cytokines that activate the helper T cell. The activated helper T cell then proliferates, dividing by mitosis to produce clonal naïve helper T cells that differentiate into subtypes with different functions (Figure \(5\)).
Activated helper T cells can differentiate into one of four distinct subtypes, summarized in Table \(2\). The differentiation process is directed by APC-secreted cytokines. Depending on which APC-secreted cytokines interact with an activated helper T cell, the cell may differentiate into a T helper 1 (TH1) cell, a T helper 2 (TH2) cell, or a memory helper T cell. The two types of helper T cells are relatively short-lived effector cells, meaning that they perform various functions of the immediate immune response. In contrast, memory helper T cells are relatively long lived; they are programmed to “remember” a specific antigen or epitope in order to mount a rapid, strong, secondary response to subsequent exposures.
TH1 cells secrete their own cytokines that are involved in stimulating and orchestrating other cells involved in adaptive and innate immunity. For example, they stimulate cytotoxic T cells, enhancing their killing of infected cells and promoting differentiation into memory cytotoxic T cells. TH1 cells also stimulate macrophages and neutrophils to become more effective in their killing of intracellular bacteria. They can also stimulate NK cells to become more effective at killing target cells.
TH2 cells play an important role in orchestrating the humoral immune response through their secretion of cytokines that activate B cells and direct B cell differentiation and antibody production. Various cytokines produced by TH2 cells orchestrate antibody class switching, which allows B cells to switch between the production of IgM, IgG, IgA, and IgE as needed to carry out specific antibody functions and to provide pathogen-specific humoral immune responses.
A third subtype of helper T cells called TH17 cells was discovered through observations that immunity to some infections is not associated with TH1 or TH2 cells. TH17 cells and the cytokines they produce appear to be specifically responsible for the body’s defense against chronic mucocutaneous infections. Patients who lack sufficient TH17 cells in the mucosa (e.g., HIV patients) may be more susceptible to bacteremia and gastrointestinal infections.1
Table \(2\): Subtypes of Helper T Cells
Subtype Functions
TH1 cells Stimulate cytotoxic T cells and produce memory cytotoxic T cells
Stimulate macrophages and neutrophils (PMNs) for more effective intracellular killing of pathogens
Stimulate NK cells to kill more effectively
TH2 cells Stimulate B cell activation and differentiation into plasma cells and memory B cells
Direct antibody class switching in B cells
TH17 cells Stimulate immunity to specific infections such as chronic mucocutaneous infections
Memory helper T cells “Remember” a specific pathogen and mount a strong, rapid secondary response upon re-exposure
Activation and Differentiation of Cytotoxic T Cells
Cytotoxic T cells (also referred to as cytotoxic T lymphocytes, or CTLs) are activated by APCs in a three-step process similar to that of helper T cells. The key difference is that the activation of cytotoxic T cells involves recognition of an antigen presented with MHC I (as opposed to MHC II) and interaction of CD8 (as opposed to CD4) with the receptor complex. After the successful co-recognition of foreign epitope and self-antigen, the production of cytokines by the APC and the cytotoxic T cell activate clonal proliferation and differentiation. Activated cytotoxic T cells can differentiate into effector cytotoxic T cells that target pathogens for destruction or memory cells that are ready to respond to subsequent exposures.
As noted, proliferation and differentiation of cytotoxic T cells is also stimulated by cytokines secreted from TH1 cells activated by the same foreign epitope. The co-stimulation that comes from these TH1 cells is provided by secreted cytokines. Although it is possible for activation of cytotoxic T cells to occur without stimulation from TH1 cells, the activation is not as effective or long-lasting.
Once activated, cytotoxic T cells serve as the effector cells of cellular immunity, recognizing and kill cells infected with intracellular pathogens through a mechanism very similar to that of NK cells. However, whereas NK cells recognize nonspecific signals of cell stress or abnormality, cytotoxic T cells recognize infected cells through antigen presentation of pathogen-specific epitopes associated with MHC I. Once an infected cell is recognized, the TCR of the cytotoxic T cell binds to the epitope and releases perforin and granzymes that destroy the infected cell (Figure \(6\)). Perforin is a protein that creates pores in the target cell, and granzymes are proteases that enter the pores and induce apoptosis. This mechanism of programmed cell death is a controlled and efficient means of destroying and removing infected cells without releasing the pathogens inside to infect neighboring cells, as might occur if the infected cells were simply lysed.
Link to Learning
In this video, you can see a cytotoxic T cell inducing apoptosis in a target cell.
Exercise \(4\)
1. Compare and contrast the activation of helper T cells and cytotoxic T cells.
2. What are the different functions of helper T cell subtypes?
3. What is the mechanism of CTL-mediated destruction of infected cells?
Superantigens and Unregulated Activation of T Cells
When T cell activation is controlled and regulated, the result is a protective response that is effective in combating infections. However, if T cell activation is unregulated and excessive, the result can be a life-threatening. Certain bacterial and viral pathogens produce toxins known as superantigens (see Virulence Factors of Bacterial and Viral Pathogens) that can trigger such an unregulated response. Known bacterial superantigens include toxic shock syndrome toxin (TSST), staphylococcal enterotoxins, streptococcal pyrogenic toxins, streptococcal superantigen, and the streptococcal mitogenic exotoxin. Viruses known to produce superantigens include Epstein-Barr virus (human herpesvirus 4), cytomegalovirus (human herpesvirus 5), and others.
The mechanism of T cell activation by superantigens involves their simultaneous binding to MHC II molecules of APCs and the variable region of the TCR β chain. This binding occurs outside of the antigen-binding cleft of MHC II, so the superantigen will bridge together and activate MHC II and TCR without specific foreign epitope recognition (Figure \(7\)). The result is an excessive, uncontrolled release of cytokines, often called a cytokine storm, which stimulates an excessive inflammatory response. This can lead to a dangerous decrease in blood pressure, shock, multi-organ failure, and potentially, death.
Exercise \(5\)
1. What are examples of superantigens?
2. How does a superantigen activate a helper T cell?
3. What effect does a superantigen have on a T cell?
Case in Point: Superantigens
Melissa, an otherwise healthy 22-year-old woman, is brought to the emergency room by her concerned boyfriend. She complains of a sudden onset of high fever, vomiting, diarrhea, and muscle aches. In her initial interview, she tells the attending physician that she is on hormonal birth control and also is two days into the menstruation portion of her cycle. She is on no other medications and is not abusing any drugs or alcohol. She is not a smoker. She is not diabetic and does not currently have an infection of any kind to her knowledge.
While waiting in the emergency room, Melissa’s blood pressure begins to drop dramatically and her mental state deteriorates to general confusion. The physician believes she is likely suffering from toxic shock syndrome (TSS). TSS is caused by the toxin TSST-1, a superantigen associated with Staphylococcus aureus, and improper tampon use is a common cause of infections leading to TSS. The superantigen inappropriately stimulates widespread T cell activation and excessive cytokine release, resulting in a massive and systemic inflammatory response that can be fatal.
Vaginal or cervical swabs may be taken to confirm the presence of the microbe, but these tests are not critical to perform based on Melissa’s symptoms and medical history. The physician prescribes rehydration, supportive therapy, and antibiotics to stem the bacterial infection. She also prescribes drugs to increase Melissa’s blood pressure. Melissa spends three days in the hospital undergoing treatment; in addition, her kidney function is monitored because of the high risk of kidney failure associated with TSS. After 72 hours, Melissa is well enough to be discharged to continue her recovery at home.
Exercise \(6\)
In what way would antibiotic therapy help to combat a superantigen?
Clinical Focus: Part 2
Olivia’s swollen lymph nodes, abdomen, and spleen suggest a strong immune response to a systemic infection in progress. In addition, little Olivia is reluctant to turn her head and appears to be experiencing severe neck pain. The physician orders a complete blood count, blood culture, and lumbar puncture. The cerebrospinal fluid (CSF) obtained appears cloudy and is further evaluated by Gram stain assessment and culturing for potential bacterial pathogens. The complete blood count indicates elevated numbers of white blood cells in Olivia’s bloodstream. The white blood cell increases are recorded at 28.5 K/µL (normal range: 6.0–17.5 K/µL). The neutrophil percentage was recorded as 60% (normal range: 23–45%). Glucose levels in the CSF were registered at 30 mg/100 mL (normal range: 50–80 mg/100 mL). The WBC count in the CSF was 1,163/mm3 (normal range: 5–20/mm3).
Exercise \(7\)
1. Based on these results, do you have a preliminary diagnosis?
2. What is a recommended treatment based on this preliminary diagnosis?
Key Concepts and Summary
• Immature T lymphocytes are produced in the red bone marrow and travel to the thymus for maturation.
• Thymic selection is a three-step process of negative and positive selection that determines which T cells will mature and exit the thymus into the peripheral bloodstream.
• Central tolerance involves negative selection of self-reactive T cells in the thymus, and peripheral toleranceinvolves anergy and regulatory T cells that prevent self-reactive immune responses and autoimmunity.
• The TCR is similar in structure to immunoglobulins, but less complex. Millions of unique epitope-binding TCRs are encoded through a process of genetic rearrangement of V, D, and J gene segments.
• T cells can be divided into three classes—helper T cells, cytotoxic T cells, and regulatory T cells—based on their expression of CD4 or CD8, the MHC molecules with which they interact for activation, and their respective functions.
• Activated helper T cells differentiate into TH1, TH2, TH17, or memory T cell subtypes. Differentiation is directed by the specific cytokines to which they are exposed. TH1, TH2, and TH17 perform different functions related to stimulation of adaptive and innate immune defenses. Memory T cells are long-lived cells that can respond quickly to secondary exposures.
• Once activated, cytotoxic T cells target and kill cells infected with intracellular pathogens. Killing requires recognition of specific pathogen epitopes presented on the cell surface using MHC I molecules. Killing is mediated by perforin and granzymes that induce apoptosis.
• Superantigens are bacterial or viral proteins that cause a nonspecific activation of helper T cells, leading to an excessive release of cytokines (cytokine storm) and a systemic, potentially fatal inflammatory response.
Footnotes
1. 1 Blaschitz C., Raffatellu M. “Th17 cytokines and the gut mucosal barrier.” J Clin Immunol. 2010 Mar; 30(2):196-203. doi: 10.1007/s10875-010-9368-7. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/18%3A_Specific_Adaptive_Host_Defenses/18.03%3A_T_Lymphocytes.txt |
Learning Objectives
• Describe the production and maturation of B cells
• Compare the structure of B-cell receptors and T-cell receptors
• Compare T-dependent and T-independent activation of B cells
• Compare the primary and secondary antibody responses
Humoral immunity refers to mechanisms of the adaptive immune defenses that are mediated by antibodies secreted by B lymphocytes, or B cells. This section will focus on B cells and discuss their production and maturation, receptors, and mechanisms of activation.
B Cell Production and Maturation
Like T cells, B cells are formed from multipotent hematopoietic stem cells (HSCs) in the bone marrow and follow a pathway through lymphoid stem cell and lymphoblast (see Figure 17.3.1). Unlike T cells, however, lymphoblasts destined to become B cells do not leave the bone marrow and travel to the thymus for maturation. Rather, eventual B cells continue to mature in the bone marrow.
The first step of B cell maturation is an assessment of the functionality of their antigen-binding receptors. This occurs through positive selection for B cells with normal functional receptors. A mechanism of negative selection is then used to eliminate self-reacting B cells and minimize the risk of autoimmunity. Negative selection of self-reacting B cells can involve elimination by apoptosis, editing or modification of the receptors so they are no longer self-reactive, or induction of anergy in the B cell. Immature B cells that pass the selection in the bone marrow then travel to the spleenfor their final stages of maturation. There they become naïve mature B cells, i.e., mature B cells that have not yet been activated.
Exercise \(1\)
Compare the maturation of B cells with the maturation of T cells.
B-Cell Receptors
Like T cells, B cells possess antigen-specific receptors with diverse specificities. Although they rely on T cells for optimum function, B cells can be activated without help from T cells. B-cell receptors (BCRs) for naïve mature B cells are membrane-bound monomeric forms of IgD and IgM. They have two identical heavy chains and two identical light chains connected by disulfide bonds into a basic “Y” shape (Figure \(1\)). The trunk of the Y-shaped molecule, the constant region of the two heavy chains, spans the B cell membrane. The two antigen-binding sites exposed to the exterior of the B cell are involved in the binding of specific pathogen epitopes to initiate the activation process. It is estimated that each naïve mature B cell has upwards of 100,000 BCRs on its membrane, and each of these BCRs has an identical epitope-binding specificity.
In order to be prepared to react to a wide range of microbial epitopes, B cells, like T cells, use genetic rearrangementof hundreds of gene segments to provide the necessary diversity of receptor specificities. The variable region of the BCR heavy chain is made up of V, D, and J segments, similar to the β chain of the TCR. The variable region of the BCR light chain is made up of V and J segments, similar to the α chain of the TCR. Genetic rearrangement of all possible combinations of V-J-D (heavy chain) and V-J (light chain) provides for millions of unique antigen-binding sites for the BCR and for the antibodies secreted after activation.
One important difference between BCRs and TCRs is the way they can interact with antigenic epitopes. Whereas TCRs can only interact with antigenic epitopes that are presented within the antigen-binding cleft of MHC I or MHC II, BCRs do not require antigen presentation with MHC; they can interact with epitopes on free antigens or with epitopesdisplayed on the surface of intact pathogens. Another important difference is that TCRs only recognize protein epitopes, whereas BCRs can recognize epitopes associated with different molecular classes (e.g., proteins, polysaccharides, lipopolysaccharides).
Activation of B cells occurs through different mechanisms depending on the molecular class of the antigen. Activation of a B cell by a protein antigen requires the B cell to function as an APC, presenting the protein epitopes with MHC II to helper T cells. Because of their dependence on T cells for activation of B cells, protein antigens are classified as T-dependent antigens. In contrast, polysaccharides, lipopolysaccharides, and other nonprotein antigens are considered T-independent antigens because they can activate B cells without antigen processing and presentation to T cells.
Exercise \(2\)
1. What types of molecules serve as the BCR?
2. What are the differences between TCRs and BCRs with respect to antigen recognition?
3. Which molecule classes are T-dependent antigens and which are T-independent antigens?
T Cell-Independent Activation of B cells
Activation of B cells without the cooperation of helper T cells is referred to as T cell-independent activation and occurs when BCRs interact with T-independent antigens. T-independent antigens (e.g., polysaccharide capsules, lipopolysaccharide) have repetitive epitope units within their structure, and this repetition allows for the cross-linkageof multiple BCRs, providing the first signal for activation (Figure \(2\)). Because T cells are not involved, the second signal has to come from other sources, such as interactions of toll-like receptors with PAMPs or interactions with factors from the complement system.
Once a B cell is activated, it undergoes clonal proliferation and daughter cells differentiate into plasma cells. Plasma cells are antibody factories that secrete large quantities of antibodies. After differentiation, the surface BCRs disappear and the plasma cell secretes pentameric IgM molecules that have the same antigen specificity as the BCRs (Figure \(2\)).
The T cell-independent response is short-lived and does not result in the production of memory B cells. Thus it will not result in a secondary response to subsequent exposures to T-independent antigens.
Exercise \(3\)
1. What are the two signals required for T cell-independent activation of B cells?
2. What is the function of a plasma cell?
T Cell-Dependent Activation of B cells
T cell-dependent activation of B cells is more complex than T cell-independent activation, but the resulting immune response is stronger and develops memory. T cell-dependent activation can occur either in response to free protein antigens or to protein antigens associated with an intact pathogen. Interaction between the BCRs on a naïve mature B cell and a free protein antigen stimulate internalization of the antigen, whereas interaction with antigens associated with an intact pathogen initiates the extraction of the antigen from the pathogen before internalization. Once internalized inside the B cell, the protein antigen is processed and presented with MHC II. The presented antigen is then recognized by helper T cells specific to the same antigen. The TCR of the helper T cell recognizes the foreign antigen, and the T cell’s CD4 molecule interacts with MHC II on the B cell. The coordination between B cells and helper T cells that are specific to the same antigen is referred to as linked recognition.
Once activated by linked recognition, TH2 cells produce and secrete cytokines that activate the B cell and cause proliferation into clonal daughter cells. After several rounds of proliferation, additional cytokines provided by the TH2 cells stimulate the differentiation of activated B cell clones into memory B cells, which will quickly respond to subsequent exposures to the same protein epitope, and plasma cells that lose their membrane BCRs and initially secrete pentameric IgM (Figure \(3\)).
After initial secretion of IgM, cytokines secreted by TH2 cells stimulate the plasma cells to switch from IgM production to production of IgG, IgA, or IgE. This process, called class switching or isotype switching, allows plasma cellscloned from the same activated B cell to produce a variety of antibody classes with the same epitope specificity. Class switching is accomplished by genetic rearrangement of gene segments encoding the constant region, which determines an antibody’s class. The variable region is not changed, so the new class of antibody retains the original epitope specificity.
Exercise \(4\)
1. What steps are required for T cell-dependent activation of B cells?
2. What is antibody class switching and why is it important?
Primary and Secondary Responses
T cell-dependent activation of B cells plays an important role in both the primary and secondary responses associated with adaptive immunity. With the first exposure to a protein antigen, a T cell-dependent primary antibody responseoccurs. The initial stage of the primary response is a lag period, or latent period, of approximately 10 days, during which no antibody can be detected in serum. This lag period is the time required for all of the steps of the primary response, including naïve mature B cell binding of antigen with BCRs, antigen processing and presentation, helper T cell activation, B cell activation, and clonal proliferation. The end of the lag period is characterized by a rise in IgM levels in the serum, as TH2 cells stimulate B cell differentiation into plasma cells. IgM levels reach their peak around 14 days after primary antigen exposure; at about this same time, TH2 stimulates antibody class switching, and IgM levels in serum begin to decline. Meanwhile, levels of IgG increase until they reach a peak about three weeks into the primary response (Figure \(4\)).
During the primary response, some of the cloned B cells are differentiated into memory B cells programmed to respond to subsequent exposures. This secondary response occurs more quickly and forcefully than the primary response. The lag period is decreased to only a few days and the production of IgG is significantly higher than observed for the primary response (Figure \(4\)). In addition, the antibodies produced during the secondary response are more effective and bind with higher affinity to the targeted epitopes. Plasma cells produced during secondary responses live longer than those produced during the primary response, so levels of specific antibody remain elevated for a longer period of time.
Exercise \(5\)
1. What events occur during the lag period of the primary antibody response?
2. Why do antibody levels remain elevated longer during the secondary antibody response?
Key Concepts and Summary
• B lymphocytes or B cells produce antibodies involved in humoral immunity. B cells are produced in the bone marrow, where the initial stages of maturation occur, and travel to the spleen for final steps of maturation into naïve mature B cells.
• B-cell receptors (BCRs) are membrane-bound monomeric forms of IgD and IgM that bind specific antigen epitopes with their Fab antigen-binding regions. Diversity of antigen binding specificity is created by genetic rearrangement of V, D, and J segments similar to the mechanism used for TCR diversity.
• Protein antigens are called T-dependent antigens because they can only activate B cells with the cooperation of helper T cells. Other molecule classes do not require T cell cooperation and are called T-independent antigens.
• T cell-independent activation of B cells involves cross-linkage of BCRs by repetitive nonprotein antigen epitopes. It is characterized by the production of IgM by plasma cells and does not produce memory B cells.
• T cell-dependent activation of B cells involves processing and presentation of protein antigens to helper T cells, activation of the B cells by cytokines secreted from activated TH2 cells, and plasma cells that produce different classes of antibodies as a result of class switching. Memory B cells are also produced.
• Secondary exposures to T-dependent antigens result in a secondary antibody response initiated by memory B cells. The secondary response develops more quickly and produces higher and more sustained levels of antibody with higher affinity for the specific antigen. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/18%3A_Specific_Adaptive_Host_Defenses/18.04%3A_B_Lymphocytes_and_Antibodies.txt |
Learning Objectives
• Compare the various kinds of artificial immunity
• Differentiate between variolation and vaccination
• Describe different types of vaccines and explain their respective advantages and disadvantages
For many diseases, prevention is the best form of treatment, and few strategies for disease prevention are as effective as vaccination. Vaccination is a form of artificial immunity. By artificially stimulating the adaptive immune defenses, a vaccine triggers memory cell production similar to that which would occur during a primary response. In so doing, the patient is able to mount a strong secondary response upon exposure to the pathogen—but without having to first suffer through an initial infection. In this section, we will explore several different kinds of artificial immunity along with various types of vaccines and the mechanisms by which they induce artificial immunity.
Classifications of Adaptive Immunity
All forms of adaptive immunity can be described as either active or passive. Active immunity refers to the activation of an individual’s own adaptive immune defenses, whereas passive immunity refers to the transfer of adaptive immune defenses from another individual or animal. Active and passive immunity can be further subdivided based on whether the protection is acquired naturally or artificially.
Natural active immunity is adaptive immunity that develops after natural exposure to a pathogen (Figure \(1\)). Examples would include the lifelong immunity that develops after recovery from a chickenpox or measles infection (although an acute infection is not always necessary to activate adaptive immunity). The length of time that an individual is protected can vary substantially depending upon the pathogen and antigens involved. For example, activation of adaptive immunity by protein spike structures during an intracellular viral infection can activate lifelong immunity, whereas activation by carbohydrate capsule antigens during an extracellular bacterial infection may activate shorter-term immunity.
Natural passive immunity involves the natural passage of antibodies from a mother to her child before and after birth. IgG is the only antibody class that can cross the placenta from mother’s blood to the fetal blood supply. Placental transfer of IgG is an important passive immune defense for the infant, lasting up to six months after birth. Secretory IgA can also be transferred from mother to infant through breast milk.
Artificial passive immunity refers to the transfer of antibodies produced by a donor (human or animal) to another individual. This transfer of antibodies may be done as a prophylactic measure (i.e., to prevent disease after exposure to a pathogen) or as a strategy for treating an active infection. For example, artificial passive immunity is commonly used for post-exposure prophylaxis against rabies, hepatitis A, hepatitis B, and chickenpox (in high risk individuals). Active infections treated by artificial passive immunity include cytomegalovirus infections in immunocompromised patients and Ebola virus infections. In 1995, eight patients in the Democratic Republic of the Congo with active Ebola infections were treated with blood transfusions from patients who were recovering from Ebola. Only one of the eight patients died (a 12.5% mortality rate), which was much lower than the expected 80% mortality rate for Ebola in untreated patients.1 Artificial passive immunity is also used for the treatment of diseases caused by bacterial toxins, including tetanus, botulism, and diphtheria.
Artificial active immunity is the foundation for vaccination. It involves the activation of adaptive immunity through the deliberate exposure of an individual to weakened or inactivated pathogens, or preparations consisting of key pathogen antigens.
Exercise \(1\)
1. What is the difference between active and passive immunity?
2. What kind of immunity is conferred by a vaccine?
Herd Immunity
The four kinds of immunity just described result from an individual’s adaptive immune system. For any given disease, an individual may be considered immune or susceptible depending on his or her ability to mount an effective immune response upon exposure. Thus, any given population is likely to have some individuals who are immune and other individuals who are susceptible. If a population has very few susceptible individuals, even those susceptible individuals will be protected by a phenomenon called herd immunity. Herd immunity has nothing to do with an individual’s ability to mount an effective immune response; rather, it occurs because there are too few susceptible individuals in a population for the disease to spread effectively.
Vaccination programs create herd immunity by greatly reducing the number of susceptible individuals in a population. Even if some individuals in the population are not vaccinated, as long as a certain percentage is immune (either naturally or artificially), the few susceptible individuals are unlikely to be exposed to the pathogen. However, because new individuals are constantly entering populations (for example, through birth or relocation), vaccination programs are necessary to maintain herd immunity.
Vaccination: Obligation or Choice
A growing number of parents are choosing not to vaccinate their children. They are dubbed “antivaxxers,” and the majority of them believe that vaccines are a cause of autism (or other disease conditions), a link that has now been thoroughly disproven. Others object to vaccines on religious or moral grounds (e.g., the argument that Gardasil vaccination against HPV may promote sexual promiscuity), on personal ethical grounds (e.g., a conscientious objection to any medical intervention), or on political grounds (e.g., the notion that mandatory vaccinations are a violation of individual liberties).2
It is believed that this growing number of unvaccinated individuals has led to new outbreaks of whooping cough and measles. We would expect that herd immunity would protect those unvaccinated in our population, but herd immunity can only be maintained if enough individuals are being vaccinated.
Vaccination is clearly beneficial for public health. But from the individual parent’s perspective the view can be murkier. Vaccines, like all medical interventions, have associated risks, and while the risks of vaccination may be extremely low compared to the risks of infection, parents may not always understand or accept the consensus of the medical community. Do such parents have a right to withhold vaccination from their children? Should they be allowed to put their children—and society at large—at risk?
Many governments insist on childhood vaccinations as a condition for entering public school, but it has become easy in most states to opt out of the requirement or to keep children out of the public system. Since the 1970s, West Virginia and Mississippi have had in place a stringent requirement for childhood vaccination, without exceptions, and neither state has had a case of measles since the early 1990s. California lawmakers recently passed a similar law in response to a measles outbreak in 2015, making it much more difficult for parents to opt out of vaccines if their children are attending public schools. Given this track record and renewed legislative efforts, should other states adopt similarly strict requirements?
What role should health-care providers play in promoting or enforcing universal vaccination? Studies have shown that many parents’ minds can be changed in response to information delivered by health-care workers, but is it the place of health-care workers to try to persuade parents to have their children vaccinated? Some health-care providers are understandably reluctant to treat unvaccinated patients. Do they have the right to refuse service to patients who decline vaccines? Do insurance companies have the right to deny coverage to antivaxxers? These are all ethical questions that policymakers may be forced to address as more parents skirt vaccination norms.
Variolation and Vaccination
Thousands of years ago, it was first recognized that individuals who survived a smallpox infection were immune to subsequent infections. The practice of inoculating individuals to actively protect them from smallpox appears to have originated in the 10th century in China, when the practice of variolation was described (Figure \(2\)). Variolation refers to the deliberate inoculation of individuals with infectious material from scabs or pustules of smallpox victims. Infectious materials were either injected into the skin or introduced through the nasal route. The infection that developed was usually milder than naturally acquired smallpox, and recovery from the milder infection provided protection against the more serious disease.
Although the majority of individuals treated by variolation developed only mild infections, the practice was not without risks. More serious and sometimes fatal infections did occur, and because smallpox was contagious, infections resulting from variolation could lead to epidemics. Even so, the practice of variolation for smallpox prevention spread to other regions, including India, Africa, and Europe.
Although variolation had been practiced for centuries, the English physician Edward Jenner (1749–1823) is generally credited with developing the modern process of vaccination. Jenner observed that milkmaids who developed cowpox, a disease similar to smallpox but milder, were immune to the more serious smallpox. This led Jenner to hypothesize that exposure to a less virulent pathogen could provide immune protection against a more virulent pathogen, providing a safer alternative to variolation. In 1796, Jenner tested his hypothesis by obtaining infectious samples from a milkmaid’s active cowpox lesion and injecting the materials into a young boy (Figure \(3\)). The boy developed a mild infection that included a low-grade fever, discomfort in his axillae (armpit) and loss of appetite. When the boy was later infected with infectious samples from smallpox lesions, he did not contract smallpox.3 This new approach was termed vaccination, a name deriving from the use of cowpox (Latin vacca meaning “cow”) to protect against smallpox. Today, we know that Jenner’s vaccine worked because the cowpox virus is genetically and antigenically related to the Variola viruses that caused smallpox. Exposure to cowpox antigens resulted in a primary response and the production of memory cells that identical or related epitopes of Variola virus upon a later exposure to smallpox.
The success of Jenner’s smallpox vaccination led other scientists to develop vaccines for other diseases. Perhaps the most notable was Louis Pasteur, who developed vaccines for rabies, cholera, and anthrax. During the 20th and 21st centuries, effective vaccines were developed to prevent a wide range of diseases caused by viruses (e.g., chickenpox and shingles, hepatitis, measles, mumps, polio, and yellow fever) and bacteria (e.g., diphtheria, pneumococcal pneumonia, tetanus, and whooping cough).
Exercise \(2\)
1. What is the difference between variolation and vaccination for smallpox?
2. Explain why vaccination is less risky than variolation.
Classes of Vaccines
For a vaccine to provide protection against a disease, it must expose an individual to pathogen-specific antigens that will stimulate a protective adaptive immune response. By its very nature, this entails some risk. As with any pharmaceutical drug, vaccines have the potential to cause adverse effects. However, the ideal vaccine causes no severe adverse effects and poses no risk of contracting the disease that it is intended to prevent. Various types of vaccines have been developed with these goals in mind. These different classes of vaccines are described in the next section and summarized in Table \(1\).
Live Attenuated Vaccines
Live attenuated vaccines expose an individual to a weakened strain of a pathogen with the goal of establishing a subclinical infection that will activate the adaptive immune defenses. Pathogens are attenuated to decrease their virulence using methods such as genetic manipulation (to eliminate key virulence factors) or long-term culturing in an unnatural host or environment (to promote mutations and decrease virulence).
By establishing an active infection, live attenuated vaccines stimulate a more comprehensive immune response than some other types of vaccines. Live attenuated vaccines activate both cellular and humoral immunity and stimulate the development of memory for long-lasting immunity. In some cases, vaccination of one individual with a live attenuated pathogen can even lead to natural transmission of the attenuated pathogen to other individuals. This can cause the other individuals to also develop an active, subclinical infection that activates their adaptive immune defenses.
Disadvantages associated with live attenuated vaccines include the challenges associated with long-term storage and transport as well as the potential for a patient to develop signs and symptoms of disease during the active infection (particularly in immunocompromised patients). There is also a risk of the attenuated pathogen reverting back to full virulence. Table \(1\) lists examples of live attenuated vaccines.
Inactivated Vaccines
Inactivated vaccines contain whole pathogens that have been killed or inactivated with heat, chemicals, or radiation. For inactivated vaccines to be effective, the inactivation process must not affect the structure of key antigens on the pathogen.
Because the pathogen is killed or inactive, inactivated vaccines do not produce an active infection, and the resulting immune response is weaker and less comprehensive than that provoked by a live attenuated vaccine. Typically the response involves only humoral immunity, and the pathogen cannot be transmitted to other individuals. In addition, inactivated vaccines usually require higher doses and multiple boosters, possibly causing inflammatory reactions at the site of injection.
Despite these disadvantages, inactivated vaccines do have the advantages of long-term storage stability and ease of transport. Also, there is no risk of causing severe active infections. However, inactivated vaccines are not without their side effects. Table \(1\) lists examples of inactivated vaccines.
Subunit Vaccines
Whereas live attenuated and inactive vaccines expose an individual to a weakened or dead pathogen, subunit vaccines only expose the patient to the key antigens of a pathogen—not whole cells or viruses. Subunit vaccines can be produced either by chemically degrading a pathogen and isolating its key antigens or by producing the antigens through genetic engineering. Because these vaccines contain only the essential antigens of a pathogen, the risk of side effects is relatively low. Table \(1\) lists examples of subunit vaccines.
Toxoid Vaccines
Like subunit vaccines, toxoid vaccines do not introduce a whole pathogen to the patient; they contain inactivated bacterial toxins, called toxoids. Toxoid vaccines are used to prevent diseases in which bacterial toxins play an important role in pathogenesis. These vaccines activate humoral immunity that neutralizes the toxins. Table \(1\) lists examples of toxoid vaccines.
Conjugate Vaccines
A conjugate vaccine is a type of subunit vaccine that consists of a protein conjugated to a capsule polysaccharide. Conjugate vaccines have been developed to enhance the efficacy of subunit vaccines against pathogens that have protective polysaccharide capsules that help them evade phagocytosis, causing invasive infections that can lead to meningitis and other serious conditions. The subunit vaccines against these pathogens introduce T-independent capsular polysaccharide antigens that result in the production of antibodies that can opsonize the capsule and thus combat the infection; however, children under the age of two years do not respond effectively to these vaccines. Children do respond effectively when vaccinated with the conjugate vaccine, in which a protein with T-dependent antigens is conjugated to the capsule polysaccharide. The conjugated protein-polysaccharide antigen stimulates production of antibodies against both the protein and the capsule polysaccharide. Table \(1\) lists examples of conjugate vaccines.
Table \(1\): Classes of Vaccines
Class Description Advantages Disadvantages Examples
Live attenuated Weakened strain of whole pathogen Cellular and humoral immunity Difficult to store and transport Chickenpox, German measles, measles, mumps, tuberculosis, typhoid fever, yellow fever
Long-lasting immunity Risk of infection in immunocompromised patients
Transmission to contacts Risk of reversion
Inactivated Whole pathogen killed or inactivated with heat, chemicals, or radiation Ease of storage and transport Weaker immunity (humoral only) Cholera, hepatitis A, influenza, plague, rabies
No risk of severe active infection Higher doses and more boosters required
Subunit Immunogenic antigens Lower risk of side effects Limited longevity Anthrax, hepatitis B, influenza, meningitis, papillomavirus, pneumococcal pneumonia, whooping cough
Multiple doses required
No protection against antigenic variation
Toxoid Inactivated bacterial toxin Humoral immunity to neutralize toxin Does not prevent infection Botulism, diphtheria, pertussis, tetanus
Conjugate Capsule polysaccharide conjugated to protein T-dependent response to capsule Costly to produce
Meningitis
(Haemophilus influenzae, Streptococcus pneumoniae, Neisseria meningitides)
No protection against antigenic variation
Better response in young children May interfere with other vaccines
Exercise \(3\)
1. What is the risk associated with a live attenuated vaccine?
2. Why is a conjugated vaccine necessary in some cases?
DNA Vaccines
DNA vaccines represent a relatively new and promising approach to vaccination. A DNA vaccine is produced by incorporating genes for antigens into a recombinant plasmid vaccine. Introduction of the DNA vaccine into a patient leads to uptake of the recombinant plasmid by some of the patient’s cells, followed by transcription and translation of antigens and presentation of these antigens with MHC I to activate adaptive immunity. This results in the stimulation of both humoral and cellular immunity without the risk of active disease associated with live attenuated vaccines.
Although most DNA vaccines for humans are still in development, it is likely that they will become more prevalent in the near future as researchers are working on engineering DNA vaccines that will activate adaptive immunity against several different pathogens at once. First-generation DNA vaccines tested in the 1990s looked promising in animal models but were disappointing when tested in human subjects. Poor cellular uptake of the DNA plasmids was one of the major problems impacting their efficacy. Trials of second-generation DNA vaccines have been more promising thanks to new techniques for enhancing cellular uptake and optimizing antigens. DNA vaccines for various cancers and viral pathogens such as HIV, HPV, and hepatitis B and C are currently in development.
Some DNA vaccines are already in use. In 2005, a DNA vaccine against West Nile virus was approved for use in horses in the United States. Canada has also approved a DNA vaccine to protect fish from infectious hematopoietic necrosis virus.4 A DNA vaccine against Japanese encephalitis virus was approved for use in humans in 2010 in Australia.
Clinical Focus: Resolution
Based on Olivia’s symptoms, her physician made a preliminary diagnosis of bacterial meningitis without waiting for positive identification from the blood and CSF samples sent to the lab. Olivia was admitted to the hospital and treated with intravenous broad-spectrum antibiotics and rehydration therapy. Over the next several days, her condition began to improve, and new blood samples and lumbar puncture samples showed an absence of microbes in the blood and CSF with levels of white blood cells returning to normal. During this time, the lab produced a positive identification of Neisseria meningitidis, the causative agent of meningococcal meningitis, in her original CSF sample.
N. meningitidis produces a polysaccharide capsule that serves as a virulence factor. N. meningitidis tends to affect infants after they begin to lose the natural passive immunity provided by maternal antibodies. At one year of age, Olivia’s maternal IgG antibodies would have disappeared, and she would not have developed memory cells capable of recognizing antigens associated with the polysaccharide capsule of the N. meningitidis. As a result, her adaptive immune system was unable to produce protective antibodies to combat the infection, and without antibiotics she may not have survived. Olivia’s infection likely would have been avoided altogether had she been vaccinated. A conjugate vaccine to prevent meningococcal meningitis is available and approved for infants as young as two months of age. However, current vaccination schedules in the United States recommend that the vaccine be administered at age 11–12 with a booster at age 16.
Link to Learning
In countries with developed public health systems, many vaccines are routinely administered to children and adults. Vaccine schedules are changed periodically, based on new information and research results gathered by public health agencies. In the United States, the CDC publishes schedules and other updated information about vaccines.
Key Concepts and Summary
• Adaptive immunity can be divided into four distinct classifications: natural active immunity, natural passive immunity, artificial passive immunity, and artificial active immunity.
• Artificial active immunity is the foundation for vaccination and vaccine development. Vaccination programs not only confer artificial immunity on individuals, but also foster herd immunity in populations.
• Variolation against smallpox originated in the 10th century in China, but the procedure was risky because it could cause the disease it was intended to prevent. Modern vaccination was developed by Edward Jenner, who developed the practice of inoculating patients with infectious materials from cowpox lesions to prevent smallpox.
• Live attenuated vaccines and inactivated vaccines contain whole pathogens that are weak, killed, or inactivated. Subunit vaccines, toxoid vaccines, and conjugate vaccines contain acellular components with antigens that stimulate an immune response.
Footnotes
1. 1 K. Mupapa, M. Massamba, K. Kibadi, K. Kivula, A. Bwaka, M. Kipasa, R. Colebunders, J. J. Muyembe-Tamfum. “Treatment of Ebola Hemorrhagic Fever with Blood Transfusions from Convalescent Patients.” Journal of Infectious Diseases 179 Suppl. (1999): S18–S23.
2. 2 Elizabeth Yale. “Why Anti-Vaccination Movements Can Never Be Tamed.” Religion & Politics, July 22, 2014. religionandpolitics.org/2014/...never-be-tamed.
3. 3 N. J. Willis. “Edward Jenner and the Eradication of Smallpox.” Scottish Medical Journal 42 (1997): 118–121.
4. 4 M. Alonso and J. C. Leong. “Licensed DNA Vaccines Against Infectious Hematopoietic Necrosis Virus (IHNV).” Recent Patents on DNA & Gene Sequences (Discontinued) 7 no. 1 (2013): 62–65, issn 1872-2156/2212-3431. doi 10.2174/1872215611307010009.
5. 5 S.B. Halstead and S. J. Thomas. “New Japanese Encephalitis Vaccines: Alternatives to Production in Mouse Brain.” Expert Review of Vaccines 10 no. 3 (2011): 355–64. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/18%3A_Specific_Adaptive_Host_Defenses/18.05%3A_Vaccines.txt |
18.1: Architecture of the Immune System
Adaptive immunity is defined by two important characteristics: specificity and memory. Specificity refers to the adaptive immune system’s ability to target specific pathogens, and memory refers to its ability to quickly respond to pathogens to which it has previously been exposed. For example, when an individual recovers from chickenpox, the body develops a memory of the infection that will specifically protect it from the causative agent if it is exposed to the virus again later.
Multiple Choice
Antibodies are produced by ________.
1. plasma cells
2. T cells
3. bone marrow
4. B cells
Answer
A
Cellular adaptive immunity is carried out by ________.
1. B cells
2. T cells
3. bone marrow
4. neutrophils
Answer
B
A single antigen molecule may be composed of many individual ________.
1. T-cell receptors
2. B-cell receptors
3. MHC II
4. epitopes
Answer
D
Which class of molecules is the most antigenic?
1. polysaccharides
2. lipids
3. proteins
4. carbohydrates
Answer
C
Matching
Match the antibody class with its description.
___IgA A. This class of antibody is the only one that can cross the placenta.
___IgD B. This class of antibody is the first to appear after activation of B cells.
___IgE C. This class of antibody is involved in the defense against parasitic infections and involved in allergic responses.
___IgG D. This class of antibody is found in very large amounts in mucus secretions.
___IgM E. This class of antibody is not secreted by B cells but is expressed on the surface of naïve B cells.
Answer
d, e, c, a, b
Fill in the Blank
There are two critically important aspects of adaptive immunity. The first is specificity, while the second is ________.
Answer
memory
________ immunity involves the production of antibody molecules that bind to specific antigens.
Answer
Humoral
The heavy chains of an antibody molecule contain ________ region segments, which help to determine its class or isotype.
Answer
constant
The variable regions of the heavy and light chains form the ________ sites of an antibody.
Answer
antigen-binding
Short Answer
What is the difference between humoral and cellular adaptive immunity?
What is the difference between an antigen and a hapten?
Describe the mechanism of antibody-dependent cell-mediated cytotoxicity.
18.2: Antigens, Antigen Presenting Cells, and Major Histocompatibility Complexes
Major histocompatibility complex (MHC) molecules are expressed on the surface of healthy cells, identifying them as normal and “self” to natural killer (NK) cells. MHC molecules also play an important role in the presentation of foreign antigens, which is a critical step in the activation of T cells and thus an important mechanism of the adaptive immune system.
Multiple Choice
MHC I molecules present
1. processed foreign antigens from proteasomes.
2. processed self-antigens from phagolysosome.
3. antibodies.
4. T cell antigens.
Answer
A
MHC II molecules present
1. processed self-antigens from proteasomes.
2. processed foreign antigens from phagolysosomes.
3. antibodies.
4. T cell receptors.
Answer
B
Which type of antigen-presenting molecule is found on all nucleated cells?
1. MHC II
2. MHC I
3. antibodies
4. B-cell receptors
Answer
B
Which type of antigen-presenting molecule is found only on macrophages, dendritic cells, and B cells?
1. MHC I
2. MHC II
3. T-cell receptors
4. B-cell receptors
Answer
B
Fill in the Blank
MHC molecules are used for antigen ________ to T cells.
Answer
presentation
MHC II molecules are made up of two subunits (α and β) of approximately equal size, whereas MHC I molecules consist of a larger α subunit and a smaller subunit called ________.
Answer
β2 microglobulin
Critical Thinking
Which mechanism of antigen presentation would be used to present antigens from a cell infected with a virus?
Which pathway of antigen presentation would be used to present antigens from an extracellular bacterial infection?
18.3: T Lymphocytes
The antibodies involved in humoral immunity often bind pathogens and toxins before they can attach to and invade host cells. Thus, humoral immunity is primarily concerned with fighting pathogens in extracellular spaces. However, pathogens that have already gained entry to host cells are largely protected from the humoral antibody-mediated defenses. Cellular immunity, on the other hand, targets and eliminates intracellular pathogens through the actions of T lymphocytes, or T cells.
Multiple Choice
What is a superantigen?
1. a protein that is highly efficient at stimulating a single type of productive and specific T cell response
2. a protein produced by antigen-presenting cells to enhance their presentation capabilities
3. a protein produced by T cells as a way of increasing the antigen activation they receive from antigen-presenting cells
4. a protein that activates T cells in a nonspecific and uncontrolled manner
Answer
D
To what does the TCR of a helper T cell bind?
1. antigens presented with MHC I molecules
2. antigens presented with MHC II molecules
3. free antigen in a soluble form
4. haptens only
Answer
B
Cytotoxic T cells will bind with their TCR to which of the following?
1. antigens presented with MHC I molecules
2. antigens presented with MHC II molecules
3. free antigen in a soluble form
4. haptens only
Answer
A
A ________ molecule is a glycoprotein used to identify and distinguish white blood cells.
1. T-cell receptor
2. B-cell receptor
3. MHC I
4. cluster of differentiation
Answer
D
Name the T helper cell subset involved in antibody production.
1. TH1
2. TH2
3. TH17
4. CTL
Answer
B
Fill in the Blank
A ________ T cell will become activated by presentation of foreign antigen associated with an MHC I molecule.
Answer
cytotoxic
A ________ T cell will become activated by presentation of foreign antigen in association with an MHC II molecule.
Answer
helper
A TCR is a protein dimer embedded in the plasma membrane of a T cell. The ________ region of each of the two protein chains is what gives it the capability to bind to a presented antigen.
Answer
variable
Peripheral tolerance mechanisms function on T cells after they mature and exit the ________.
Answer
thymus
Both ________ and effector T cells are produced during differentiation of activated T cells.
Answer
memory
Short Answer
What is the basic difference in effector function between helper and cytotoxic T cells?
What necessary interactions are required for activation of helper T cells and activation/effector function of cytotoxic T cells?
18.4: B Lymphocytes and Antibodies
Humoral immunity refers to mechanisms of the adaptive immune defenses that are mediated by antibodies secreted by B lymphocytes, or B cells. This section focuses on B cells and discusses their production and maturation, receptors, and mechanisms of activation.
Multiple Choice
Which of the following would be a T-dependent antigen?
1. lipopolysaccharide
2. glycolipid
3. protein
4. carbohydrate
Answer
C
Which of the following would be a BCR?
1. CD4
2. MHC II
3. MHC I
4. IgD
Answer
D
Which of the following does not occur during the lag period of the primary antibody response?
1. activation of helper T cells
2. class switching to IgG
3. presentation of antigen with MHC II
4. binding of antigen to BCRs
Answer
B
Fill in the Blank
________ antigens can stimulate B cells to become activated but require cytokine assistance delivered by helper T cells.
Answer
T-dependent
T-independent antigens can stimulate B cells to become activated and secrete antibodies without assistance from helper T cells. These antigens possess ________ antigenic epitopes that cross-link BCRs.
Answer
repetitive
Critical Thinking
A patient lacks the ability to make functioning T cells because of a genetic disorder. Would this patient’s B cells be able to produce antibodies in response to an infection? Explain your answer.
18.5: Vaccines
By artificially stimulating the adaptive immune defenses, a vaccine triggers memory cell production similar to that which would occur during a primary response. In so doing, the patient is able to mount a strong secondary response upon exposure to the pathogen—but without having to first suffer through an initial infection. In this section, we explore several different kinds of artificial immunity along with various types of vaccines and their mechanisms for inducing artificial immunity.
Multiple Choice
A patient is bitten by a dog with confirmed rabies infection. After treating the bite wound, the physician injects the patient with antibodies that are specific for the rabies virus to prevent the development of an active infection. This is an example of:
1. Natural active immunity
2. Artificial active immunity
3. Natural passive immunity
4. Artificial passive immunity
Answer
D
A patient gets a cold, and recovers a few days later. The patient's classmates come down with the same cold roughly a week later, but the original patient does not get the same cold again. This is an example of:
1. Natural active immunity
2. Artificial active immunity
3. Natural passive immunity
4. Artificial passive immunity
Answer
A
Matching
Match each type of vaccine with the corresponding example.
___inactivated vaccine A. Weakened influenza virions that can only replicate in the slightly lower temperatures of the nasal passages are sprayed into the nose. They do not cause serious flu symptoms, but still produce an active infection that induces a protective adaptive immune response.
___live attenuated vaccine B. Tetanus toxin molecules are harvested and chemically treated to render them harmless. They are then injected into a patient’s arm.
___toxoid vaccine C. Influenza virus particles grown in chicken eggs are harvested and chemically treated to render them noninfectious. These immunogenic particles are then purified and packaged and administered as an injection.
___subunit vaccine D. The gene for hepatitis B virus surface antigen is inserted into a yeast genome. The modified yeast is grown and the virus protein is produced, harvested, purified, and used in a vaccine.
Answer
C, A, B, D
Fill in the Blank
A(n) ________ pathogen is in a weakened state; it is still capable of stimulating an immune response but does not cause a disease.
Answer
attenuated
________ immunity occurs when antibodies from one individual are harvested and given to another to protect against disease or treat active disease.
Answer
Artificial passive
In the practice of ________, scabs from smallpox victims were used to immunize susceptible individuals against smallpox.
Answer
variolation
Short answer
Briefly compare the pros and cons of inactivated versus live attenuated vaccines. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/18%3A_Specific_Adaptive_Host_Defenses/18.E%3A_Specific_Adaptive_Host_Defenses_%28Exercises%29.txt |
An allergic reaction is an immune response to a type of antigen called an allergen. Allergens can be found in many different items, from peanuts and insect stings to latex and some drugs. Unlike other kinds of antigens, allergens are not necessarily associated with pathogenic microbes, and many allergens provoke no immune response at all in most people.
Allergic responses vary in severity. Some are mild and localized, like hay fever or hives, but others can result in systemic, life-threatening reactions. Anaphylaxis, for example, is a rapidly developing allergic reaction that can cause a dangerous drop in blood pressure and severe swelling of the throat that may close off the airway.
Allergies are just one example of how the immune system—the system normally responsible for preventing disease—can actually cause or mediate disease symptoms. In this chapter, we will further explore allergies and other disorders of the immune system, including hypersensitivity reactions, autoimmune diseases, transplant rejection, and diseases associated with immunodeficiency.
• 19.1: Hypersensitivities
An allergy is an adaptive immune response, sometimes life-threatening, to an allergen. Hypersensitivity reactions are classified by their immune mechanism.
• 19.2: Autoimmune Disorders
Autoimmune diseases result from a breakdown in immunological tolerance. The actual induction event(s) for autoimmune states are largely unknown. Some autoimmune diseases attack specific organs, whereas others are more systemic. Organ-specific autoimmune diseases include celiac disease, Graves disease, Hashimoto thyroiditis, type I diabetes mellitus, and Addison disease.
• 19.3: Organ Transplantation and Rejection
Grafts and transplants can be classified as autografts, isografts, allografts, or xenografts based on the genetic differences between the donor’s and recipient’s tissues. Genetic differences, especially among the MHC (HLA) genes, will dictate the likelihood that rejection of the transplanted tissue will occur. Transplant recipients usually require immunosuppressive therapy to avoid rejection, even with good genetic matching.
• 19.4: Immunodeficiency
Primary immunodeficiencies are caused by genetic abnormalities; secondary immunodeficiencies are acquired through disease, diet, or environmental exposures. Primary immunodeficiencies may result from flaws in phagocyte killing of innate immunity, or impairment of T cells and B cells. Primary immunodeficiencies include chronic granulomatous disease, X-linked agammaglobulinemia, selective IgA deficiency, and severe combined immunodeficiency disease.
• 19.5: Cancer Immunobiology and Immunotherapy
When control of the cell cycle is lost, the affected cells rapidly divide and often lose the ability to differentiate into the cell type appropriate for their location in the body. In addition, they lose contact inhibition and can start to grow on top of each other. This can result in formation of a tumor. It is important to make a distinction here: The term “cancer” is used to describe the diseases resulting from loss of cell-cycle regulation and subsequent cell proliferation.
• 19.E: Diseases of the Immune System (Exercises)
Thumbnail: Allergens in plant pollen, shown here in a colorized electron micrograph, may trigger allergic rhinitis or hay fever in sensitive individuals. (Public Domain/modified from original; Dartmouth Electron Microscope Facility, Dartmouth College via Wikimedia Commons).
19: Diseases of the Immune System
Learning Objectives
• Identify and compare the distinguishing characteristics, mechanisms, and major examples of type I, II, III, and IV hypersensitivities
Clinical Focus: Part 1
Kerry, a 40-year-old airline pilot, has made an appointment with her primary care physician to discuss a rash that develops whenever she spends time in the sun. As she explains to her physician, it does not seem like sunburn. She is careful not to spend too much time in the sun and she uses sunscreen. Despite these precautions, the rash still appears, manifesting as red, raised patches that get slightly scaly. The rash persists for 7 to 10 days each time, and it seems to largely go away on its own. Lately, the rashes have also begun to appear on her cheeks and above her eyes on either side of her forehead.
Exercise \(1\)
1. Is Kerry right to be concerned, or should she simply be more careful about sun exposure?
2. Are there conditions that might be brought on by sun exposure that Kerry’s physician should be considering?
In Adaptive Specific Host Defenses, we discussed the mechanisms by which adaptive immune defenses, both humoral and cellular, protect us from infectious diseases. However, these same protective immune defenses can also be responsible for undesirable reactions called hypersensitivity reactions. Hypersensitivity reactions are classified by their immune mechanism.
• Type I hypersensitivity reactions involve immunoglobulin E (IgE) antibody against soluble antigen, triggering mast cell degranulation.
• Type II hypersensitivity reactions involve IgG and IgM antibodies directed against cellular antigens, leading to cell damage mediated by other immune system effectors.
• Type III hypersensitivity reactions involve the interactions of IgG, IgM, and, occasionally, IgA1 antibodies with antigen to form immune complexes. Accumulation of immune complexes in tissue leads to tissue damage mediated by other immune system effectors.
• Type IV hypersensitivity reactions are T-cell–mediated reactions that can involve tissue damage mediated by activated macrophages and cytotoxic T cells.
Type I Hypersensitivities
When a presensitized individual is exposed to an allergen, it can lead to a rapid immune response that occurs almost immediately. Such a response is called an allergy and is classified as a type I hypersensitivity. Allergens may be seemingly harmless substances such as animal dander, molds, or pollen. Allergens may also be substances considered innately more hazardous, such as insect venom or therapeutic drugs. Food intolerances can also yield allergic reactions as individuals become sensitized to foods such as peanuts or shellfish (Figure \(1\)). Regardless of the allergen, the first exposure activates a primary IgE antibody response that sensitizes an individual to type I hypersensitivity reaction upon subsequent exposure.
For susceptible individuals, a first exposure to an allergen activates a strong TH2 cell response (Figure \(2\)). Cytokines interleukin (IL)-4 and IL-13 from the TH2 cells activate B cells specific to the same allergen, resulting in clonal proliferation, differentiation into plasma cells, and antibody-class switch from production of IgM to production of IgE. The fragment crystallizable (Fc) regions of the IgE antibodies bind to specific receptors on the surface of mast cellsthroughout the body. It is estimated that each mast cell can bind up to 500,000 IgE molecules, with each IgE molecule having two allergen-specific fragment antigen-binding (Fab) sites available for binding allergen on subsequent exposures. By the time this occurs, the allergen is often no longer present and there is no allergic reaction, but the mast cells are primed for a subsequent exposure and the individual is sensitized to the allergen.
On subsequent exposure, allergens bind to multiple IgE molecules on mast cells, cross-linking the IgE molecules. Within minutes, this cross-linking of IgE activates the mast cells and triggers degranulation, a reaction in which the contents of the granules in the mast cell are released into the extracellular environment. Preformed components that are released from granules include histamine, serotonin, and bradykinin (Table \(1\)). The activated mast cells also release newly formed lipid mediators (leukotrienes and prostaglandins from membrane arachadonic acid metabolism) and cytokines such as tumor necrosis factor (Table \(2\)).
Table \(1\): Selected Preformed Components of Mast Cell Granules
Granule Component Activity
Heparin Stimulates the generation of bradykinin, which causes increased vascular permeability, vasodilation, bronchiole constriction, and increased mucus secretion
Histamine Causes smooth-muscle contraction, increases vascular permeability, increases mucus and tear formation
Serotonin Increases vascular permeability, causes vasodilation and smooth-muscle contraction
The chemical mediators released by mast cells collectively cause the inflammation and signs and symptoms associated with type I hypersensitivity reactions. Histamine stimulates mucus secretion in nasal passages and tear formation from lacrimal glands, promoting the runny nose and watery eyes of allergies. Interaction of histamine with nerve endings causes itching and sneezing. The vasodilation caused by several of the mediators can result in hives, headaches, angioedema (swelling that often affects the lips, throat, and tongue), and hypotension (low blood pressure). Bronchiole constriction caused by some of the chemical mediators leads to wheezing, dyspnea (difficulty breathing), coughing, and, in more severe cases, cyanosis (bluish color to the skin or mucous membranes). Vomiting can result from stimulation of the vomiting center in the cerebellum by histamine and serotonin. Histamine can also cause relaxation of intestinal smooth muscles and diarrhea.
Table \(2\): Selected Newly Formed Chemical Mediators of Inflammation and Allergic Response
Chemical Mediator Activity
Leukotriene Causes smooth-muscle contraction and mucus secretion, increases vascular permeability
Prostaglandin Causes smooth-muscle contraction and vasodilation
TNF-α (cytokine) Causes inflammation and stimulates cytokine production by other cell types
Type I hypersensitivity reactions can be either localized or systemic. Localized type I hypersensitivity reactions include hay fever rhinitis, hives, and asthma (Table \(3\)). Systemic type I hypersensitivity reactions are referred to as anaphylaxisor anaphylactic shock. Although anaphylaxis shares many symptoms common with the localized type I hypersensitivity reactions, the swelling of the tongue and trachea, blockage of airways, dangerous drop in blood pressure, and development of shock can make anaphylaxis especially severe and life-threatening. In fact, death can occur within minutes of onset of signs and symptoms.
Late-phase reactions in type I hypersensitivities may develop 4–12 hours after the early phase and are mediated by eosinophils, neutrophils, and lymphocytes that have been recruited by chemotactic factors released from mast cells. Activation of these recruited cells leads to the release of more chemical mediators that cause tissue damage and late-phase symptoms of swelling and redness of the skin, coughing, wheezing, and nasal discharge.
Individuals who possess genes for maladaptive traits, such as intense type I hypersensitivity reactions to otherwise harmless components of the environment, would be expected to suffer reduced reproductive success. With this kind of evolutionary selective pressure, such traits would not be expected to persist in a population. This suggests that type I hypersensitivities may have an adaptive function. There is evidence that the IgE produced during type I hypersensitivity reactions is actually meant to counter helminth infections2. Helminths are one of few organisms that possess proteins that are targeted by IgE. In addition, there is evidence that helminth infections at a young age reduce the likelihood of type I hypersensitivities to innocuous substances later in life. Thus it may be that allergies are an unfortunate consequence of strong selection in the mammalian lineage or earlier for a defense against parasitic worms.
Table \(3\): Type I Hypersensitivities
Common Name Cause Signs and Symptoms
Allergy-induced asthma Inhalation of allergens Constriction of bronchi, labored breathing, coughing, chills, body aches
Anaphylaxis Systemic reaction to allergens Hives, itching, swelling of tongue and throat, nausea, vomiting, low blood pressure, shock
Hay fever Inhalation of mold or pollen Runny nose, watery eyes, sneezing
Hives (urticaria) Food or drug allergens, insect stings Raised, bumpy skin rash with itching; bumps may converge into large raised areas
Exercise \(2\)
1. What are the cells that cause a type I hypersensitivity reaction?
2. Describe the differences between immediate and late-phase type I hypersensitivity reactions.
3. List the signs and symptoms of anaphylaxis.
The Hygiene Hypothesis
In most modern societies, good hygiene is associated with regular bathing, and good health with cleanliness. But some recent studies suggest that the association between health and clean living may be a faulty one. Some go so far as to suggest that children should be encouraged to play in the dirt—or even eat dirt3—for the benefit of their health. This recommendation is based on the so-called hygiene hypothesis, which proposes that childhood exposure to antigens from a diverse range of microbes leads to a better-functioning immune system later in life.
The hygiene hypothesis was first suggested in 1989 by David Strachan4, who observed an inverse relationship between the number of older children in a family and the incidence of hay fever. Although hay fever in children had increased dramatically during the mid-20th century, incidence was significantly lower in families with more children. Strachan proposed that the lower incidence of allergies in large families could be linked to infections acquired from older siblings, suggesting that these infections made children less susceptible to allergies. Strachan also argued that trends toward smaller families and a greater emphasis on cleanliness in the 20th century had decreased exposure to pathogens and thus led to higher overall rates of allergies, asthma, and other immune disorders.
Other researchers have observed an inverse relationship between the incidence of immune disorders and infectious diseases that are now rare in industrialized countries but still common in less industrialized countries.5 In developed nations, children under the age of 5 years are not exposed to many of the microbes, molecules, and antigens they almost certainly would have encountered a century ago. The lack of early challenges to the immune system by organisms with which humans and their ancestors evolved may result in failures in immune system functioning later in life.
Type II (Cytotoxic) Hypersensitivities
Immune reactions categorized as type II hypersensitivities, or cytotoxic hypersensitivities, are mediated by IgG and IgM antibodies binding to cell-surface antigens or matrix-associated antigens on basement membranes. These antibodies can either activate complement, resulting in an inflammatory response and lysis of the targeted cells, or they can be involved in antibody-dependent cell-mediated cytotoxicity (ADCC) with cytotoxic T cells.
In some cases, the antigen may be a self-antigen, in which case the reaction would also be described as an autoimmune disease. (Autoimmune diseases are described in Autoimmune Disorders). In other cases, antibodies may bind to naturally occurring, but exogenous, cell-surface molecules such as antigens associated with blood typing found on red blood cells (RBCs). This leads to the coating of the RBCs by antibodies, activation of the complement cascade, and complement-mediated lysis of RBCs, as well as opsonization of RBCs for phagocytosis. Two examples of type II hypersensitivity reactions involving RBCs are hemolytic transfusion reaction (HTR) and hemolytic disease of the newborn (HDN). These type II hypersensitivity reactions, which will be discussed in greater detail, are summarized in Table \(4\).
Immunohematology is the study of blood and blood-forming tissue in relation to the immune response. Antibody-initiated responses against blood cells are type II hypersensitivities, thus falling into the field of immunohematology. For students first learning about immunohematology, understanding the immunological mechanisms involved is made even more challenging by the complex nomenclature system used to identify different blood-group antigens, often called blood types. The first blood-group antigens either used alphabetical names or were named for the first person known to produce antibodies to the red blood cell antigen (e.g., Kell, Duffy, or Diego). However, in 1980, the International Society of Blood Transfusion (ISBT) Working Party on Terminology created a standard for blood-group terminology in an attempt to more consistently identify newly discovered blood group antigens. New antigens are now given a number and assigned to a blood-group system, collection, or series. However, even with this effort, blood-group nomenclature is still inconsistent.
Table \(4\): Common Type II Hypersensitivities
Common Name Cause Signs and Symptoms
Hemolytic disease of the newborn (HDN) IgG from mother crosses the placenta, targeting the fetus’ RBCs for destruction Anemia, edema, enlarged liver or spleen, hydrops (fluid in body cavity), leading to death of newborn in severe cases
Hemolytic transfusion reactions (HTR) IgG and IgM bind to antigens on transfused RBCs, targeting donor RBCs for destruction Fever, jaundice, hypotension, disseminated intravascular coagulation, possibly leading to kidney failure and death
ABO Blood Group Incompatibility
The recognition that individuals have different blood types was first described by Karl Landsteiner (1868–1943) in the early 1900s, based on his observation that serum from one person could cause a clumping of RBCs from another. These studies led Landsteiner to the identification of four distinct blood types. Subsequent research by other scientists determined that the four blood types were based on the presence or absence of surface glycoproteins “A” and “B,” and this provided the foundation for the ABO blood group system that is still in use today (Figure \(3\)). The functions of these antigens are unknown, but some have been associated with normal biochemical functions of the cell. Furthermore, ABO blood types are inherited as alleles (one from each parent), and they display patterns of dominant and codominant inheritance. The alleles for A and B blood types are codominant to each other, and both are dominant over blood type O. Therefore, individuals with genotypes of AA or AO have type A blood and express the A glycoprotein antigen on the surface of their RBCs. People with genotypes of BB or BO have type B blood and express the B glycoprotein antigen on the surface of their RBCs. Those with a genotype of AB have type AB blood and express both A and B glycoprotein antigens on the surface of their RBCs. Finally, individuals with a genotype of OO have type O blood and lack A and B glycoproteins on the surface of their RBCs.
It is important to note that the RBCs of all four ABO blood types share a common protein receptor molecule, and it is the addition of specific carbohydrates to the protein receptors that determines A, B, and AB blood types. The genes that are inherited for the A, B, and AB blood types encode enzymes that add the carbohydrate component to the protein receptor. Individuals with O blood type still have the protein receptor but lack the enzymes that would add carbohydrates that would make their red blood cell type A, B, or AB.
IgM antibodies in plasma that cross-react with blood group antigens not present on an individual’s own RBCs are called isohemagglutinins (Figure \(3\)). Isohemagglutinins are produced within the first few weeks after birth and persist throughout life. These antibodies are produced in response to exposure to environmental antigens from food and microorganisms. A person with type A blood has A antigens on the surface of their RBCs and will produce anti-B antibodies to environmental antigens that resemble the carbohydrate component of B antigens. A person with type B blood has B antigens on the surface of their RBCs and will produce anti-A antibodies to environmental antigens that are similar to the carbohydrate component of A antigens. People with blood type O lack both A and B antigens on their RBCs and, therefore, produce both anti-A and anti-B antibodies. Conversely, people with AB blood type have both A and B antigens on their RBCs and, therefore, lack anti-A and anti-B antibodies.
A patient may require a blood transfusion because they lack sufficient RBCs (anemia) or because they have experienced significant loss of blood volume through trauma or disease. Although the blood transfusion is given to help the patient, it is essential that the patient receive a transfusion with matching ABO blood type. A transfusion with an incompatible ABO blood type may lead to a strong, potentially lethal type II hypersensitivity cytotoxic response called hemolytic transfusion reaction (HTR) (Figure \(4\)).
For instance, if a person with type B blood receives a transfusion of type A blood, their anti-A antibodies will bind to and agglutinate the transfused RBCs. In addition, activation of the classical complement cascade will lead to a strong inflammatory response, and the complement membrane attack complex (MAC) will mediate massive hemolysis of the transfused RBCs. The debris from damaged and destroyed RBCs can occlude blood vessels in the alveoli of the lungs and the glomeruli of the kidneys. Within 1 to 24 hours of an incompatible transfusion, the patient experiences fever, chills, pruritus (itching), urticaria (hives), dyspnea, hemoglobinuria (hemoglobin in the urine), and hypotension (low blood pressure). In the most serious reactions, dangerously low blood pressure can lead to shock, multi-organ failure, and death of the patient.
Hospitals, medical centers, and associated clinical laboratories typically use hemovigilance systems to minimize the risk of HTRs due to clerical error. Hemovigilance systems are procedures that track transfusion information from the donor source and blood products obtained to the follow-up of recipient patients. Hemovigilance systems used in many countries identify HTRs and their outcomes through mandatory reporting (e.g., to the Food and Drug Administration in the United States), and this information is valuable to help prevent such occurrences in the future. For example, if an HTR is found to be the result of laboratory or clerical error, additional blood products collected from the donor at that time can be located and labeled correctly to avoid additional HTRs. As a result of these measures, HTR-associated deaths in the United States occur in about one per 2 million transfused units.6
Rh Factors
Many different types of erythrocyte antigens have been discovered since the description of the ABO red cell antigens. The second most frequently described RBC antigens are Rh factors, named after the rhesus macaque (Macaca mulatta) factors identified by Karl Landsteiner and Alexander Weiner in 1940. The Rh system of RBC antigens is the most complex and immunogenic blood group system, with more than 50 specificities identified to date. Of all the Rh antigens, the one designated Rho (Weiner) or D (Fisher-Race) is the most immunogenic. Cells are classified as Rh positive (Rh+) if the Rho/D antigen is present or as Rh negative (Rh−) if the Rho/D antigen is absent. In contrast to the carbohydrate molecules that distinguish the ABO blood groups and are the targets of IgM isohemagglutinins in HTRs, the Rh factor antigens are proteins. As discussed in B Lymphocytes and Humoral Immunity, protein antigens activate B cells and antibody production through a T-cell–dependent mechanism, and the TH2 cells stimulate class switching from IgM to other antibody classes. In the case of Rh factor antigens, TH2 cells stimulate class switching to IgG, and this has important implications for the mechanism of HDN.
Like ABO incompatibilities, blood transfusions from a donor with the wrong Rh factor antigens can cause a type II hypersensitivity HTR. However, in contrast to the IgM isohemagglutinins produced early in life through exposure to environmental antigens, production of anti-Rh factor antibodies requires the exposure of an individual with Rh− blood to Rh+ positive RBCs and activation of a primary antibody response. Although this primary antibody response can cause an HTR in the transfusion patient, the hemolytic reaction would be delayed up to 2 weeks during the extended lag period of a primary antibody response (B Lymphocytes and Humoral Immunity). However, if the patient receives a subsequent transfusion with Rh+ RBCs, a more rapid HTR would occur with anti-Rh factor antibody already present in the blood. Furthermore, the rapid secondary antibody response would provide even more anti-Rh factor antibodies for the HTR.
Rh factor incompatibility between mother and fetus can also cause a type II hypersensitivity hemolytic reaction, referred to as hemolytic disease of the newborn (HDN) (Figure \(5\)). If an Rh− woman carries an Rh+ baby to term, the mother’s immune system can be exposed to Rh+ fetal red blood cells. This exposure will usually occur during the last trimester of pregnancy and during the delivery process. If this exposure occurs, the Rh+ fetal RBCs will activate a primary adaptive immune response in the mother, and anti-Rh factor IgG antibodies will be produced. IgG antibodies are the only class of antibody that can cross the placenta from mother to fetus; however, in most cases, the first Rh+ baby is unaffected by these antibodies because the first exposure typically occurs late enough in the pregnancy that the mother does not have time to mount a sufficient primary antibody response before the baby is born.
If a subsequent pregnancy with an Rh+ fetus occurs, however, the mother’s second exposure to the Rh factor antigens causes a strong secondary antibody response that produces larger quantities of anti-Rh factor IgG. These antibodies can cross the placenta from mother to fetus and cause HDN, a potentially lethal condition for the baby (Figure \(5\)).
Prior to the development of techniques for diagnosis and prevention, Rh factor incompatibility was the most common cause of HDN, resulting in thousands of infant deaths each year worldwide.7 For this reason, the Rh factors of prospective parents are regularly screened, and treatments have been developed to prevent HDN caused by Rh incompatibility. To prevent Rh factor-mediated HDN, human Rho(D) immune globulin (e.g., RhoGAM) is injected intravenously or intramuscularly into the mother during the 28th week of pregnancy and within 72 hours after delivery. Additional doses may be administered after events that may result in transplacental hemorrhage (e.g., umbilical blood sampling, chorionic villus sampling, abdominal trauma, amniocentesis). This treatment is initiated during the first pregnancy with an Rh+ fetus. The anti-Rh antibodies in Rho(D) immune globulin will bind to the Rh factor of any fetal RBCs that gain access to the mother’s bloodstream, preventing these Rh+ cells from activating the mother’s primary antibody response. Without a primary anti-Rh factor antibody response, the next pregnancy with an Rh+ will have minimal risk of HDN. However, the mother will need to be retreated with Rho(D) immune globulin during that pregnancy to prevent a primary anti-Rh antibody response that could threaten subsequent pregnancies.
Link to Learning
Use this interactive Blood Typing Game to reinforce your knowledge of blood typing.
Exercise \(3\)
1. What happens to cells that possess incompatible antigens in a type II hypersensitivity reaction?
2. Describe hemolytic disease of the newborn and explain how it can be prevented.
Clinical Focus: Part 2
Kerry’s primary care physician is not sure why Kerry seems to develop rashes after spending time in the sun, so she orders a urinalysis and basic blood tests. The results reveal that Kerry has proteinuria (abnormal protein levels in the urine), hemoglobinuria (excess hemoglobin in the urine), and a low hematocrit (RBC count). These tests suggest that Kerry is suffering from a mild bout of hemolytic anemia. The physician suspects that the problem might be autoimmune, so she refers Kerry to a rheumatologist for additional testing and diagnosis.
Exercise \(4\)
Rheumatologists specialize in musculoskeletal diseases such as arthritis, osteoporosis, and joint pain. Why might Kerry’s physician refer her to this particular type of specialist even though she is exhibiting none of these symptoms?
Type III Hypersensitivities
Type III hypersensitivities are immune-complex reactions that were first characterized by Nicolas Maurice Arthus (1862–1945) in 1903. To produce antibodies for experimental procedures, Arthus immunized rabbits by injecting them with serum from horses. However, while immunizing rabbits repeatedly with horse serum, Arthus noticed a previously unreported and unexpected localized subcutaneous hemorrhage with edema at the site of injection. This reaction developed within 3 to10 hours after injection. This localized reaction to non-self serum proteins was called an Arthus reaction. An Arthus reaction occurs when soluble antigens bind with IgG in a ratio that results in the accumulation of antigen-antibody aggregates called immune complexes.
A unique characteristic of type III hypersensitivity is antibody excess (primarily IgG), coupled with a relatively low concentration of antigen, resulting in the formation of small immune complexes that deposit on the surface of the epithelial cells lining the inner lumen of small blood vessels or on the surfaces of tissues (Figure \(6\)). This immune complex accumulation leads to a cascade of inflammatory events that include the following:
1. IgG binding to antibody receptors on localized mast cells, resulting in mast-cell degranulation
2. Complement activation with production of pro-inflammatory C3a and C5a (see Chemical Defenses)
3. Increased blood-vessel permeability with chemotactic recruitment of neutrophils and macrophages
Because these immune complexes are not an optimal size and are deposited on cell surfaces, they cannot be phagocytosed in the usual way by neutrophils and macrophages, which, in turn, are often described as “frustrated.” Although phagocytosis does not occur, neutrophil degranulation results in the release of lysosomal enzymes that cause extracellular destruction of the immune complex, damaging localized cells in the process. Activation of coagulation pathways also occurs, resulting in thrombi (blood clots) that occlude blood vessels and cause ischemia that can lead to vascular necrosis and localized hemorrhage.
Systemic type III hypersensitivity (serum sickness) occurs when immune complexes deposit in various body sites, resulting in a more generalized systemic inflammatory response. These immune complexes involve non-self proteins such as antibodies produced in animals for artificial passive immunity (see Vaccines), certain drugs, or microbial antigens that are continuously released over time during chronic infections (e.g., subacute bacterial endocarditis, chronic viral hepatitis). The mechanisms of serum sickness are similar to those described in localized type III hypersensitivity but involve widespread activation of mast cells, complement, neutrophils, and macrophages, which causes tissue destruction in areas such as the kidneys, joints, and blood vessels. As a result of tissue destruction, symptoms of serum sickness include chills, fever, rash, vasculitis, and arthritis. Development of glomerulonephritis or hepatitis is also possible.
Autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis can also involve damaging type III hypersensitivity reactions when auto-antibodies form immune complexes with self antigens. These conditions are discussed in Autoimmune Disorders.
Exercise \(5\)
1. Why is antibody excess important in type III hypersensitivity?
2. Describe the differences between the Arthus reaction and serum sickness.
Diphtheria Antitoxin
Antibacterial sera are much less commonly used now than in the past, having been replaced by toxoid vaccines. However, a diphtheria antitoxin produced in horses is one example of such a treatment that is still used in some parts of the world. Although it is not licensed by the FDA for use in the United States, diphtheria antitoxin can be used to treat cases of diphtheria, which are caused by the bacterium Corynebacterium diphtheriae.8 The treatment is not without risks, however. Serum sickness can occur when the patient develops an immune response to non-self horse proteins. Immune complexes are formed between the horse proteins and circulating antibodies when the two exist in certain proportions. These immune complexes can deposit in organs, causing damage such as arthritis, nephritis, rash, and fever. Serum sickness is usually transient with no permanent damage unless the patient is chronically exposed to the antigen, which can then result in irreversible damage to body sites such as joints and kidneys. Over time, phagocytic cells such as macrophages are able to clear the horse serum antigens, which results in improvement of the patient’s condition and a decrease in symptoms as the immune response dissipates.
Clinical Focus: Part 3
Kerry does not make it to the rheumatologist. She has a seizure as she is leaving her primary care physician’s office. She is quickly rushed to the emergency department, where her primary care physician relates her medical history and recent test results. The emergency department physician calls in the rheumatologist on staff at the hospital for consultation. Based on the symptoms and test results, the rheumatologist suspects that Kerry has lupus and orders a pair of blood tests: an antinuclear antibody test (ANA) to look for antibodies that bind to DNA and another test that looks for antibodies that bind to a self-antigen called the Smith antigen (Sm).
Exercise \(6\)
Based on the blood tests ordered, what type of reaction does the rheumatologist suspect is causing Kerry’s seizure?
Type IV Hypersensitivities
Type IV hypersensitivities are not mediated by antibodies like the other three types of hypersensitivities. Rather, type IV hypersensitivities are regulated by T cells and involve the action of effector cells. These types of hypersensitivities can be organized into three subcategories based on T-cell subtype, type of antigen, and the resulting effector mechanism (Table \(5\)).
In the first type IV subcategory, CD4 TH1-mediated reactions are described as delayed-type hypersensitivities (DTH). The sensitization step involves the introduction of antigen into the skin and phagocytosis by local antigen presenting cells (APCs). The APCs activate helper T cells, stimulating clonal proliferation and differentiation into memory TH1 cells. Upon subsequent exposure to the antigen, these sensitized memory TH1 cells release cytokines that activate macrophages, and activated macrophages are responsible for much of the tissue damage. Examples of this TH1-mediated hypersensitivity are observed in tuberculin the Mantoux skin test and contact dermatitis, such as occurs in latex allergy reactions.
In the second type IV subcategory, CD4 TH2-mediated reactions result in chronic asthma or chronic allergic rhinitis. In these cases, the soluble antigen is first inhaled, resulting in eosinophil recruitment and activation with the release of cytokines and inflammatory mediators.
In the third type IV subcategory, CD8 cytotoxic T lymphocyte (CTL)-mediated reactions are associated with tissue transplant rejection and contact dermatitis (Figure \(7\)). For this form of cell-mediated hypersensitivity, APCs process and present the antigen with MHC I to naïve CD8 T cells. When these naïve CD8 T cells are activated, they proliferate and differentiate into CTLs. Activated TH1 cells can also enhance the activation of the CTLs. The activated CTLs then target and induce granzyme-mediated apoptosis in cells presenting the same antigen with MHC I. These target cells could be “self” cells that have absorbed the foreign antigen (such as with contact dermatitis due to poison ivy), or they could be transplanted tissue cells displaying foreign antigen from the donor.
Table \(5\): Type IV Hypersensitivities
Subcategory Antigen Effector Mechanism Examples
1 Soluble antigen Activated macrophages damage tissue and promote inflammatory response Contact dermatitis (e.g., exposure to latex) and delayed-type hypersensitivity (e.g., tuberculin reaction)
2 Soluble antigen Eosinophil recruitment and activation release cytokines and pro-inflammatory chemicals Chronic asthma and chronic allergic rhinitis
3 Cell-associated antigen CTL-mediated cytotoxicity Contact dermatitis (e.g., contact with poison ivy) and tissue-transplant rejection
Exercise \(7\)
1. Describe the three subtypes of type IV hypersensitivity.
2. Explain how T cells contribute to tissue damage in type IV hypersensitivity.
Using Delayed Hypersensitivity to Test for TB
Austrian pediatrician Clemans von Pirquet (1874–1929) first described allergy mechanisms, including type III serum sickness.9 His interest led to the development of a test for tuberculosis (TB), using the tuberculin antigen, based on earlier work identifying the TB pathogen performed by Robert Koch. Pirquet’s method involved scarification, which results in simultaneous multiple punctures, using a device with an array of needles to break the skin numerous times in a small area. The device Pirquet used was similar to the tine testdevice with four needles seen in Figure \(8\).
The tips of all the needles in the array are coated with tuberculin, a protein extract of TB bacteria, effectively introducing the tuberculin into the skin. One to 3 days later, the area can be examined for a delayed hypersensitivity reaction, signs of which include swelling and redness.
As you can imagine, scarification was not a pleasant experience,10 and the numerous skin punctures put the patient at risk of developing bacterial infection of the skin. Mantoux modified Pirquet’s test to use a single subcutaneous injection of purified tuberculin material. A positive test, which is indicated by a delayed localized swelling at the injection site, does not necessarily mean that the patient is currently infected with active TB. Because type IV (delayed-type) hypersensitivity is mediated by reactivation of memory T cells, such cells may have been created recently (due to an active current infection) or years prior (if a patient had TB and had spontaneously cleared it, or if it had gone into latency). However, the test can be used to confirm infection in cases in which symptoms in the patient or findings on a radiograph suggest its presence.
Hypersensitivity Pneumonitis
Some disease caused by hypersensitivities are not caused exclusively by one type. For example, hypersensitivity pneumonitis (HP), which is often an occupational or environmental disease, occurs when the lungs become inflamed due to an allergic reaction to inhaled dust, endospores, bird feathers, bird droppings, molds, or chemicals. HP goes by many different names associated with various forms of exposure (Figure \(9\)). HP associated with bird droppings is sometimes called pigeon fancier’s lung or poultry worker’s lung—both common in bird breeders and handlers. Cheese handler’s disease, farmer’s lung, sauna takers' disease, and hot-tub lung are other names for HP associated with exposure to molds in various environments.
Pathology associated with HP can be due to both type III (mediated by immune complexes) and type IV (mediated by TH1 cells and macrophages) hypersensitivities. Repeated exposure to allergens can cause alveolitis due to the formation of immune complexes in the alveolar wall of the lung accompanied by fluid accumulation, and the formation of granulomas and other lesions in the lung as a result of TH1-mediated macrophage activation. Alveolitis with fluid and granuloma formation results in poor oxygen perfusion in the alveoli, which, in turn, can cause symptoms such as coughing, dyspnea, chills, fever, sweating, myalgias, headache, and nausea. Symptoms may occur as quickly as 2 hours after exposure and can persist for weeks if left untreated.
Exercise \(8\)
Explain why hypersensitivity pneumonitis is considered an occupational disease.
Figure \(10\) summarizes the mechanisms and effects of each type of hypersensitivity discussed in this section.
Diagnosis of Hypersensitivities
Diagnosis of type I hypersensitivities is a complex process requiring several diagnostic tests in addition to a well-documented patient history. Serum IgE levels can be measured, but elevated IgE alone does not confirm allergic disease. As part of the process to identify the antigens responsible for a type I reaction allergy, testing through a prick puncture skin test (PPST) or an intradermal test can be performed. PPST is carried out with the introduction of allergens in a series of superficial skin pricks on the patient’s back or arms (Figure \(11\)). PPSTs are considered to be the most convenient and least expensive way to diagnose allergies, according to the US Joint Council of Allergy and the European Academy of Allergy and Immunology. The second type of testing, the intradermal test, requires injection into the dermis with a small needle. This needle, also known as a tuberculin needle, is attached to a syringe containing a small amount of allergen. Both the PPST and the intradermal tests are observed for 15–20 minutes for a wheal-flare reaction to the allergens. Measurement of any wheal (a raised, itchy bump) and flare (redness) within minutes indicates a type I hypersensitivity, and the larger the wheal-flare reaction, the greater the patient’s sensitivity to the allergen.
Type III hypersensitivities can often be misdiagnosed because of their nonspecific inflammatory nature. The symptoms are easily visible, but they may be associated with any of a number of other diseases. A strong, comprehensive patient history is crucial to proper and accurate diagnosis. Tests used to establish the diagnosis of hypersensitivity pneumonitis (resulting from type III hypersensitivity) include bronchoalveolar lavage (BAL), pulmonary function tests, and high-resolution computed tomography (HRCT).
Exercise \(9\)
1. Describe the prick puncture skin test.
2. Explain why type III hypersensitivities can be difficult to diagnose
Treatments of Hypersensitivities
Allergic reactions can be treated in various ways. Prevention of allergic reactions can be achieved by desensitization(hyposensitization) therapy, which can be used to reduce the hypersensitivity reaction through repeated injections of allergens. Extremely dilute concentrations of known allergens (determined from the allergen tests) are injected into the patient at prescribed intervals (e.g., weekly). The quantity of allergen delivered by the shots is slowly increased over a buildup period until an effective dose is determined and that dose is maintained for the duration of treatment, which can last years. Patients are usually encouraged to remain in the doctor’s office for 30 minutes after receiving the injection in case the allergens administered cause a severe systemic reaction. Doctors’ offices that administer desensitization therapy must be prepared to provide resuscitation and drug treatment in the case of such an event.
Desensitization therapy is used for insect sting allergies and environmental allergies. The allergy shots elicit the production of different interleukins and IgG antibody responses instead of IgE. When excess allergen-specific IgG antibodies are produced and bind to the allergen, they can act as blocking antibodies to neutralize the allergen before it can bind IgE on mast cells. There are early studies using oral therapy for desensitization of food allergies that are promising.1112 These studies involve feeding children who have allergies tiny amounts of the allergen (e.g., peanut flour) or related proteins over time. Many of the subjects show reduced severity of reaction to the food allergen after the therapy.
There are also therapies designed to treat severe allergic reactions. Emergency systemic anaphylaxis is treated initially with an epinephrine injection, which can counteract the drop in blood pressure. Individuals with known severe allergies often carry a self-administering auto-injector that can be used in case of exposure to the allergen (e.g., an insect sting or accidental ingestion of a food that causes a severe reaction). By self-administering an epinephrine shot (or sometimes two), the patient can stem the reaction long enough to seek medical attention. Follow-up treatment generally involves giving the patient antihistamines and slow-acting corticosteroids for several days after the reaction to prevent potential late-phase reactions. However, the effects of antihistamine and corticosteroid treatment are not well studied and are used based on theoretical considerations.
Treatment of milder allergic reactions typically involves antihistamines and other anti-inflammatory drugs. A variety of antihistamine drugs are available, in both prescription and over-the-counter strengths. There are also antileukotriene and antiprostaglandin drugs that can be used in tandem with antihistamine drugs in a combined (and more effective) therapy regime.
Treatments of type III hypersensitivities include preventing further exposure to the antigen and the use of anti-inflammatory drugs. Some conditions can be resolved when exposure to the antigen is prevented. Anti-inflammatory corticosteroid inhalers can also be used to diminish inflammation to allow lung lesions to heal. Systemic corticosteroid treatment, oral or intravenous, is also common for type III hypersensitivities affecting body systems. Treatment of hypersensitivity pneumonitis includes avoiding the allergen, along with the possible addition of prescription steroids such as prednisone to reduce inflammation.
Treatment of type IV hypersensitivities includes antihistamines, anti-inflammatory drugs, analgesics, and, if possible, eliminating further exposure to the antigen.
Exercise \(10\)
1. Describe desensitization therapy.
2. Explain the role of epinephrine in treatment of hypersensitivity reactions.
Key Concepts and Summary
• An allergy is an adaptive immune response, sometimes life-threatening, to an allergen.
• Type I hypersensitivity requires sensitization of mast cells with IgE, involving an initial IgE antibody response and IgE attachment to mast cells. On second exposure to an allergen, cross-linking of IgE molecules on mast cells triggers degranulation and release of preformed and newly formed chemical mediators of inflammation. Type I hypersensitivity may be localized and relatively minor (hives and hay fever) or system-wide and dangerous (systemic anaphylaxis).
• Type II hypersensitivities result from antibodies binding to antigens on cells and initiating cytotoxic responses. Examples include hemolytic transfusion reaction and hemolytic disease of the newborn.
• Type III hypersensitivities result from formation and accumulation of immune complexes in tissues, stimulating damaging inflammatory responses.
• Type IV hypersensitivities are not mediated by antibodies, but by helper T-cell activation of macrophages, eosinophils, and cytotoxic T cells.
Footnotes
1. 1 D.S. Strayer et al (eds). Rubin’s Pathology: Clinicopathologic Foundations of Medicine. 7th ed. 2Philadelphia, PA: Lippincott, Williams & Wilkins, 2014.
2. 2 C.M. Fitzsimmons et al. “Helminth Allergens, Parasite-Specific IgE, and Its Protective Role in Human Immunity.” Frontier in Immunology 5 (2015):47.
3. 3 S.T. Weiss. “Eat Dirt—The Hygiene Hypothesis and Allergic Diseases.” New England Journal of Medicine 347 no. 12 (2002):930–931.
4. 4 D.P. Strachan “Hay Fever, Hygiene, and Household Size.” British Medical Journal 299 no. 6710 (1989):1259.
5. 5 H. Okada et al. “The ‘Hygiene Hypothesis’ for Autoimmune and Allergic Diseases: An Update.” Clinical & Experimental Immunology 160 no. 1 (2010):1–9.
6. 6 E.C. Vamvakas, M.A. Blajchman. “Transfusion-Related Mortality: The Ongoing Risks of Allogeneic Blood Transfusion and the Available Strategies for Their Prevention.” Blood 113 no. 15 (2009):3406–3417.
7. 7 G. Reali. “Forty Years of Anti-D Immunoprophylaxis.” Blood Transfusion 5 no. 1 (2007):3–6.
8. 8 Centers for Disease Control and Prevention. “Diphtheria Antitoxin.” http://www.cdc.gov/diphtheria/dat.html. Accessed March 25, 2016.
9. 9 B. Huber “100 Jahre Allergie: Clemens von Pirquet–sein Allergiebegriff und das ihm zugrunde liegende Krankheitsverständnis.” Wiener Klinische Wochenschrift 118 no. 19–20 (2006):573–579.
10. 10 C.A. Stewart. “The Pirquet Test: Comparison of the Scarification and the Puncture Methods of Application.” Archives of Pediatrics & Adolescent Medicine 35 no. 3 (1928):388–391.
11. 11 C.L. Schneider et al. “A Pilot Study of Omalizumab to Facilitate Rapid Oral Desensitization in High-Risk Peanut-Allergic Patients.” Journal of Allergy and Clinical Immunology 132 no. 6 (2013):1368–1374.
12. 12 P. Varshney et al. “A Randomized Controlled Study of Peanut Oral Immunotherapy: Clinical Desensitization and Modulation of the Allergic Response.” Journal of Allergy and Clinical Immunology 127 no. 3 (2011):654–660. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/19%3A_Diseases_of_the_Immune_System/19.01%3A_Hypersensitivities.txt |
Learning Objectives
• Explain why autoimmune disorders develop
• Provide a few examples of organ-specific and systemic autoimmune diseases
In 1970, artist Walt Kelly developed a poster promoting Earth Day, featuring a character from Pogo, his daily newspaper comic strip. In the poster, Pogo looks out across a litter-strewn forest and says wryly, “We have met the enemy and he is us.” Pogo was not talking about the human immune system, but he very well could have been. Although the immune system protects the body by attacking invading “enemies” (pathogens), in some cases, the immune system can mistakenly identify the body’s own cells as the enemy, resulting in autoimmune disease.
Autoimmune diseases are those in which the body is attacked by its own specific adaptive immune response. In normal, healthy states, the immune system induces tolerance, which is a lack of an anti-self immune response. However, with autoimmunity, there is a loss of immune tolerance, and the mechanisms responsible for autoimmune diseases include type II, III, and IV hypersensitivity reactions. Autoimmune diseases can have a variety of mixed symptoms that flare up and disappear, making diagnosis difficult.
The causes of autoimmune disease are a combination of the individual's genetic makeup and the effect of environmental influences, such as sunlight, infections, medications, and environmental chemicals. However, the vagueness of this list reflects our poor understanding of the etiology of these diseases. Except in a very few specific diseases, the initiation event(s) of most autoimmune states has not been fully characterized.
There are several possible causes for the origin of autoimmune diseases and autoimmunity is likely due to several factors. Evidence now suggests that regulatory T and B cells play an essential role in the maintenance of tolerance and prevention of autoimmune responses. The regulatory T cells are especially important for inhibiting autoreactive T cells that are not eliminated during thymic selection and escape the thymus (see T Lymphocytes and Cellular Immunity). In addition, antigen mimicry between pathogen antigens and our own self antigens can lead to cross-reactivity and autoimmunity. Hidden self-antigens may become exposed because of trauma, drug interactions, or disease states, and trigger an autoimmune response. All of these factors could contribute to autoimmunity. Ultimately, damage to tissues and organs in the autoimmune disease state comes as a result of inflammatory responses that are inappropriate; therefore, treatment often includes immunosuppressive drugs and corticosteroids.
Organ-Specific Autoimmune Diseases
Some autoimmune diseases are considered organ specific, meaning that the immune system targets specific organs or tissues. Examples of organ-specific autoimmune diseases include celiac disease, Graves disease, Hashimoto thyroiditis, type I diabetes mellitus, and Addison disease.
Celiac Disease
Celiac disease is largely a disease of the small intestine, although other organs may be affected. People in their 30s and 40s, and children are most commonly affected, but celiac disease can begin at any age. It results from a reaction to proteins, commonly called gluten, found mainly in wheat, barley, rye, and some other grains. The disease has several genetic causes (predispositions) and poorly understood environmental influences. On exposure to gluten, the body produces various autoantibodies and an inflammatory response. The inflammatory response in the small intestine leads to a reduction in the depth of the microvilli of the mucosa, which hinders absorption and can lead to weight loss and anemia. The disease is also characterized by diarrhea and abdominal pain, symptoms that are often misdiagnosed as irritable bowel syndrome.
Diagnosis of celiac disease is accomplished from serological tests for the presence of primarily IgA antibodies to components of gluten, the transglutinaminase enzyme, and autoantibodies to endomysium, a connective tissue surrounding muscle fibers. Serological tests are typically followed up with endoscopy and biopsy of the duodenal mucosa. Serological screening surveys have found about 1% of individuals in the United Kingdom are positive even though they do not all display symptoms.1 This early recognition allows for more careful monitoring and prevention of severe disease.
Celiac disease is treated with complete removal of gluten-containing foods from the diet, which results in improved symptoms and reduced risk of complications. Other theoretical approaches include breeding grains that do not contain the immunologically reactive components or developing dietary supplements that contain enzymes that break down the protein components that cause the immune response.2
Disorders of the Thyroid
Graves disease is the most common cause of hyperthyroidism in the United States. Symptoms of Graves disease result from the production of thyroid-stimulating immunoglobulin (TSI) also called TSH-receptor antibody. TSI targets and binds to the receptor for thyroid stimulating hormone (TSH), which is naturally produced by the pituitary gland. TSI may cause conflicting symptoms because it may stimulate the thyroid to make too much thyroid hormone or block thyroid hormone production entirely, making diagnosis more difficult. Signs and symptoms of Graves disease include heat intolerance, rapid and irregular heartbeat, weight loss, goiter (a swollen thyroid gland, protruding under the skin of the throat [Figure \(1\)]) and exophthalmia (bulging eyes) often referred to as Graves ophthalmopathy (Figure \(2\)).
The most common cause of hypothyroidism in the United States is Hashimoto thyroiditis, also called chronic lymphocytic thyroiditis. Patients with Hashimoto thyroiditis often develop a spectrum of different diseases because they are more likely to develop additional autoimmune diseases such as Addison disease (discussed later in this section), type 1 diabetes, rheumatoid arthritis, and celiac disease. Hashimoto thyroiditis is a TH1 cell-mediated disease that occurs when the thyroid gland is attacked by cytotoxic lymphocytes, macrophages, and autoantibodies. This autoimmune response leads to numerous symptoms that include goiter (Figure \(1\)), cold intolerance, muscle weakness, painful and stiff joints, depression, and memory loss.
Type 1 Diabetes
Juvenile diabetes, or type 1 diabetes mellitus, is usually diagnosed in children and young adults. It is a T-cell-dependent autoimmune disease characterized by the selective destruction of the β cells of the islets of Langerhans in the pancreas by CD4 TH1-mediated CD8 T cells, anti-β-cell antibodies, and macrophage activity. There is also evidence that viral infections can have either a potentiating or inhibitory role in the development of type 1 diabetes (T1D) mellitus. The destruction of the β cells causes a lack of insulin production by the pancreas. In T1D, β-cell destruction may take place over several years, but symptoms of hyperglycemia, extreme increase in thirst and urination, weight loss, and extreme fatigue usually have a sudden onset, and diagnosis usually does not occur until most β cells have already been destroyed.
Autoimmune Addison Disease
Destruction of the adrenal glands (the glands lying above the kidneys that produce glucocorticoids, mineralocorticoids, and sex steroids) is the cause of Addison disease, also called primary adrenal insufficiency (PAI). Today, up to 80% of Addison disease cases are diagnosed as autoimmune Addison disease (AAD), which is caused by an autoimmune response to adrenal tissues disrupting adrenal function. Disruption of adrenal function causes impaired metabolic processes that require normal steroid hormone levels, causing signs and symptoms throughout the body. There is evidence that both humoral and CD4 TH1-driven CD8 T-cell–mediated immune mechanisms are directed at the adrenal cortex in AAD. There is also evidence that the autoimmune response is associated with autoimmune destruction of other endocrine glands as well, such as the pancreas and thyroid, conditions collectively referred to as autoimmune polyendocrine syndromes (APS). In up to 80% of patients with AAD, antibodies are produced to three enzymes involved in steroid synthesis: 21-hydroxylase (21-OH), 17α-hydroxylase, and cholesterol side-chain–cleaving enzyme.3The most common autoantibody found in AAD is to 21-OH, and antibodies to any of the key enzymes for steroid production are diagnostic for AAD. The adrenal cortex cells are targeted, destroyed, and replaced with fibrous tissue by immune-mediated inflammation. In some patients, at least 90% of the adrenal cortex is destroyed before symptoms become diagnostic.
Symptoms of AAD include weakness, nausea, decreased appetite, weight loss, hyperpigmentation (Figure \(3\)), hyperkalemia (elevated blood potassium levels), hyponatremia (decreased blood sodium levels), hypoglycemia(decreased levels of blood sugar), hypotension (decreased blood pressure), anemia, lymphocytosis (decreased levels of white blood cells), and fatigue. Under extreme stress, such as surgery, accidental trauma, or infection, patients with AAD may experience an adrenal crisis that causes the patient to vomit, experience abdominal pain, back or leg cramps, and even severe hypotension leading to shock.
Exercise \(1\)
1. What are the names of autoimmune diseases that interfere with hormone gland function?
2. Describe how the mechanisms of Graves disease and Hashimoto thyroiditis differ.
3. Name the cells that are destroyed in type 1 diabetes mellitus and describe the result.
Systemic Autoimmune Diseases
Whereas organ-specific autoimmune diseases target specific organs or tissues, systemic autoimmune diseases are more generalized, targeting multiple organs or tissues throughout the body. Examples of systemic autoimmune diseases include multiple sclerosis, myasthenia gravis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.
Multiple Sclerosis
Multiple sclerosis (MS) is an autoimmune central nervous system disease that affects the brain and spinal cord. Lesions in multiple locations within the central nervous system are a hallmark of multiple sclerosis and are caused by infiltration of immune cells across the blood-brain barrier. The immune cells include T cells that promote inflammation, demyelination, and neuron degeneration, all of which disrupt neuronal signaling. Symptoms of MS include visual disturbances; muscle weakness; difficulty with coordination and balance; sensations such as numbness, prickling, or “pins and needles”; and cognitive and memory problems.
Myasthenia Gravis
Autoantibodies directed against acetylcholine receptors (AChRs) in the synaptic cleft of neuromuscular junctions lead to myasthenia gravis (Figure \(4\)). Anti-AChR antibodies are high-affinity IgGs and their synthesis requires activated CD4 T cells to interact with and stimulate B cells. Once produced, the anti-AChR antibodies affect neuromuscular transmission by at least three mechanisms:
• Complement binding and activation at the neuromuscular junction
• Accelerated AChR endocytosis of molecules cross-linked by antibodies
• Functional AChR blocking, which prevents normal acetylcholine attachment to, and activation of, AChR
Regardless of the mechanism, the effect of anti-AChR is extreme muscle weakness and potentially death through respiratory arrest in severe cases.
Psoriasis
Psoriasis is a skin disease that causes itchy or sore patches of thick, red skin with silvery scales on elbows, knees, scalp, back, face, palms, feet, and sometimes other areas. Some individuals with psoriasis also get a form of arthritis called psoriatic arthritis, in which the joints can become inflamed. Psoriasis results from the complex interplay between keratinocytes, dendritic cells, and T cells, and the cytokines produced by these various cells. In a process called cell turnover, skin cells that grow deep in the skin rise to the surface. Normally, this process takes a month. In psoriasis, as a result of cytokine activation, cell turnover happens in just a few days. The thick inflamed patches of skin that are characteristic of psoriasis develop because the skin cells rise too fast.
Rheumatoid Arthritis
The most common chronic inflammatory joint disease is rheumatoid arthritis (RA) (Figure \(5\)) and it is still a major medical challenge because of unsolved questions related to the environmental and genetic causes of the disease. RA involves type III hypersensitivity reactions and the activation of CD4 T cells, resulting in chronic release of the inflammatory cytokines IL-1, IL-6, and tumor necrosis factor-α (TNF-α). The activated CD4 T cells also stimulate the production of rheumatoid factor (RF) antibodies and anticyclic citrullinated peptide antibodies (anti-CCP) that form immune complexes. Increased levels of acute-phase proteins, such as C-reactive protein (CRP), are also produced as part of the inflammatory process and participate in complement fixation with the antibodies on the immune complexes. The formation of immune complexes and reaction to the immune factors cause an inflammatory process in joints, particularly in the hands, feet, and legs. Diagnosis of RA is based on elevated levels of RF, anti-CCP, quantitative CRP, and the erythrocyte sedimentation rate (ESR) (modified Westergren). In addition, radiographs, ultrasound, or magnetic resonance imaging scans can identify joint damage, such as erosions, a loss of bone within the joint, and narrowing of joint space.
Systemic Lupus Erythematosus
The damage and pathology of systemic lupus erythematosus (SLE) is caused by type III hypersensitivity reactions. Autoantibodies produced in SLE are directed against nuclear and cytoplasmic proteins. Anti-nuclear antibodies (ANAs) are present in more than 95% of patients with SLE,4 with additional autoantibodies including anti-double–stranded DNA (ds-DNA) and anti-Sm antibodies (antibodies to small nuclear ribonucleoprotein). Anti-ds-DNA and anti-Sm antibodies are unique to patients with SLE; thus, their presence is included in the classification criteria of SLE. Cellular interaction with autoantibodies leads to nuclear and cellular destruction, with components released after cell death leading to the formation of immune complexes.
Because autoantibodies in SLE can target a wide variety of cells, symptoms of SLE can occur in many body locations. However, the most common symptoms include fatigue, fever with no other cause, hair loss, and a sunlight-sensitive "butterfly" or wolf-mask (lupus) rash that is found in about 50% of people with SLE (Figure \(6\)). The rash is most often seen over the cheeks and bridge of the nose, but can be widespread. Other symptoms may appear depending on affected areas. The joints may be affected, leading to arthritis of the fingers, hands, wrists, and knees. Effects on the brain and nervous system can lead to headaches, numbness, tingling, seizures, vision problems, and personality changes. There may also be abdominal pain, nausea, vomiting, arrhythmias, shortness of breath, and blood in the sputum. Effects on the skin can lead to additional areas of skin lesions, and vasoconstriction can cause color changes in the fingers when they are cold (Raynaud phenomenon). Effects on the kidneys can lead to edema in the legs and weight gain. A diagnosis of SLE depends on identification of four of 11 of the most common symptoms and confirmed production of an array of autoantibodies unique to SLE. A positive test for ANAs alone is not diagnostic.
Exercise \(2\)
1. List the ways antibodies contribute to the pathogenesis of myasthenia gravis.
2. Explain why rheumatoid arthritis is considered a type III hypersensitivity.
3. Describe the symptoms of systemic lupus erythematosus and explain why they affect so many different parts of the body.
4. What is recognized as an antigen in myasthenia gravis?
Table \(1\) summarizes the causes, signs, and symptoms of select autoimmune diseases. Table \(1\): Select Autoimmune Diseases
Disease Cause Signs and Symptoms
Addison disease Destruction of adrenal gland cells by cytotoxic T cells Weakness, nausea, hypotension, fatigue; adrenal crisis with severe pain in abdomen, lower back, and legs; circulatory system collapse, kidney failure
Celiac disease Antibodies to gluten become autoantibodies that target cells of the small intestine Severe diarrhea, abdominal pain, anemia, malnutrition
Diabetes mellitus (type I) Cytotoxic T-cell destruction of the insulin-producing β cells of the pancreas Hyperglycemia, extreme increase in thirst and urination, weight loss, extreme fatigue
Graves disease Autoantibodies target thyroid-stimulating hormone receptors, resulting in overstimulation of the thyroid Hyperthyroidism with rapid and irregular heartbeat, heat intolerance, weight loss, goiter, exophthalmia
Hashimoto thyroiditis Thyroid gland is attacked by cytotoxic T cells, lymphocytes, macrophages, and autoantibodies Thyroiditis with goiter, cold intolerance, muscle weakness, painful and stiff joints, depression, memory loss
Multiple sclerosis (MS) Cytotoxic T-cell destruction of the myelin sheath surrounding nerve axons in the central nervous system Visual disturbances, muscle weakness, impaired coordination and balance, numbness, prickling or “pins and needles” sensations, impaired cognitive function and memory
Myasthenia gravis Autoantibodies directed against acetylcholine receptors within the neuromuscular junction Extreme muscle weakness eventually leading to fatal respiratory arrest
Psoriasis Cytokine activation of keratinocytes causes rapid and excessive epidermal cell turnover Itchy or sore patches of thick, red skin with silvery scales; commonly affects elbows, knees, scalp, back, face, palms, feet
Rheumatoid arthritis Autoantibodies, immune complexes, complement activation, phagocytes, and T cells damage membranes and bone in joints Joint inflammation, pain and disfigurement, chronic systemic inflammation
Systemic lupus erythematosus (SLE) Autoantibodies directed against nuclear and cytoplasmic molecules form immune complexes that deposit in tissues. Phagocytic cells and complement activation cause tissue damage and inflammation Fatigue, fever, joint pain and swelling, hair loss, anemia, clotting, a sunlight-sensitive "butterfly" rash, skin lesions, photosensitivity, decreased kidney function, memory loss, confusion, depression
Key Concepts and Summary
• Autoimmune diseases result from a breakdown in immunological tolerance. The actual induction event(s) for autoimmune states are largely unknown.
• Some autoimmune diseases attack specific organs, whereas others are more systemic.
• Organ-specific autoimmune diseases include celiac disease, Graves disease, Hashimoto thyroiditis, type I diabetes mellitus, and Addison disease.
• Systemic autoimmune diseases include multiple sclerosis, myasthenia gravis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.
• Treatments for autoimmune diseases generally involve anti-inflammatory and immunosuppressive drugs.
Footnotes
1. 1 D.A. Van Heel, J. West. “Recent Advances in Coeliac Disease.” Gut 55 no. 7 (2006):1037—1046.
2. 2 ibid.
3. 3 P. Martorell et al. “Autoimmunity in Addison’s Disease.” Netherlands Journal of Medicine 60 no. 7 (2002):269—275.
4. 4 C.C. Mok, C.S. Lau. “Pathogenesis of Systemic Lupus Erythematosus.” Journal of Clinical Pathology 56 no. 7 (2003):481—490. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/19%3A_Diseases_of_the_Immune_System/19.02%3A_Autoimmune_Disorders.txt |
Learning Objectives
• Explain why human leukocyte antigens (HLAs) are important in tissue transplantation
• Explain the types of grafts possible and their potential for interaction with the immune system
• Describe what occurs during graft-versus-host disease (GVHD)
A graft is the transplantation of an organ or tissue to a different location, with the goal of replacing a missing or damaged organ or tissue. Grafts are typically moved without their attachments to the circulatory system and must reestablish these, in addition to the other connections and interactions with their new surrounding tissues. There are different types of grafts depending on the source of the new tissue or organ. Tissues that are transplanted from one genetically distinct individual to another within the same species are called allografts. An interesting variant of the allograft is an isograft, in which tissue from one twin is transplanted to another. As long as the twins are monozygotic(therefore, essentially genetically identical), the transplanted tissue is virtually never rejected. If tissues are transplanted from one area on an individual to another area on the same individual (e.g., a skin graft on a burn patient), it is known as an autograft. If tissues from an animal are transplanted into a human, this is called a xenograft.
Transplant Rejection
The different types of grafts described above have varying risks for rejection (Table \(1\)). Rejection occurs when the recipient’s immune system recognizes the donor tissue as foreign (non-self), triggering an immune response. The major histocompatibility complex markers MHC I and MHC II, more specifically identified as human leukocyte antigens (HLAs), play a role in transplant rejection. The HLAs expressed in tissue transplanted from a genetically different individual or species may be recognized as non-self molecules by the host’s dendritic cells. If this occurs, the dendritic cells will process and present the foreign HLAs to the host’s helper T cells and cytotoxic T cells, thereby activating them. Cytotoxic T cells then target and kill the grafted cells through the same mechanism they use to kill virus-infected cells; helper T cells may also release cytokines that activate macrophages to kill graft cells.
Table \(1\): Types of Tissue and Organ Grafts and Their Complications
Graft Procedure Complications
Autograft From self to self No rejection concerns
Isograft From identical twin to twin Little concern of rejection
Allograft From relative or nonrelative to individual Rejection possible
Xenograft From animal to human Rejection possible
With the three highly polymorphic MHC I genes in humans (HLA-A, HLA-B, and HLA-C) determining compatibility, each with many alleles segregating in a population, odds are extremely low that a randomly chosen donor will match a recipient's six-allele genotype (the two alleles at each locus are expressed codominantly). This is why a parent or a sibling may be the best donor in many situations—a genetic match between the MHC genes is much more likely and the organ is much less likely to be rejected.
Although matching all of the MHC genes can lower the risk for rejection, there are a number of additional gene products that also play a role in stimulating responses against grafted tissue. Because of this, no non-self grafted tissue is likely to completely avoid rejection. However, the more similar the MHC gene match, the more likely the graft is to be tolerated for a longer time. Most transplant recipients, even those with tissues well matched to their MHC genes, require treatment with immunosuppressant drugs for the rest of their lives. This can make them more vulnerable than the general population to complications from infectious diseases. It can also result in transplant-related malignancies because the body’s normal defenses against cancer cells are being suppressed.
Exercise \(1\)
1. What part of the immune response is responsible for graft rejection?
2. Explain why blood relatives are preferred as organ donors.
3. Describe the role of immunosuppression in transplantation.
Graft-versus-Host Disease
A form of rejection called graft-versus-host disease (GVHD) primarily occurs in recipients of bone marrow transplants and peripheral blood stem cells. GHVD presents a unique situation because the transplanted tissue is capable of producing immune cells; APCs in the donated bone marrow may recognize the host cells as non-self, leading to activation of the donor cytotoxic T cells. Once activated, the donor’s T cells attack the recipient cells, causing acute GVHD.
Acute GVHD typically develops within weeks after a bone marrow transplant, causing tissue damage affecting the skin, gastrointestinal tract, liver, and eyes. In addition, acute GVHD may also lead to a cytokine storm, an unregulated secretion of cytokines that may be fatal. In addition to acute GVHD, there is also the risk for chronic GVHD developing months after the bone marrow transplant. The mechanisms responsible for chronic GVHD are not well understood.
To minimize the risk of GVHD, it is critically important to match the HLAs of the host and donor as closely as possible in bone marrow transplants. In addition, the donated bone marrow is processed before grafting to remove as many donor APCs and T cells as possible, leaving mostly hematopoietic stem cells.
Exercise \(\PageIndex{}\)2
1. Why does GVHD occur in specifically in bone marrow transplants?
2. What cells are responsible for GVHD?
The Future of Transplantation
Historically speaking, the practice of transplanting tissues—and the complications that can accompany such procedures—is a relatively recent development. It was not until 1954 that the first successful organ transplantation between two humans was achieved. Yet the field of organ transplantation has progressed rapidly since that time.
Nonetheless, the practice of transplanting non-self tissues may soon become obsolete. Scientists are now attempting to develop methods by which new organs may be grown in vitro from an individual’s own harvested cells to replace damaged or abnormal ones. Because organs produced in this way would contain the individual’s own cells, they could be transplanted into the individual without risk for rejection.
An alternative approach that is gaining renewed research interest is genetic modification of donor animals, such as pigs, to provide transplantable organs that do not elicit an immune response in the recipient. The approach involves excising the genes in the pig (in the embryo) that are most responsible for the rejection reaction after transplantation. Finding these genes and effectively removing them is a challenge, however. So too is identifying and neutralizing risks from viral sequences that might be embedded in the pig genome, posing a risk for infection in the human recipient.
Clinical Focus: Resolution
Kerry's tests come back positive, confirming a diagnosis of lupus, a disease that occurs 10 times more frequently in women than men. SLE cannot be cured, but there are various therapies available for reducing and managing its symptoms. Specific therapies are prescribed based on the particular symptoms presenting in the patient. Kerry's rheumatologist starts her therapy with a low dose of corticosteroids to reduce her rashes. She also prescribes a low dose of hydroxychloroquine, an anti-inflammatory drug that is used to treat inflammation in patients with RA, childhood arthritis, SLE, and other autoimmune diseases. Although the mechanism of action of hydroxychloroquine is not well defined, it appears that this drug interferes with the processes of antigen processing and activation of autoimmunity. Because of its mechanism, the effects of hydroxychloroquine are not as immediate as that of other anti-inflammatory drugs, but it is still considered a good companion therapy for SLE. Kerry’s doctor also advises her to limit her exposure to sunlight, because photosensitivity to sunlight may precipitate rashes.
Over the next 6 months, Kerry follows her treatment plan and her symptoms do not return. However, future flare-ups are likely to occur. She will need to continue her treatment for the rest of her life and seek medical attention whenever new symptoms develop.
Key Concepts and Summary
• Grafts and transplants can be classified as autografts, isografts, allografts, or xenografts based on the genetic differences between the donor’s and recipient’s tissues.
• Genetic differences, especially among the MHC (HLA) genes, will dictate the likelihood that rejection of the transplanted tissue will occur.
• Transplant recipients usually require immunosuppressive therapy to avoid rejection, even with good genetic matching. This can create additional problems when immune responses are needed to fight off infectious agents and prevent cancer.
• Graft-versus-host disease can occur in bone marrow transplants, as the mature T cells in the transplant itself recognize the recipient’s tissues as foreign.
• Transplantation methods and technology have improved greatly in recent decades and may move into new areas with the use of stem cell technology to avoid the need for genetic matching of MHC molecules. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/19%3A_Diseases_of_the_Immune_System/19.03%3A_Organ_Transplantation_and_Rejection.txt |
Learning Objectives
• Compare the causes of primary and secondary immunodeficiencies
• Describe treatments for primary and secondary immunodeficiencies
Immunodeficiencies are inherited (primary) or acquired (secondary) disorders in which elements of host immune defenses are either absent or functionally defective. In developed countries, most immunodeficiencies are inherited, and they are usually first seen in the clinic as recurrent or overwhelming infections in infants. However, on a global scale, malnutrition is the most common cause of immunodeficiency and would be categorized as an acquired immunodeficiency. Acquired immunodeficiencies are more likely to develop later in life, and the pathogenic mechanisms of many remain obscure.
Primary Immunodeficiency
Primary immunodeficiencies, which number more than 250, are caused by inherited defects of either nonspecific innate or specific adaptive immune defenses. In general, patients born with primary immunodeficiency (PI) commonly have an increased susceptibility to infection. This susceptibility can become apparent shortly after birth or in early childhood for some individuals, whereas other patients develop symptoms later in life. Some primary immunodeficiencies are due to a defect of a single cellular or humoral component of the immune system; others may result from defects of more than one component. Examples of primary immunodeficiencies include chronic granulomatous disease, X-linked agammaglobulinemia, selective IgA deficiency, and severe combined immunodeficiency disease.
Chronic Granulomatous Disease
The causes of chronic granulomatous disease (CGD) are defects in the NADPH oxidase system of phagocytic cells, including neutrophils and macrophages, that prevent the production of superoxide radicals in phagolysosomes. The inability to produce superoxide radicals impairs the antibacterial activity of phagocytes. As a result, infections in patients with CGD persist longer, leading to a chronic local inflammation called a granuloma. Microorganisms that are the most common causes of infections in patients with CGD include Aspergillus spp., Staphylococcus aureus, Chromobacterium violaceum, Serratia marcescens, and Salmonella typhimurium.
X-Linked Agammaglobulinemia
Deficiencies in B cells due to defective differentiation lead to a lack of specific antibody production known as X-linked agammaglobulinemia. In 1952, Ogden C. Bruton (1908–2003) described the first immunodeficiency in a boy whose immune system failed to produce antibodies. This defect is inherited on the X chromosome and is characterized by the absence of immunoglobulin in the serum; it is called Bruton X-linked agammaglobulinemia (XLA). The defective gene, BTK, in XLA is now known to encode a tyrosine kinase called Bruton tyrosine kinase (Btk). In patients whose B cells are unable to produce sufficient amounts of Btk, the B-cell maturation and differentiation halts at the pre-B-cell stage of growth. B-cell maturation and differentiation beyond the pre-B-cell stage of growth is required for immunoglobulin production. Patients who lack antibody production suffer from recurrent infections almost exclusively due to extracellular pathogens that cause pyogenic infections: Haemophilus influenzae, Streptococcus pneumoniae, S. pyogenes, and S. aureus. Because cell-mediated immunity is not impaired, these patients are not particularly vulnerable to infections caused by viruses or intracellular pathogens.
Selective IgA Deficiency
The most common inherited form of immunoglobulin deficiency is selective IgA deficiency, affecting about one in 800 people. Individuals with selective IgA deficiency produce normal levels of IgG and IgM, but are not able to produce secretory IgA. IgA deficiency predisposes these individuals to lung and gastrointestinal infections for which secretory IgA is normally an important defense mechanism. Infections in the lungs and gastrointestinal tract can involve a variety of pathogens, including H. influenzae, S. pneumoniae, Moraxella catarrhalis, S. aureus, Giardia lamblia, or pathogenic strains of Escherichia coli.
Severe Combined Immunodeficiency
Patients who suffer from severe combined immunodeficiency (SCID) have B-cell and T-cell defects that impair T-cell dependent antibody responses as well as cell-mediated immune responses. Patients with SCID also cannot develop immunological memory, so vaccines provide them no protection, and live attenuated vaccines (e.g., for varicella-zoster, measles virus, rotavirus, poliovirus) can actually cause the infection they are intended to prevent. The most common form is X-linked SCID, which accounts for nearly 50% of all cases and occurs primarily in males. Patients with SCID are typically diagnosed within the first few months of life after developing severe, often life-threatening, opportunistic infection by Candida spp., Pneumocystis jirovecii, or pathogenic strains of E. coli.
Without treatment, babies with SCID do not typically survive infancy. In some cases, a bone marrow transplant may successfully correct the defects in lymphocyte development that lead to the SCID phenotype, by replacing the defective component. However, this treatment approach is not without risks, as demonstrated by the famous case of David Vetter (1971–1984), better known as “Bubble Boy” (Figure \(1\)). Vetter, a patient with SCID who lived in a protective plastic bubble to prevent exposure to opportunistic microbes, received a bone marrow transplant from his sister. Because of a latent Epstein-Barr virus infection in her bone marrow, however, he developed mononucleosis and died of Burkitt lymphoma at the age of 12 years.
Exercise \(1\)
1. What is the fundamental cause of a primary immunodeficiency?
2. Explain why patients with chronic granulomatous disease are especially susceptible to bacterial infections.
3. Explain why individuals with selective IgA deficiency are susceptible to respiratory and gastrointestinal infections.
Secondary Immunodeficiency
A secondary immunodeficiency occurs as a result an acquired impairment of function of B cells, T cells, or both. Secondary immunodeficiencies can be caused by:
• Systemic disorders such as diabetes mellitus, malnutrition, hepatitis, or HIV infection
• Immunosuppressive treatments such as cytotoxic chemotherapy, bone marrow ablation before transplantation, or radiation therapy
• Prolonged critical illness due to infection, surgery, or trauma in the very young, elderly, or hospitalized patients
Unlike primary immunodeficiencies, which have a genetic basis, secondary immunodeficiencies are often reversible if the underlying cause is resolved. Patients with secondary immunodeficiencies develop an increased susceptibility to an otherwise benign infection by opportunistic pathogens such as Candida spp., P. jirovecii, and Cryptosporidium.
HIV infection and the associated acquired immunodeficiency syndrome (AIDS) are the best-known secondary immunodeficiencies. AIDS is characterized by profound CD4 T-cell lymphopenia (decrease in lymphocytes). The decrease in CD4 T cells is the result of various mechanisms, including HIV-induced pyroptosis (a type of apoptosis that stimulates an inflammatory response), viral cytopathic effect, and cytotoxicity to HIV-infected cells.
The most common cause of secondary immunodeficiency worldwide is severe malnutrition, which affects both innate and adaptive immunity. More research and information are needed for the more common causes of secondary immunodeficiency; however, the number of new discoveries in AIDS research far exceeds that of any other single cause of secondary immunodeficiency. AIDS research has paid off extremely well in terms of discoveries and treatments; increased research into the most common cause of immunodeficiency, malnutrition, would likely be as beneficial.
Exercise \(2\)
1. What is the most common cause of secondary immunodeficiencies?
2. Explain why secondary immunodeficiencies can sometimes be reversed.
An Immunocompromised Host
Benjamin, a 50-year-old male patient who has been receiving chemotherapy to treat his chronic myelogenous leukemia (CML), a disease characterized by massive overproduction of nonfunctional, malignant myelocytic leukocytes that crowd out other, healthy leukocytes, is seen in the emergency department. He is complaining of a productive, wet cough, dyspnea, and fatigue. On examination, his pulse is 120 beats per minute (bpm) (normal range is 60–100 bpm) and weak, and his blood pressure is 90/60 mm Hg (normal is 120/80 mm Hg). During auscultation, a distinct crackling can be heard in his lungs as he breathes, and his pulse-oximeter level (a measurement of blood-oxygen saturation) is 80% (normal is 95%–100%). He has a fever; his temperature is 38.9 °C (102 °F). Sputum cultures and blood samples are obtained and sent to the lab, but Benjamin goes into respiratory distress and dies before the results can be obtained.
Benjamin’s death was a result of a combination of his immune system being compromised by his leukemia and his chemotherapy treatment further weakening his ability to mount an immune response. CML (and leukemia in general) and corresponding chemotherapy cause a decrease in the number of leukocytes capable of normal function, leading to secondary immunodeficiency. This increases the risk for opportunistic bacterial, viral, protozoal, and fungal infections that could include Staphylococcus, enteroviruses, Pneumocystis, Giardia, or Candida. Benjamin’s symptoms were suggestive of bacterial pneumonia, but his leukemia and chemotherapy likely complicated and contributed to the severity of the pneumonia, resulting in his death. Because his leukemia was overproducing certain white blood cells, and those overproduced white blood cells were largely nonfunctional or abnormal in their function, he did not have the proper immune system blood cells to help him fight off the infection.
Table \(1\) summarizes primary and secondary immunodeficiencies, their effects on immune function, and typical outcomes. Table \(1\): Primary and Secondary Immunodeficiencies
Disease Effect on Immune Function Outcomes
Primary immunodeficiencies Chronic granulomatous disease Impaired killing of bacteria within the phagolysosome of neutrophils and macrophages Chronic infections and granulomas
Selective IgA deficiency Inability to produce secretory IgA Predisposition to lung and gastrointestinal infections
Severe combined immunodeficiency disease (SCID) Deficient humoral and cell-mediated immune responses Early development of severe and life-threatening opportunistic infections
X-linked agammaglobulinemia Flawed differentiation of B cells and absence of specific antibodies Recurrent infections almost exclusively due to pathogens that cause pyogenic infections
Secondary immunodeficiencies Immunosuppressive therapies (e.g., chemotherapy, radiotherapy) Impaired humoral and/or cell-mediated immune responses Opportunistic infections, rare cancers
Malnutrition Impaired humoral and/or cell-mediated immune responses Opportunistic infections, rare cancers
Viral infection (e.g., HIV) Impaired cell-mediated immune responses due to CD4 T-cell lymphopenia Opportunistic infections, rare cancers
Key Concepts and Summary
• Primary immunodeficiencies are caused by genetic abnormalities; secondary immunodeficiencies are acquired through disease, diet, or environmental exposures.
• Primary immunodeficiencies may result from flaws in phagocyte killing of innate immunity, or impairment of T cells and B cells.
• Primary immunodeficiencies include chronic granulomatous disease, X-linked agammaglobulinemia, selective IgA deficiency, and severe combined immunodeficiency disease.
• Secondary immunodeficiencies result from environmentally induced defects in B cells and/or T cells.
• Causes for secondary immunodeficiencies include malnutrition, viral infection, diabetes, prolonged infections, and chemical or radiation exposure. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/19%3A_Diseases_of_the_Immune_System/19.04%3A_Immunodeficiency.txt |
Learning Objectives
• Explain how the adaptive specific immune response responds to tumors
• Discuss the risks and benefits of tumor vaccines
Cancer involves a loss of the ability of cells to control their cell cycle, the stages each eukaryotic cell goes through as it grows and then divides. When this control is lost, the affected cells rapidly divide and often lose the ability to differentiate into the cell type appropriate for their location in the body. In addition, they lose contact inhibition and can start to grow on top of each other. This can result in formation of a tumor. It is important to make a distinction here: The term “cancer” is used to describe the diseases resulting from loss of cell-cycle regulation and subsequent cell proliferation. But the term “tumor” is more general. A “tumor” is an abnormal mass of cells, and a tumor can be benign (not cancerous) or malignant (cancerous).
Traditional cancer treatment uses radiation and/or chemotherapy to destroy cancer cells; however, these treatments can have unwanted side effects because they harm normal cells as well as cancer cells. Newer, promising therapies attempt to enlist the patient’s immune system to target cancer cells specifically. It is known that the immune system can recognize and destroy cancerous cells, and some researchers and immunologists also believe, based on the results of their experiments, that many cancers are eliminated by the body’s own defenses before they can become a health problem. This idea is not universally accepted by researchers, however, and needs further investigation for verification.
Cell-Mediated Response to Tumors
Cell-mediated immune responses can be directed against cancer cells, many of which do not have the normal complement of self-proteins, making them a target for elimination. Abnormal cancer cells may also present tumor antigens. These tumor antigens are not a part of the screening process used to eliminate lymphocytes during development; thus, even though they are self-antigens, they can stimulate and drive adaptive immune responses against abnormal cells.
Presentation of tumor antigens can stimulate naïve helper T cells to become activated by cytokines such as IL-12 and differentiate into TH1 cells. TH1 cells release cytokines that can activate natural killer (NK) cells and enhance the killing of activated cytotoxic T cells. Both NK cells and cytotoxic T cells can recognize and target cancer cells, and induce apoptosis through the action of perforins and granzymes. In addition, activated cytotoxic T cells can bind to cell-surface proteins on abnormal cells and induce apoptosis by a second killing mechanism called the CD95 (Fas) cytotoxic pathway.
Despite these mechanisms for removing cancerous cells from the body, cancer remains a common cause of death. Unfortunately, malignant tumors tend to actively suppress the immune response in various ways. In some cancers, the immune cells themselves are cancerous. In leukemia, lymphocytes that would normally facilitate the immune response become abnormal. In other cancers, the cancerous cells can become resistant to induction of apoptosis. This may occur through the expression of membrane proteins that shut off cytotoxic T cells or that induce regulatory T cells that can shut down immune responses.
The mechanisms by which cancer cells alter immune responses are still not yet fully understood, and this is a very active area of research. As scientists’ understanding of adaptive immunity improves, cancer therapies that harness the body’s immune defenses may someday be more successful in treating and eliminating cancer.
Exercise \(1\)
1. How do cancer cells suppress the immune system?
2. Describe how the immune system recognizes and destroys cancer cells.
Cancer Vaccines
There are two types of cancer vaccines: preventive and therapeutic. Preventive vaccines are used to prevent cancer from occurring, whereas therapeutic vaccines are used to treat patients with cancer. Most preventive cancer vaccines target viral infections that are known to lead to cancer. These include vaccines against human papillomavirus (HPV)and hepatitis B, which help prevent cervical and liver cancer, respectively.
Most therapeutic cancer vaccines are in the experimental stage. They exploit tumor-specific antigens to stimulate the immune system to selectively attack cancer cells. Specifically, they aim to enhance TH1 function and interaction with cytotoxic T cells, which, in turn, results in more effective attack on abnormal tumor cells. In some cases, researchers have used genetic engineering to develop antitumor vaccines in an approach similar to that used for DNA vaccines (see Micro Connections: DNA vaccines). The vaccine contains a recombinant plasmid with genes for tumor antigens; theoretically, the tumor gene would not induce new cancer because it is not functional, but it could trick the immune system into targeting the tumor gene product as a foreign invader.
The first FDA-approved therapeutic cancer vaccine was sipuleucel-T (Provenge), approved in 2010 to treat certain cases of prostate cancer.1 This unconventional vaccine is custom designed using the patient’s own cells. APCs are removed from the patient and cultured with a tumor-specific molecule; the cells are then returned to the patient. This approach appears to enhance the patient’s immune response against the cancer cells. Another therapeutic cancer vaccine (talimogene laherparepvec, also called T-VEC or Imlygic) was approved by the FDA in 2015 for treatment of melanoma, a form of skin cancer. This vaccine contains a virus that is injected into tumors, where it infects and lyses the tumor cells. The virus also induces a response in lesions or tumors besides those into which the vaccine is injected, indicating that it is stimulating a more general (as opposed to local) antitumor immune response in the patient.
Exercise \(2\)
1. Explain the difference between preventative and therapeutic cancer vaccines.
2. Describe at least two different approaches to developing therapeutic anti-cancer vaccines.
Using Viruses to Cure Cancer
Viruses typically destroy the cells they infect—a fact responsible for any number of human diseases. But the cell-killing powers of viruses may yet prove to be the cure for some types of cancer, which is generally treated by attempting to rid the body of cancerous cells. Several clinical trials are studying the effects of viruses targeted at cancer cells. Reolysin, a drug currently in testing phases, uses reoviruses (respiratory enteric orphan viruses) that can infect and kill cells that have an activated Ras-signaling pathway, a common mutation in cancerous cells. Viruses such as rubeola (the measles virus) can also be genetically engineered to aggressively attack tumor cells. These modified viruses not only bind more specifically to receptors overexpressed on cancer cells, they also carry genes driven by promoters that are only turned on within cancer cells. Herpesvirus and others have also been modified in this way.
Key Concepts and Summary
• Cancer results from a loss of control of the cell cycle, resulting in uncontrolled cell proliferation and a loss of the ability to differentiate.
• Adaptive and innate immune responses are engaged by tumor antigens, self-molecules only found on abnormal cells. These adaptive responses stimulate helper T cells to activate cytotoxic T cells and NK cells of innate immunity that will seek and destroy cancer cells.
• New anticancer therapies are in development that will exploit natural adaptive immunity anticancer responses. These include external stimulation of cytotoxic T cells and therapeutic vaccines that assist or enhance the immune response.
Footnotes
1. 1 National Institutes of Health, National Cancer Institute. "Cancer Vaccines." www.cancer.gov/about-cancer/c...-fact-sheet#q8. Accessed on May 20, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/19%3A_Diseases_of_the_Immune_System/19.05%3A_Cancer_Immunobiology_and_Immunotherapy.txt |
19.1: Hypersensitivities
An allergy is an adaptive immune response, sometimes life-threatening, to an allergen. Hypersensitivity reactions are classified by their immune mechanism.
Multiple Choice
Which of the following is the type of cell largely responsible for type I hypersensitivity responses?
1. erythrocyte
2. mast cell
3. T lymphocyte
4. antibody
Answer
B
Type I hypersensitivities require which of the following initial priming events to occur?
1. sensitization
2. secondary immune response
3. cellular trauma
4. degranulation
Answer
A
Which of the following are the main mediators/initiators of type II hypersensitivity reactions?
1. antibodies
2. mast cells
3. erythrocytes
4. histamines
Answer
A
Inflammatory molecules are released by mast cells in type I hypersensitivities; type II hypersensitivities, however, are characterized by which of the following?
1. cell lysis (cytotoxicity)
2. strong antibody reactions against antigens
3. leukotriene release upon stimulation
4. localized tissue reactions, such as hives
Answer
A
An immune complex is an aggregate of which of the following?
1. antibody molecules
2. antigen molecules
3. antibody and antigen molecules
4. histamine molecules
Answer
C
Which of the following is a common treatment for type III hypersensitivity reactions?
1. anti-inflammatory steroid treatments
2. antihistamine treatments
3. hyposensitization injections of allergens
4. RhoGAM injections
Answer
A
Which of the following induces a type III hypersensitivity?
1. release of inflammatory molecules from mast cells
2. accumulation of immune complexes in tissues and small blood vessels
3. destruction of cells bound by antigens
4. destruction of cells bound by antibodies
Answer
B
Which one of the following is not an example of a type IV hypersensitivity?
1. latex allergy
2. Contact dermatitis (e.g., contact with poison ivy)
3. a positive tuberculin skin test
4. hemolytic disease of the newborn
Answer
D
Fill in the Blank
Antibodies involved in type I hypersensitivities are of the ________ class.
Answer
IgE
Allergy shots work by shifting antibody responses to produce ________ antibodies.
Answer
IgG
A person who is blood type A would have IgM hemagglutinin antibodies against type ________ red blood cells in their plasma.
Answer
B
The itchy and blistering rash that develops with contact to poison ivy is caused by a type ________ hypersensitivity reaction.
Answer
IV
Short Answer
Although both type I and type II hypersensitivities involve antibodies as immune effectors, different mechanisms are involved with these different hypersensitivities. Differentiate the two.
What types of antibodies are most common in type III hypersensitivities, and why?
Critical Thinking
Patients are frequently given instructions to avoid allergy medications for a period of time prior to allergy testing. Why would this be important?
In some areas of the world, a tuberculosis vaccine known as bacillus Calmette-Guérin (BCG) is used. It is not used in the United States. Every person who has received this vaccine and mounted a protective response will have a positive reaction in a tuberculin skin test. Why? What does this mean for the usefulness of this skin test in those countries where this vaccine is used?
19.2: Autoimmune Disorders
Autoimmune diseases result from a breakdown in immunological tolerance. The actual induction event(s) for autoimmune states are largely unknown. Some autoimmune diseases attack specific organs, whereas others are more systemic. Organ-specific autoimmune diseases include celiac disease, Graves disease, Hashimoto thyroiditis, type I diabetes mellitus, and Addison disease.
Multiple Choice
Which of the following is an example of an organ-specific autoimmune disease?
1. rheumatoid arthritis
2. psoriasis
3. Addison disease
4. myasthenia gravis
Answer
C
Which of the following is an example of a systemic autoimmune disease?
1. Hashimoto thyroiditis
2. type I diabetes mellitus
3. Graves disease
4. myasthenia gravis
Answer
D
Fill in the Blank
The thyroid-stimulating immunoglobulin that acts like thyroid-stimulating hormone and causes Graves disease is an antibody to the ________.
Answer
thyroid-stimulating hormone receptor
19.3: Organ Transplantation and Rejection
Grafts and transplants can be classified as autografts, isografts, allografts, or xenografts based on the genetic differences between the donor’s and recipient’s tissues. Genetic differences, especially among the MHC (HLA) genes, will dictate the likelihood that rejection of the transplanted tissue will occur. Transplant recipients usually require immunosuppressive therapy to avoid rejection, even with good genetic matching.
Matching
Match the graft with its description.
___autograft A. donor is a different species than the recipient
___allograft B. donor and recipient are the same individual
___xenograft C. donor is an identical twin of the recipient
___isograft D. donor is the same species as the recipient, but genetically different
Answer
B, D, A, C
Fill in the Blank
For a transplant to have the best chances of avoiding rejection, the genes coding for the ________ molecules should be closely matched between donor and recipient.
Answer
MHC
Because it is a “transplant” that can include APCs and T cells from the donor, a bone marrow transplant may induce a very specific type of rejection known as ________ disease.
Answer
graft-versus-host
Short Answer
Why is a parent usually a better match for transplanted tissue to a donor than a random individual of the same species?
19.4: Immunodeficiency
Primary immunodeficiencies are caused by genetic abnormalities; secondary immunodeficiencies are acquired through disease, diet, or environmental exposures. Primary immunodeficiencies may result from flaws in phagocyte killing of innate immunity, or impairment of T cells and B cells. Primary immunodeficiencies include chronic granulomatous disease, X-linked agammaglobulinemia, selective IgA deficiency, and severe combined immunodeficiency disease.
Multiple Choice
Which of the following is a genetic disease that results in lack of production of antibodies?
1. agammaglobulinemia
2. myasthenia gravis
3. HIV/AIDS
4. chronic granulomatous disease
Answer
A
Which of the following is a genetic disease that results in almost no adaptive immunity due to lack of B and/ or T cells?
1. agammaglobulinemia
2. severe combined immunodeficiency
3. HIV/AIDS
4. chronic granulomatous disease
Answer
B
All but which one of the following are examples of secondary immunodeficiencies?
1. HIV/AIDS
2. malnutrition
3. chronic granulomatous disease
4. immunosuppression due to measles infection
Answer
C
Fill in the Blank
Diseases due to ________ abnormalities are termed primary immunodeficiencies.
Answer
genetic
A secondary immunodeficiency is ________, rather than genetic.
Answer
acquired
Short Answer
Compare the treatments for primary and secondary immunodeficiencies.
19.5: Cancer Immunobiology and Immunotherapy
When control of the cell cycle is lost, the affected cells rapidly divide and often lose the ability to differentiate into the cell type appropriate for their location in the body. In addition, they lose contact inhibition and can start to grow on top of each other. This can result in formation of a tumor. It is important to make a distinction here: The term “cancer” is used to describe the diseases resulting from loss of cell-cycle regulation and subsequent cell proliferation.
Multiple Choice
Cancer results when a mutation leads to which of the following?
1. cell death
2. apoptosis
3. loss of cell-cycle control
4. shutdown of the cell cycle
Answer
C
Tumor antigens are ________ that are inappropriately expressed and found on abnormal cells.
1. self antigens
2. foreign antigens
3. antibodies
4. T-cell receptors
Answer
A
Fill in the Blank
A ________ cancer vaccine is one that stops the disease from occurring in the first place.
Answer
preventive
A ________ cancer vaccine is one that will help to treat the disease after it has occurred.
Answer
therapeutic
Short Answer
How can tumor antigens be effectively targeted without inducing an autoimmune (anti-self) response? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/19%3A_Diseases_of_the_Immune_System/19.E%3A_Diseases_of_the_Immune_System_%28Exercises%29.txt |
Many laboratory tests are designed to confirm a presumptive diagnosis by detecting antibodies specific to a suspected pathogen. Unfortunately, many such tests are time-consuming and expensive. That is now changing, however, with the development of new, miniaturized technologies that are fast and inexpensive. For example, researchers at Columbia University are developing a “lab-on-a-chip” technology that will test a single drop of blood for 15 different infectious diseases, including HIV and syphilis, in a matter of minutes.1 The blood is pulled through tiny capillaries into reaction chambers where the patient’s antibodies mix with reagents. A chip reader that attaches to a cell phone analyzes the results and sends them to the patient’s healthcare provider. Currently the device is being field tested in Rwanda to check pregnant women for chronic diseases. Researchers estimate that the chip readers will sell for about \$100 and individual chips for \$1.2
• 20.1: Practical Applications of Monoclonal and Polyclonal Antibodies
In addition to being crucial for our normal immune response, antibodies provide powerful tools for research and diagnostic purposes. The high specificity of antibodies makes them an excellent tool for detecting and quantifying a broad array of targets, from drugs to serum proteins to microorganisms. With in vitro assays, antibodies can be used to precipitate soluble antigens, agglutinate cells, and neutralize drugs, toxins, and viruses.
• 20.2: Detecting Antigen-Antibody Complexes in vitro
Laboratory tests to detect antibodies and antigens outside of the body (e.g., in a test tube) are called in vitro assays. When both antibodies and their corresponding antigens are present in a solution, we can often observe a precipitation reaction in which large complexes (lattices) form and settle out of solution. In the next several sections, we will discuss several common in vitro assays.
• 20.3: Agglutination Assays
In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination. Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or microtiter plate.
• 20.4: Enzyme Immunoassays (EIA) and Enzyme-Linked Immunosorbent Assays (ELISA)
Enzyme immunoassays (EIA) are used to visualize and quantify antigens. They use an antibody conjugated to an enzyme to bind the antigen, and the enzyme converts a substrate into an observable end product. The substrate may be either a chromogen or a fluorogen. Immunostaining is an EIA technique for visualizing cells in a tissue (immunohistochemistry) or examining intracellular structures (immunocytochemistry). Direct ELISA is used to quantify an antigen in solution.
• 20.5: Fluorescent Auto-Antibody Techniques
Rapid visualization of bacteria from a clinical sample such as a throat swab or sputum can be achieved through fluorescent antibody (FA) techniques that attach a fluorescent marker (fluorogen) to the constant region of an antibody, resulting in a reporter molecule that is quick to use, easy to see or measure, and able to bind to target markers with high specificity. We can also label cells, allowing us to precisely quantify particular subsets of cells or even purify them for further research.
• 20.E: Laboratory Analysis of the Immune Response (Exercises)
Footnotes
1. 1 Chin, Curtis D. et al., “Mobile Device for Disease Diagnosis and Data Tracking in Resource-Limited Settings,” Clinical Chemistry 59, no. 4 (2013): 629-40.
2. 2 Evarts, H., “Fast, Low-Cost Device Uses the Cloud to Speed Up Testing for HIV and More,” January 24, 2013. Accessed July 14, 2016. http://engineering.columbia.edu/fast...g-hiv-and-more.
Thumbnail: Enzyme-linked antibodies against CD8 were used to stain the CD8 cells in this preparation of bone marrow using a chromogen. (credit: modification of work by Yamashita M, Fujii Y, Ozaki K, Urano Y, Iwasa M, Nakamura S, Fujii S, Abe M, Sato Y, Yoshino T).
20: Laboratory Analysis of the Immune Response
Learning Objectives
• Compare the method of development, use, and characteristics of monoclonal and polyclonal antibodies
• Explain the nature of antibody cross-reactivity and why this is less of a problem with monoclonal antibodies
Clinical Focus: Part 1
In an unfortunate incident, a healthcare worker struggling with addiction was caught stealing syringes of painkillers and replacing them with syringes filled with unknown substances. The hospital immediately fired the employee and had him arrested; however, two patients that he had worked with later tested positive for HIV.
While there was no proof that the infections originated from the tainted syringes, the hospital’s public health physician took immediate steps to determine whether any other patients had been put at risk. Although the worker had only been employed for a short time, it was determined that he had come into contact with more than 1300 patients. The hospital decided to contact all of these patients and have them tested for HIV.
Exercise \(1\)
1. Why does the hospital feel it is necessary to test every patient for HIV?
2. What types of tests can be used to determine if a patient has HIV?
In addition to being crucial for our normal immune response, antibodies provide powerful tools for research and diagnostic purposes. The high specificity of antibodies makes them an excellent tool for detecting and quantifying a broad array of targets, from drugs to serum proteins to microorganisms. With in vitro assays, antibodies can be used to precipitate soluble antigens, agglutinate (clump) cells, opsonize and kill bacteria with the assistance of complement, and neutralize drugs, toxins, and viruses.
An antibody’s specificity results from the antigen-binding site formed within the variable regions—regions of the antibody that have unique patterns of amino acids that can only bind to target antigens with a molecular sequence that provides complementary charges and noncovalent bonds. There are limitations to antibody specificity, however. Some antigens are so chemically similar that cross-reactivity occurs; in other words, antibodies raised against one antigen bind to a chemically similar but different antigen. Consider an antigen that consists of a single protein with multiple epitopes (Figure \(1\)). This single protein may stimulate the production of many different antibodies, some of which may bind to chemically identical epitopes on other proteins.
Cross-reactivity is more likely to occur between antibodies and antigens that have low affinity or avidity. Affinity, which can be determined experimentally, is a measure of the binding strength between an antibody's binding site and an epitope, whereas avidity is the total strength of all the interactions in an antibody-antigen complex (which may have more than one bonding site). Avidity is influenced by affinity as well as the structural arrangements of the epitope and the variable regions of the antibody. If an antibody has a high affinity/avidity for a specific antigen, it is less likely to cross-react with an antigen for which it has a lower affinity/avidity.
Exercise \(2\)
1. What property makes antibodies useful for research and clinical diagnosis?
2. What is cross-reactivity and why does it occur?
Producing Polyclonal Antibodies
Antibodies used for research and diagnostic purposes are often obtained by injecting a lab animal such as a rabbit or a goat with a specific antigen. Within a few weeks, the animal’s immune system will produce high levels of antibodies specific for the antigen. These antibodies can be harvested in an antiserum, which is whole serum collected from an animal following exposure to an antigen. Because most antigens are complex structures with multiple epitopes, they result in the production of multiple antibodies in the lab animal. This so-called polyclonal antibody response is also typical of the response to infection by the human immune system. Antiserum drawn from an animal will thus contain antibodies from multiple clones of B cells, with each B cell responding to a specific epitope on the antigen (Figure \(2\)).
Lab animals are usually injected at least twice with antigen when being used to produce antiserum. The second injection will activate memory cells that make class IgG antibodies against the antigen. The memory cells also undergo affinity maturation, resulting in a pool of antibodies with higher average affinity. Affinity maturation occurs because of mutations in the immunoglobulin gene variable regions, resulting in B cells with slightly altered antigen-binding sites. On re-exposure to the antigen, those B cells capable of producing antibody with higher affinity antigen-binding sites will be stimulated to proliferate and produce more antibody than their lower-affinity peers. An adjuvant, which is a chemical that provokes a generalized activation of the immune system that stimulates greater antibody production, is often mixed with the antigen prior to injection.
Antiserum obtained from animals will not only contain antibodies against the antigen artificially introduced in the laboratory, but it will also contain antibodies to any other antigens to which the animal has been exposed during its lifetime. For this reason, antisera must first be “purified” to remove other antibodies before using the antibodies for research or diagnostic assays.
Clinical Uses of Polyclonal Antisera
Polyclonal antisera are used in many clinical tests that are designed to determine whether a patient is producing antibodies in response to a particular pathogen. While these tests are certainly powerful diagnostic tools, they have their limitations, because they are an indirect means of determining whether a particular pathogen is present. Tests based on a polyclonal response can sometimes lead to a false-positive result—in other words, a test that confirms the presence of an antigen that is, in fact, not present. Antibody-based tests can also result in a false-negative result, which occurs when the test fails to detect an antibody that is, in fact, present.
The accuracy of antibody tests can be described in terms of test sensitivity and test specificity. Test sensitivity is the probability of getting a positive test result when the patient is indeed infected. If a test has high sensitivity, the probability of a false negative is low. Test specificity, on the other hand, is the probability of getting a negative test result when the patient is not infected. If a test has high specificity, the probability of a false positive is low.
False positives often occur due to cross-reactivity, which can occur when epitopes from a different pathogen are similar to those found on the pathogen being tested for. For this reason, antibody-based tests are often used only as screening tests; if the results are positive, other confirmatory tests are used to make sure that the results were not a false positive.
For example, a blood sample from a patient suspected of having hepatitis C can be screened for the virus using antibodies that bind to antigens on hepatitis C virus. If the patient is indeed infected with hepatitis C virus, the antibodies will bind to the antigens, yielding a positive test result. If the patient is not infected with hepatitic C virus, the antibodies will generally not bind to anything and the test should be negative; however, a false positive may occur if the patient has been previously infected by any of a variety of pathogens that elicit antibodies that cross-react with the hepatitis C virus antigens. Antibody tests for hepatitis C have high sensitivity (a low probability of a false negative) but low specificity (a high probability of a false positive). Thus, patients who test positive must have a second, confirmatory test to rule out the possibility of a false positive. The confirmatory test is a more expensive and time-consuming test that directly tests for the presence of hepatitis C viral RNA in the blood. Only after the confirmatory test comes back positive can the patient be definitively diagnosed with a hepatitis C infection. Antibody-based tests can result in a false negative if, for any reason, the patient’s immune system has not produced detectable levels of antibodies. For some diseases, it may take several weeks following infection before the immune system produces enough antibodies to cross the detection threshold of the assay. In immunocompromised patients, the immune system may not be capable of producing a detectable level of antibodies.
Another limitation of using antibody production as an indicator of disease is that antibodies in the blood will persist long after the infection has been cleared. Depending on the type of infection, antibodies will be present for many months; sometimes, they may be present for the remainder of the patient’s life. Thus, a positive antibody-based test only means that the patient was infected at some point in time; it does not prove that the infection is active.
In addition to their role in diagnosis, polyclonal antisera can activate complement, detect the presence of bacteria in clinical and food industry settings, and perform a wide array of precipitation reactions that can detect and quantify serum proteins, viruses, or other antigens. However, with the many specificities of antibody present in a polyclonal antiserum, there is a significant likelihood that the antiserum will cross-react with antigens to which the individual was never exposed. Therefore, we must always account for the possibility of false-positive results when working with a polyclonal antiserum.
Exercise \(3\)
1. What is a false positive and what are some reasons that false positives occur?
2. What is a false negative and what are some reasons that false positives occur?
3. If a patient tests negative on a highly sensitive test, what is the likelihood that the person is infected with the pathogen?
Producing Monoclonal Antibodies
Some types of assays require better antibody specificity and affinity than can be obtained using a polyclonal antiserum. To attain this high specificity, all of the antibodies must bind with high affinity to a single epitope. This high specificity can be provided by monoclonal antibodies (mAbs). Table \(1\) compares some of the important characteristics of monoclonal and polyclonal antibodies.
Unlike polyclonal antibodies, which are produced in live animals, monoclonal antibodies are produced in vitro using tissue-culture techniques. mAbs are produced by immunizing an animal, often a mouse, multiple times with a specific antigen. B cells from the spleen of the immunized animal are then removed. Since normal B cells are unable to proliferate forever, they are fused with immortal, cancerous B cells called myeloma cells, to yield hybridoma cells. All of the cells are then placed in a selective medium that allows only the hybridomas to grow; unfused myeloma cells cannot grow, and any unfused B cells die off. The hybridomas, which are capable of growing continuously in culture while producing antibodies, are then screened for the desired mAb. Those producing the desired mAb are grown in tissue culture; the culture medium is harvested periodically and mAbs are purified from the medium. This is a very expensive and time-consuming process. It may take weeks of culturing and many liters of media to provide enough mAbs for an experiment or to treat a single patient. mAbs are expensive (Figure \(3\)).
Table \(1\): Characteristics of Polyclonal and Monoclonal Antibodies
Monoclonal Antibodies Polyclonal Antibodies
Expensive production Inexpensive production
Long production time Rapid production
Large quantities of specific antibodies Large quantities of nonspecific antibodies
Recognize a single epitope on an antigen Recognize multiple epitopes on an antigen
Production is continuous and uniform once the hybridoma is made Different batches vary in composition
Clinical Uses of Monoclonal Antibodies
Since the most common methods for producing monoclonal antibodies use mouse cells, it is necessary to create humanized monoclonal antibodies for human clinical use. Mouse antibodies cannot be injected repeatedly into humans, because the immune system will recognize them as being foreign and will respond to them with neutralizing antibodies. This problem can be minimized by genetically engineering the antibody in the mouse B cell. The variable regions of the mouse light and heavy chain genes are ligated to human constant regions, and the chimeric gene is then transferred into a host cell. This allows production of a mAb that is mostly “human” with only the antigen-binding site being of mouse origin.
Humanized mAbs have been successfully used to treat cancer with minimal side effects. For example, the humanized monoclonal antibody drug Herceptin has been helpful for the treatment of some types of breast cancer. There have also been a few preliminary trials of humanized mAb for the treatment of infectious diseases, but none of these treatments are currently in use. In some cases, mAbs have proven too specific to treat infectious diseases, because they recognize some serovars of a pathogen but not others. Using a cocktail of multiple mAbs that target different strains of the pathogen can address this problem. However, the great cost associated with mAb production is another challenge that has prevented mAbs from becoming practical for use in treating microbial infections.1
One promising technology for inexpensive mAbs is the use of genetically engineered plants to produce antibodies (or plantibodies). This technology transforms plant cells into antibody factories rather than relying on tissue culture cells, which are expensive and technically demanding. In some cases, it may even be possible to deliver these antibodies by having patients eat the plants rather than by extracting and injecting the antibodies. For example, in 2013, a research group cloned antibody genes into plants that had the ability to neutralize an important toxin from bacteria that can cause severe gastrointestinal disease.2 Eating the plants could potentially deliver the antibodies directly to the toxin.
Exercise \(4\)
1. How are humanized monoclonal antibodies produced?
2. What does the “monoclonal” of monoclonal antibodies mean?
Using Monoclonal Antibodies to Combat Ebola
During the 2014–2015 Ebola outbreak in West Africa, a few Ebola-infected patients were treated with ZMapp, a drug that had been shown to be effective in trials done in rhesus macaques only a few months before.3 ZMapp is a combination of three mAbs produced by incorporating the antibody genes into tobacco plants using a viral vector. By using three mAbs, the drug is effective across multiple strains of the virus. Unfortunately, there was only enough ZMapp to treat a tiny number of patients.
While the current technology is not adequate for producing large quantities of ZMapp, it does show that plantibodies—plant-produced mAbs—are feasible for clinical use, potentially cost effective, and worth further development. The last several years have seen an explosion in the number of new mAb-based drugs for the treatment of cancer and infectious diseases; however, the widespread use of such drugs is currently inhibited by their exorbitant cost, especially in underdeveloped parts of the world, where a single dose might cost more than the patient’s lifetime income. Developing methods for cloning antibody genes into plants could reduce costs dramatically.
Key Concepts and Summary
• Antibodies bind with high specificity to antigens used to challenge the immune system, but they may also show cross-reactivity by binding to other antigens that share chemical properties with the original antigen.
• Injection of an antigen into an animal will result in a polyclonal antibody response in which different antibodies are produced that react with the various epitopes on the antigen.
• Polyclonal antisera are useful for some types of laboratory assays, but other assays require more specificity. Diagnostic tests that use polyclonal antisera are typically only used for screening because of the possibility of false-positive and false-negative results.
• Monoclonal antibodies provide higher specificity than polyclonal antisera because they bind to a single epitope and usually have high affinity.
• Monoclonal antibodies are typically produced by culturing antibody-secreting hybridomas derived from mice. mAbs are currently used to treat cancer, but their exorbitant cost has prevented them from being used more widely to treat infectious diseases. Still, their potential for laboratory and clinical use is driving the development of new, cost-effective solutions such as plantibodies.
Footnotes
1. 1 Saylor, Carolyn, Ekaterina Dadachova and Arturo Casadevall, “Monoclonal Antibody-Based Therapies for Microbial Diseases,” Vaccine 27 (2009): G38-G46.
2. 2 Nakanishi, Katsuhiro et al., “Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1,” PloS One 8, no. 11 (2013): e80712.
3. 3 Qiu, Xiangguo et al., “Reversion of Advanced Ebola Virus Disease in Nonhuman Primates with ZMapp,” Nature 514 (2014): 47–53. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/20%3A_Laboratory_Analysis_of_the_Immune_Response/20.01%3A_Practical_Applications_of_Monoclonal_and_Polyclonal_Antibodies.txt |
Learning Objectives
• Describe various types of assays used to find antigen-antibody complexes
• Describe the circumstances under which antigen-antibody complexes precipitate out of solution
• Explain how antibodies in patient serum can be used to diagnose disease
Laboratory tests to detect antibodies and antigens outside of the body (e.g., in a test tube) are called in vitro assays. When both antibodies and their corresponding antigens are present in a solution, we can often observe a precipitation reaction in which large complexes (lattices) form and settle out of solution. In the next several sections, we will discuss several common in vitro assays.
Precipitin Reactions
A visible antigen-antibody complex is called a precipitin, and in vitro assays that produce a precipitin are called precipitin reactions. A precipitin reaction typically involves adding soluble antigens to a test tube containing a solution of antibodies. Each antibody has two arms, each of which can bind to an epitope. When an antibody binds to two antigens, the two antigens become bound together by the antibody. A lattice can form as antibodies bind more and more antigens together, resulting in a precipitin (Figure \(1\)). Most precipitin tests use a polyclonal antiserum rather than monoclonal antibodies because polyclonal antibodies can bind to multiple epitopes, making lattice formation more likely. Although mAbs may bind some antigens, the binding will occur less often, making it much less likely that a visible precipitin will form.
The amount of precipitation also depends on several other factors. For example, precipitation is enhanced when the antibodies have a high affinity for the antigen. While most antibodies bind antigen with high affinity, even high-affinity binding uses relatively weak noncovalent bonds, so that individual interactions will often break and new interactions will occur.
In addition, for precipitin formation to be visible, there must be an optimal ratio of antibody to antigen. The optimal ratio is not likely to be a 1:1 antigen-to-antibody ratio; it can vary dramatically, depending on the number of epitopes on the antigen and the class of antibody. Some antigens may have only one or two epitopes recognized by the antiserum, whereas other antigens may have many different epitopes and/or multiple instances of the same epitope on a single antigen molecule.
Figure \(2\) illustrates how the ratio of antigen and antibody affects the amount of precipitation. To achieve the optimal ratio, antigen is slowly added to a solution containing antibodies, and the amount of precipitin is determined qualitatively. Initially, there is not enough antigen to produce visible lattice formation; this is called the zone of antibody excess. As more antigen is added, the reaction enters the equivalence zone (or zone of equivalence), where both the optimal antigen-antibody interaction and maximal precipitation occur. If even more antigen were added, the amount of antigen would become excessive and actually cause the amount of precipitation to decline.
Exercise \(1\)
1. What is a precipitin?
2. Why do polyclonal antisera produce a better precipitin reaction?
Precipitin Ring Test
A variety of techniques allow us to use precipitin formation to quantify either antigen concentration or the amount of antibody present in an antiserum. One such technique is the precipitin ring test (Figure \(3\)), which is used to determine the relative amount of antigen-specific antibody in a sample of serum. To perform this test, a set of test tubes is prepared by adding an antigen solution to the bottom of each tube. Each tube receives the same volume of solution, and the concentration of antigens is constant (e.g., 1 mg/mL). Next, glycerol is added to the antigen solution in each test tube, followed by a serial dilution of the antiserum. The glycerol prevents mixing of the antiserum with the antigen solution, allowing antigen-antibody binding to take place only at the interface of the two solutions. The result is a visible ring of precipitin in the tubes that have an antigen-antibody ratio within the equivalence zone. This highest dilution with a visible ring is used to determine the titer of the antibodies. The titer is the reciprocal of the highest dilution showing a positive result, expressed as a whole number. In Figure \(3\), the titer is 16.
While a measurement of titer does not tell us in absolute terms how much antibody is present, it does give a measure of biological activity, which is often more important than absolute amount. In this example, it would not be useful to know what mass of IgG were present in the antiserum, because there are many different specificities of antibody present; but it is important for us to know how much of the antibody activity in a patient’s serum is directed against the antigen of interest (e.g., a particular pathogen or allergen).
Ouchterlony Assay
While the precipitin ring test provides insights into antibody-antigen interactions, it also has some drawbacks. It requires the use of large amounts of serum, and great care must be taken to avoid mixing the solutions and disrupting the ring. Performing a similar test in an agar gel matrix can minimize these problems. This type of assay is variously called double immunodiffusion or the Ouchterlony assay for Orjan Ouchterlony,1 who first described the technique in 1948.
When agar is highly purified, it produces a clear, colorless gel. Holes are punched in the gel to form wells, and antigen and antisera are added to neighboring wells. Proteins are able to diffuse through the gel, and precipitin arcs form between the wells at the zone of equivalence. Because the precipitin lattice is too large to diffuse through the gel, the arcs are firmly locked in place and easy to see (Figure \(4\)).
Although there are now more sensitive and quantitative methods of detecting antibody-antigen interactions, the Ouchterlony test provides a rapid and qualitative way of determining whether an antiserum has antibodies against a particular antigen. The Ouchterlony test is particularly useful when looking for cross-reactivity. We can check an antiserum against a group of closely related antigens and see which combinations form precipitin arcs.
Radial Immunodiffusion Assay
The radial immunodiffusion (RID) assay is similar to the Ouchterlony assay but is used to precisely quantify antigen concentration rather than to compare different antigens. In this assay, the antiserum is added to tempered agar (liquid agar at slightly above 45 °C), which is poured into a small petri dish or onto a glass slide and allowed to cool. Wells are cut in the cooled agar, and antigen is then added to the wells and allowed to diffuse. As the antigen and antibody interact, they form a zone of precipitation. The square of the diameter of the zone of precipitation is directly proportional to the concentration of antigen. By measuring the zones of precipitation produced by samples of known concentration (see the outer ring of samples in Figure \(5\)), we can prepare a standard curve for determining the concentration of an unknown solution. The RID assay is a also useful test for determining the concentration of many serum proteins such as the C3 and C4 complement proteins, among others.
Exercise \(2\)
1. Why does a precipitin ring form in a precipitin ring test, and what are some reasons why a ring might not form?
2. Compare and contrast the techniques used in an Ouchterlony assay and a radial immunodiffusion assay.
Flocculation Assays
A flocculation assay is similar to a precipitin reaction except that it involves insoluble antigens such as lipids. A flocculant is similar to a precipitin in that there is a visible lattice of antigen and antibody, but because lipids are insoluble in aqueous solution, they cannot precipitate. Instead of precipitation, flocculation (foaming) is observed in the test tube fluid.
Using Flocculation to Test for Syphilis
Syphilis is a sexually transmitted infection that can cause severe, chronic disease in adults. In addition, it is readily passed from infected mothers to their newborns during pregnancy and childbirth, often resulting in stillbirth or serious long-term health problems for the infant. Unfortunately, syphilis can also be difficult to diagnose in expectant mothers, because it is often asymptomatic, especially in women. In addition, the causative agent, the bacterium Treponema pallidum, is both difficult to grow on conventional lab media and too small to see using routine microcopy. For these reasons, presumptive diagnoses of syphilis are generally confirmed indirectly in the laboratory using tests that detect antibodies to treponemal antigens.
In 1906, German scientist August von Wassermann (1866–1925) introduced the first test for syphilis that relied on detecting anti-treponemal antibodies in the patient’s blood. The antibodies detected in the Wassermann test were antiphospholipid antibodies that are nonspecific to T. pallidum. Their presence can assist in the diagnosis of syphilis, but because they are nonspecific, they can also lead to false-positive results in patients with other diseases and autoimmune conditions. The original Wasserman test has been modified over the years to minimize false-positives and is now known as the Venereal Disease Research Lab test, better known by its acronym, the VDRL test.
To perform the VDRL test, patient serum or cerebral spinal fluid is placed on a slide with a mixture of cardiolipin (an antigenic phospholipid found in the mitochondrial membrane of various pathogens), lecithin, and cholesterol. The lecithin and cholesterol stabilize the reaction and diminish false positives. Anti-treponemal antibodies from an infected patient’s serum will bind cardiolipin and form a flocculant. Although the VDRL test is more specific than the original Wassermann assay, false positives may still occur in patients with autoimmune diseases that cause extensive cell damage (e.g., systemic lupus erythematosus).
Neutralization Assay
To cause infection, viruses must bind to receptors on host cells. Antiviral antibodies can neutralize viral infections by coating the virions, blocking the binding (Figure 18.1.6). This activity neutralizes virions and can result in the formation of large antibody-virus complexes (which are readily removed by phagocytosis) or by antibody binding to the virus and blocking its binding to host cell receptors. This neutralization activity is the basis of neutralization assays, sensitive assays used for diagnoses of viral infections.
When viruses infect cells, they often cause damage (cytopathic effects) that may include lysis of the host cells. Cytopathic effects can be visualized by growing host cells in a petri dish, covering the cells with a thin layer of agar, and then adding virus (see Isolation, Culture, and Identification of Viruses). The virus will diffuse very slowly through the agar. A virus will enter a host cell, proliferate (causing cell damage), be released from the dead host cell, and then move to neighboring cells. As more and more cells die, plaques of dead cells will form (Figure \(6\)).
During the course of a viral infection, the patient will mount an antibody response to the virus, and we can quantify those antibodies using a plaque reduction assay. To perform the assay, a serial dilution is carried out on a serum sample. Each dilution is then mixed with a standardized amount of the suspect virus. Any virus-specific antibodies in the serum will neutralize some of the virus. The suspensions are then added to host cells in culture to allow any nonneutralized virus to infect the cells and form plaques after several days. The titer is defined as the reciprocal of the highest dilution showing a 50% reduction in plaques. Titer is always expressed as a whole number. For example, if a 1/64 dilution was the highest dilution to show 50% plaque reduction, then the titer is 64.
The presence of antibodies in the patient’s serum does not tell us whether the patient is currently infected or was infected in the past. Current infections can be identified by waiting two weeks and testing another serum sample. A four-fold increase in neutralizing titer in this second sample indicates a new infection.
Exercise \(3\)
In a neutralization assay, if a patient’s serum has high numbers of antiviral antibodies, would you expect to see more or fewer plaques?
Immunoelectrophoresis
When a patient has elevated protein levels in the blood or is losing protein in the urine, a clinician will often order a polyacrylamide gel electrophoresis (PAGE) assay (see Visualizing and Characterizing DNA, RNA, and Protein). This assay compares the relative abundance of the various types of serum proteins. Abnormal protein electrophoresis patterns can be further studied using immunoelectrophoresis (IEP). The IEP begins by running a PAGE. Antisera against selected serum proteins are added to troughs running parallel to the electrophoresis track, forming precipitin arcs similar to those seen in an Ouchterlony assay (Figure \(7\)). This allows the identification of abnormal immunoglobulin proteins in the sample.
IEP is particularly useful in the diagnosis of multiple myeloma, a cancer of antibody-secreting cells. Patients with multiple myeloma cannot produce healthy antibodies; instead they produce abnormal antibodies that are monoclonal proteins (M proteins). Thus, patients with multiple myeloma will present with elevated serum protein levels that show a distinct band in the gamma globulin region of a protein electrophoresis gel and a sharp spike (in M protein) on the densitometer scan rather than the normal broad smear (Figure \(7\)). When antibodies against the various types of antibody heavy and light chains are used to form precipitin arcs, the M protein will cause distinctly skewed arcs against one class of heavy chain and one class of light chain as seen in Figure \(7\).
Protein Electrophoresis and the Characterization of Immunoglobulin Structure
The advent of electrophoresis ultimately led to researching and understanding the structure of antibodies. When Swedish biochemist Arne Tiselius (1902–1971) published the first protein electrophoresis results in 1937,2 he could identify the protein albumin (the smallest and most abundant serum protein) by the sharp band it produced in the gel. The other serum proteins could not be resolved in a simple protein electrophoresis, so he named the three broad bands, with many proteins in each band, alpha, beta, and gamma globulins. Two years later, American immunologist Elvin Kabat (1914–2000) traveled to Sweden to work with Tiselius using this new technique and showed that antibodies migrated as gamma globulins.3 With this new understanding in hand, researchers soon learned that multiple myeloma, because it is a cancer of antibody-secreting cells, could be tentatively diagnosed by the presence of a large M spike in the gamma-globulin region by protein electrophoresis. Prior to this discovery, studies on immunoglobulin structure had been minimal, because of the difficulty of obtaining pure samples to study. Sera from multiple myeloma patients proved to be an excellent source of highly enriched monoclonal immunoglobulin, providing the raw material for studies over the next 20-plus years that resulted in the elucidation of the structure of immunoglobulin.
Exercise \(4\)
In general, what does an immunoelectrophoresis assay accomplish?
Immunoblot Assay: The Western Blot
After performing protein gel electrophoresis, specific proteins can be identified in the gel using antibodies. This technique is known as the western blot. Following separation of proteins by PAGE, the protein antigens in the gel are transferred to and immobilized on a nitrocellulose membrane. This membrane can then be exposed to a primary antibody produced to specifically bind to the protein of interest. A second antibody equipped with a molecular beacon will then bind to the first. These secondary antibodies are coupled to another molecule such as an enzyme or a fluorophore (a molecule that fluoresces when excited by light). When using antibodies coupled to enzymes, a chromogenic substrate for the enzyme is added. This substrate is usually colorless but will develop color in the presence of the antibody. The fluorescence or substrate coloring identifies the location of the specific protein in the membrane to which the antibodies are bound (Figure \(9\)).
Typically, polyclonal antibodies are used for western blot assays. They are more sensitive than mAbs because of their ability to bind to various epitopes of the primary antigen, and the signal from polyclonal antibodies is typically stronger than that from mAbs. Monoclonal antibodies can also be used; however, they are much more expensive to produce and are less sensitive, since they are only able to recognize one specific epitope.
Several variations of the western blot are useful in research. In a southwestern blot, proteins are separated by SDS-PAGE, blotted onto a nitrocellulose membrane, allowed to renature, and then probed with a fluorescently or radioactively labeled DNA probe; the purpose of the southwestern is to identify specific DNA-protein interactions. Far-western blotsare carried out to determine protein-protein interactions between immobilized proteins (separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and allowed to renature) and non-antibody protein probes. The bound non-antibody proteins that interact with the immobilized proteins in a far-western blot may be detected by radiolabeling, fluorescence, or the use of an antibody with an enzymatic molecular beacon.
Exercise \(5\)
What is the function of the enzyme in the immunoblot assay?
Complement-Mediated Immunoassay
One of the key functions of antibodies is the activation (fixation) of complement. When antibody binds to bacteria, for example, certain complement proteins recognize the bound antibody and activate the complement cascade. In response, other complement proteins bind to the bacteria where some serve as opsonins to increase the efficiency of phagocytosis and others create holes in gram-negative bacterial cell membranes, causing lysis. This lytic activity can be used to detect the presence of antibodies against specific antigens in the serum.
Red blood cells are good indicator cells to use when evaluating complement-mediated cytolysis. Hemolysis of red blood cells releases hemoglobin, which is a brightly colored pigment, and hemolysis of even a small number of red cells will cause the solution to become noticeably pink (Figure \(10\)). This characteristic plays a role in the complement fixation test, which allows the detection of antibodies against specific pathogens. The complement fixation test can be used to check for antibodies against pathogens that are difficult to culture in the lab such as fungi, viruses, or the bacteria Chlamydia.
To perform the complement fixation test, antigen from a pathogen is added to patient serum. If antibodies to the antigen are present, the antibody will bind the antigen and fix all the available complement. When red blood cells and antibodies against red blood cells are subsequently added to the mix, there will be no complement left to lyse the red cells. Thus, if the solution remains clear, the test is positive. If there are no antipathogen antibodies in the patient’s serum, the added antibodies will activate the complement to lyse the red cells, yielding a negative test (Figure \(10\)).
Link to Learning
View this video to see an outline of the steps of the complement fixation test.
Exercise \(6\)
In a complement fixation test, if the serum turns pink, does the patient have antibodies to the antigen or not? Explain.
Table \(1\) summarizes the various types of antibody-antigen assays discussed in this section.
Table \(1\): Mechanisms of Select Antibody-Antigen Assays
Type of Assay Mechanism Examples
Precipitation Antibody binds to soluble antigen, forming a visible precipitin Precipitin ring test to visualize lattice formation in solution
Immunoelectrophoresis to examine distribution of antigens following electrophoresis
Ouchterlony assay to compare diverse antigens
Radial immunodiffusion assay to quantify antigens
Flocculation Antibody binds to insoluble molecules in suspension, forming visible aggregates VDRL test for syphilis
Neutralization Antibody binds to virus, blocking viral entry into target cells and preventing formation of plaques Plaque reduction assay for detecting presence of neutralizing antibodies in patient sera
Complement activation Antibody binds to antigen, inducing complement activation and leaving no complement to lyse red blood cells Complement fixation test for patient antibodies against hard-to-culture bacteria such as Chlamydia
Key Concepts and Summary
• When present in the correct ratio, antibody and antigen will form a precipitin, or lattice that precipitates out of solution.
• A precipitin ring test can be used to visualize lattice formation in solution. The Ouchterlony assay demonstrates lattice formation in a gel. The radial immunodiffusion assay is used to quantify antigen by measuring the size of a precipitation zone in a gel infused with antibodies.
• Insoluble antigens in suspension will form flocculants when bound by antibodies. This is the basis of the VDRL test for syphilis in which anti-treponemal antibodies bind to cardiolipin in suspension.
• Viral infections can be detected by quantifying virus-neutralizing antibodies in a patient’s serum.
• Different antibody classes in plasma or serum are identified by using immunoelectrophoresis.
• The presence of specific antigens (e.g., bacterial or viral proteins) in serum can be demonstrated by western blot assays, in which the proteins are transferred to a nitrocellulose membrane and identified using labeled antibodies.
• In the complement fixation test, complement is used to detect antibodies against various pathogens.
Footnotes
1. 1 Ouchterlony, Örjan, “In Vitro Method for Testing the Toxin-Producing Capacity of Diphtheria Bacteria,” Acta Pathologica Microbiologica Scandinavica 26, no. 4 (1949): 516-24.
2. 2 Tiselius, Arne, “Electrophoresis of Serum Globulin: Electrophoretic Analysis of Normal and Immune Sera,” Biochemical Journal 31, no. 9 (1937): 1464.
3. 3 Tiselius, Arne and Elvin A. Kabat. “An Electrophoretic Study of Immune Sera and Purified Antibody Preparations,” The Journal of Experimental Medicine 69, no. 1 (1939): 119-31. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/20%3A_Laboratory_Analysis_of_the_Immune_Response/20.02%3A_Detecting_Antigen-Antibody_Complexes_in_vitro.txt |
Learning Objectives
• Compare direct and indirect agglutination
• Identify various uses of hemagglutination in the diagnosis of disease
• Explain how blood types are determined
• Explain the steps used to cross-match blood to be used in a transfusion
In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination (Figure 18.1.8). Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or microtiter plate (Figure \(1\)). Microtiter plates have an array of wells to hold small volumes of reagents and to observe reactions (e.g., agglutination) either visually or using a specially designed spectrophotometer. The wells come in many different sizes for assays involving different volumes of reagents.
Agglutination of Bacteria and Viruses
The use of agglutination tests to identify streptococcal bacteria was developed in the 1920s by Rebecca Lancefieldworking with her colleagues A.R. Dochez and Oswald Avery.1 She used antibodies to identify M protein, a virulence factor on streptococci that is necessary for the bacteria’s ability to cause strep throat. Production of antibodies against M protein is crucial in mounting a protective response against the bacteria.
Lancefield used antisera to show that different strains of the same species of streptococci express different versions of M protein, which explains why children can come down with strep throat repeatedly. Lancefield classified beta-hemolytic streptococci into many groups based on antigenic differences in group-specific polysaccharides located in the bacterial cell wall. The strains are called serovars because they are differentiated using antisera. Identifying the serovars present in a disease outbreak is important because some serovars may cause more severe disease than others.
The method developed by Lancefield is a direct agglutination assay, since the bacterial cells themselves agglutinate. A similar strategy is more commonly used today when identifying serovars of bacteria and viruses; however, to improve visualization of the agglutination, the antibodies may be attached to inert latex beads. This technique is called an indirect agglutination assay (or latex fixation assay), because the agglutination of the beads is a marker for antibody binding to some other antigen (Figure \(2\)). Indirect assays can be used to detect the presence of either antibodies or specific antigens.
To identify antibodies in a patient’s serum, the antigen of interest is attached to latex beads. When mixed with patient serum, the antibodies will bind the antigen, cross-linking the latex beads and causing the beads to agglutinate indirectly; this indicates the presence of the antibody (Figure \(3\)). This technique is most often used when looking for IgM antibodies, because their structure provides maximum cross-linking. One widely used example of this assay is a test for rheumatoid factor (RF) to confirm a diagnosis of rheumatoid arthritis. RF is, in fact, the presence of IgM antibodies that bind to the patient’s own IgG. RF will agglutinate IgG-coated latex beads.
In the reverse test, soluble antigens can be detected in a patient’s serum by attaching specific antibodies (commonly mAbs) to the latex beads and mixing this complex with the serum (Figure \(3\)).
Agglutination tests are widely used in underdeveloped countries that may lack appropriate facilities for culturing bacteria. For example, the Widal test, used for the diagnosis of typhoid fever, looks for agglutination of Salmonella enterica subspecies typhi in patient sera. The Widal test is rapid, inexpensive, and useful for monitoring the extent of an outbreak; however, it is not as accurate as tests that involve culturing of the bacteria. The Widal test frequently produces false positives in patients with previous infections with other subspecies of Salmonella, as well as false negatives in patients with hyperproteinemia or immune deficiencies.
In addition, agglutination tests are limited by the fact that patients generally do not produce detectable levels of antibody during the first week (or longer) of an infection. A patient is said to have undergone seroconversion when antibody levels reach the threshold for detection. Typically, seroconversion coincides with the onset of signs and symptoms of disease. However, in an HIV infection, for example, it generally takes 3 weeks for seroconversion to take place, and in some instances, it may take much longer.
Similar to techniques for the precipitin ring test and plaque assays, it is routine to prepare serial two-fold dilutions of the patient’s serum and determine the titer of agglutinating antibody present. Since antibody levels change over time in both primary and secondary immune responses, by checking samples over time, changes in antibody titer can be detected. For example, a comparison of the titer during the acute phase of an infection versus the titer from the convalescent phase will distinguish whether an infection is current or has occurred in the past. It is also possible to monitor how well the patient’s immune system is responding to the pathogen.
Link to Learning
Watch this video that demonstrates agglutination reactions with latex beads.
Exercise \(1\)
1. How is agglutination used to distinguish serovars from each other?
2. In a latex bead assay to test for antibodies in a patient's serum, with what are the beads coated?
3. What has happened when a patient has undergone seroconversion?
Hemagglutination
Agglutination of red blood cells is called hemagglutination. One common assay that uses hemagglutination is the direct Coombs’ test, also called the direct antihuman globulin test (DAT), which generally looks for nonagglutinating antibodies. The test can also detect complement attached to red blood cells.
The Coombs’ test is often employed when a newborn has jaundice, yellowing of the skin caused by high blood concentrations of bilirubin, a product of the breakdown of hemoglobin in the blood. The Coombs’ test is used to determine whether the child’s red blood cells have been bound by the mother’s antibodies. These antibodies would activate complement, leading to red blood cell lysis and the subsequent jaundice. Other conditions that can cause positive direct Coombs’ tests include hemolytic transfusion reactions, autoimmune hemolytic anemia, infectious mononucleosis (caused by Epstein-Barr virus), syphilis, and Mycoplasma pneumonia. A positive direct Coombs’ test may also be seen in some cancers and as an allergic reaction to some drugs (e.g., penicillin).
The antibodies bound to red blood cells in these conditions are most often IgG, and because of the orientation of the antigen-binding sites on IgG and the comparatively large size of a red blood cell, it is unlikely that any visible agglutination will occur. However, the presence of IgG bound to red blood cells can be detected by adding Coombs’ reagent, an antiserum containing antihuman IgG antibodies (that may be combined with anti-complement) (Figure \(4\)). The Coombs’ reagent links the IgG attached to neighboring red blood cells and thus promotes agglutination.
There is also an indirect Coombs’ test known as the indirect antiglobulin test (IAT). This screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum (Figure \(4\)). IAT can be used to screen pregnant women for antibodies that may cause hemolytic disease of the newborn. It can also be used prior to giving blood transfusions. More detail on how the IAT is performed is discussed below.
Antibodies that bind to red blood cells are not the only cause of hemagglutination. Some viruses also bind to red blood cells, and this binding can cause agglutination when the viruses cross-link the red blood cells. For example, influenza viruses have two different types of viral spikes called neuraminidase (N) and hemagglutinin (H), the latter named for its ability to agglutinate red blood cells (see Viruses). Thus, we can use red blood cells to detect the presence of influenza virus by direct hemagglutination assays (HA), in which the virus causes visible agglutination of red blood cells. The mumps and rubella viruses can also be detected using HA.
Most frequently, a serial dilution viral agglutination assay is used to measure the titer or estimate the amount of virus produced in cell culture or for vaccine production. A viral titer can be determined using a direct HA by making a serial dilution of the sample containing the virus, starting with a high concentration of sample that is then diluted in a series of wells. The highest dilution producing visible agglutination is the titer. The assay is carried out in a microtiter plate with V- or round-bottomed wells. In the presence of agglutinating viruses, the red blood cells and virus clump together and produce a diffuse mat over the bottom of the well. In the absence of virus, the red blood cells roll or sediment to the bottom of the well and form a dense pellet, which is why flat-bottomed wells cannot be used (Figure \(5\)).
A modification of the HA assay can be used to determine the titer of antiviral antibodies. The presence of these antibodies in a patient’s serum or in a lab-produced antiserum will neutralize the virus and block it from agglutinating the red cells, making this a viral hemagglutination inhibition assay (HIA). In this assay, patient serum is mixed with a standardized amount of virus. After a short incubation, a standardized amount of red blood cells is added and hemagglutination is observed. The titer of the patient’s serum is the highest dilution that blocks agglutination (Figure \(6\)).
Exercise \(2\)
1. What is the mechanism by which viruses are detected in a hemagglutination assay?
2. Which hemagglutination result tells us the titer of virus in a sample?
Animals in the Laboratory
Much of what we know today about the human immune system has been learned through research conducted using animals—primarily, mammals—as models. Besides research, mammals are also used for the production of most of the antibodies and other immune system components needed for immunodiagnostics. Vaccines, diagnostics, therapies, and translational medicine in general have all been developed through research with animal models.
Consider some of the common uses of laboratory animals for producing immune system components. Guinea pigs are used as a source of complement, and mice are the primary source of cells for making mAbs. These mAbs can be used in research and for therapeutic purposes. Antisera are raised in a variety of species, including horses, sheep, goats, and rabbits. When producing an antiserum, the animal will usually be injected at least twice, and adjuvants may be used to boost the antibody response. The larger animals used for making antisera will have blood harvested repeatedly over long periods of time, with little harm to the animals, but that is not usually the case for rabbits. Although we can obtain a few milliliters of blood from the ear veins of rabbits, we usually need larger volumes, which results in the deaths of the animals.
We also use animals for the study of disease. The only way to grow Treponema pallidum for the study of syphilis is in living animals. Many viruses can be grown in cell culture, but growth in cell culture tells us very little about how the immune system will respond to the virus. When working on a newly discovered disease, we still employ Koch’s postulates, which require causing disease in lab animals using pathogens from pure culture as a crucial step in proving that a particular microorganism is the cause of a disease. Studying the proliferation of bacteria and viruses in animal hosts, and how the host immune system responds, has been central to microbiological research for well over 100 years.
While the practice of using laboratory animals is essential to scientific research and medical diagnostics, many people strongly object to the exploitation of animals for human benefit. This ethical argument is not a new one—indeed, one of Charles Darwin's daughters was an active antivivisectionist (vivisection is the practice of cutting or dissecting a live animal to study it). Most scientists acknowledge that there should be limits on the extent to which animals can be exploited for research purposes. Ethical considerations have led the National Institutes of Health (NIH) to develop strict regulations on the types of research that may be performed. These regulations also include guidelines for the humane treatment of lab animals, setting standards for their housing, care, and euthanization. The NIH document “Guide for the Care and Use of Laboratory Animals” makes it clear that the use of animals in research is a privilege granted by society to researchers.
The NIH guidelines are based on the principle of the three R’s: replace, refine, and reduce. Researchers should strive to replace animal models with nonliving models, replace vertebrates with invertebrates whenever possible, or use computer-models when applicable. They should refine husbandry and experimental procedures to reduce pain and suffering, and use experimental designs and procedures that reduce the number of animals needed to obtain the desired information. To obtain funding, researchers must satisfy NIH reviewers that the research justifies the use of animals and that their use is in accordance with the guidelines.
At the local level, any facility that uses animals and receives federal funding must have an Institutional Animal Care and Use Committee (IACUC) that ensures that the NIH guidelines are being followed. The IACUC must include researchers, administrators, a veterinarian, and at least one person with no ties to the institution, that is, a concerned citizen. This committee also performs inspections of laboratories and protocols. For research involving human subjects, an Institutional Review Board (IRB) ensures that proper guidelines are followed.
Link to Learning
Visit this site to view the NIH Guide for the Care and Use of Laboratory Animals.
Blood Typing and Cross-Matching
In addition to antibodies against bacteria and viruses to which they have previously been exposed, most individuals also carry antibodies against blood types other than their own. There are presently 33 immunologically important blood-type systems, many of which are restricted within various ethnic groups or rarely result in the production of antibodies. The most important and perhaps best known are the ABO and Rh blood groups (see Figure 19.1.3).
When units of blood are being considered for transfusion, pretransfusion blood testing must be performed. For the blood unit, commercially prepared antibodies against the A, B, and Rh antigens are mixed with red blood cells from the units to initially confirm that the blood type on the unit is accurate. Once a unit of blood has been requested for transfusion, it is vitally important to make sure the donor (unit of blood) and recipient (patient) are compatible for these crucial antigens. In addition to confirming the blood type of the unit, the patient’s blood type is also confirmed using the same commercially prepared antibodies to A, B, and Rh. For example, as shown in Figure \(7\), if the donor blood is A-positive, it will agglutinate with the anti-A antiserum and with the anti-Rh antiserum. If no agglutination is observed with any of the sera, then the blood type would be O-negative.
Following determination of the blood type, immediately prior to releasing the blood for transfusion, a cross-match is performed in which a small aliquot of the donor red blood cells are mixed with serum from the patient awaiting transfusion. If the patient does have antibodies against the donor red blood cells, hemagglutination will occur. To confirm any negative test results and check for sensitized red blood cells, Coombs’ reagent may be added to the mix to facilitate visualization of the antibody-red blood cell interaction.
Under some circumstances, a minor cross-match may be performed as well. In this assay, a small aliquot of donor serum is mixed with patient red blood cells. This allows the detection of agglutinizing antibodies in the donor serum. This test is rarely necessary because transfusions generally use packed red blood cells with most of the plasma removed by centrifugation.
Red blood cells have many other antigens in addition to ABO and Rh. While most people are unlikely to have antibodies against these antigens, women who have had multiple pregnancies or patients who have had multiple transfusions may have them because of repeated exposure. For this reason, an antibody screen test is used to determine if such antibodies are present. Patient serum is checked against commercially prepared, pooled, type O red blood cells that express these antigens. If agglutination occurs, the antigen to which the patient is responding must be identified and determined not to be present in the donor unit.
Exercise \(3\)
1. If a patient's blood agglutinates with anti-B serum, what is the patient’s blood type?
2. What is a cross-match assay, and why is it performed?
Table \(1\) summarizes the various kinds of agglutination assays discussed in this section.
Table \(1\): Mechanisms of Select Antibody-Antigen Assays
Type of Assay Mechanism Example
Agglutination Direct: Antibody is used to clump bacterial cells or other large structures Serotyping bacteria
Indirect: Latex beads are coupled with antigen or antibody to look for antibody or antigen, respectively, in patient serum Confirming the presence of rheumatoid factor (IgM-binding Ig) in patient serum
Hemagglutination Direct: Some bacteria and viruses cross-link red blood cells and clump them together Diagnosing influenza, mumps, and measles
Direct Coombs’ test (DAT): Detects nonagglutinating antibodies or complement proteins on red blood cells in vivo Checking for maternal antibodies binding to neonatal red blood cells
Indirect Coombs’ test (IAT): Screens an individual for antibodies against red blood cell antigens (other than the A and B antigens) that are unbound in a patient’s serum in vitro Performing pretransfusion blood testing
Viral hemagglutination inhibition: Uses antibodies from a patient to inhibit viral agglutination Diagnosing various viral diseases by the presence of patient antibodies against the virus
Blood typing and cross-matching: Detects ABO, Rh, and minor antigens in the blood Matches donor blood to recipient immune requirements
Key Concepts and Summary
• Antibodies can agglutinate cells or large particles into a visible matrix. Agglutination tests are often done on cards or in microtiter plates that allow multiple reactions to take place side by side using small volumes of reagents.
• Using antisera against certain proteins allows identification of serovars within species of bacteria.
• Detecting antibodies against a pathogen can be a powerful tool for diagnosing disease, but there is a period of time before patients go through seroconversion and the level of antibodies becomes detectable.
• Agglutination of latex beads in indirect agglutination assays can be used to detect the presence of specific antigens or specific antibodies in patient serum.
• The presence of some antibacterial and antiviral antibodies can be confirmed by the use of the direct Coombs’ test, which uses Coombs’ reagent to cross-link antibodies bound to red blood cells and facilitate hemagglutination.
• Some viruses and bacteria will bind and agglutinate red blood cells; this interaction is the basis of the direct hemagglutination assay, most often used to determine the titer of virus in solution.
• Neutralization assays quantify the level of virus-specific antibody by measuring the decrease in hemagglutination observed after mixing patient serum with a standardized amount of virus.
• Hemagglutination assays are also used to screen and cross-match donor and recipient blood to ensure that the transfusion recipient does not have antibodies to antigens in the donated blood.
Footnotes
1. 1 Lancefield, Rebecca C., “The Antigenic Complex of Streptococcus haemoliticus. I. Demonstration of a Type-Specific Substance in Extracts of Streptococcus haemolyticus,” The Journal of Experimental Medicine 47, no. 1 (1928): 91-103. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/20%3A_Laboratory_Analysis_of_the_Immune_Response/20.03%3A_Agglutination_Assays.txt |
Learning Objectives
• Explain the differences and similarities between EIA, FEIA, and ELISA
• Describe the difference and similarities between immunohistochemistry and immunocytochemistry
• Describe the different purposes of direct and indirect ELISA
Similar to the western blot, enzyme immunoassays (EIAs) use antibodies to detect the presence of antigens. However, EIAs differ from western blots in that the assays are conducted in microtiter plates or in vivo rather than on an absorbent membrane. There are many different types of EIAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. The addition of a substrate for the enzyme allows the antigen to be visualized or quantified (Figure \(1\)).
In EIAs, the substrate for the enzyme is most often a chromogen, a colorless molecule that is converted into a colored end product. The most widely used enzymes are alkaline phosphatase and horseradish peroxidase for which appropriate substrates are readily available. In some EIAs, the substrate is a fluorogen, a nonfluorescent molecule that the enzyme converts into a fluorescent form. EIAs that utilize a fluorogen are called fluorescent enzyme immunoassays (FEIAs). Fluorescence can be detected by either a fluorescence microscope or a spectrophotometer.
The MMR Titer
The MMR vaccine is a combination vaccine that provides protection against measles, mumps, and rubella (German measles). Most people receive the MMR vaccine as children and thus have antibodies against these diseases. However, for various reasons, even vaccinated individuals may become susceptible to these diseases again later in life. For example, some children may receive only one round of the MMR vaccine instead of the recommended two. In addition, the titer of protective antibodies in an individual’s body may begin to decline with age or as the result of some medical conditions.
To determine whether the titer of antibody in an individual’s bloodstream is sufficient to provide protection, an MMR titer test can be performed. The test is a simple immunoassay that can be done quickly with a blood sample. The results of the test will indicate whether the individual still has immunity or needs another dose of the MMR vaccine.
Submitting to an MMR titer is often a pre-employment requirement for healthcare workers, especially those who will frequently be in contact with young children or immunocompromised patients. Were a healthcare worker to become infected with measles, mumps, or rubella, the individual could easily pass these diseases on to susceptible patients, leading to an outbreak. Depending on the results of the MMR titer, healthcare workers might need to be revaccinated prior to beginning work.
Immunostaining
One powerful use of EIA is immunostaining, in which antibody-enzyme conjugates enhance microscopy. Immunohistochemistry (IHC) is used for examining whole tissues. As seen in Figure \(2\), a section of tissue can be stained to visualize the various cell types. In this example, a mAb against CD8 was used to stain CD8 cells in a section of tonsil tissue. It is now possible to count the number of CD8 cells, determine their relative numbers versus the other cell types present, and determine the location of these cells within this tissue. Such data would be useful for studying diseases such as AIDS, in which the normal function of CD8 cells is crucial for slowing disease progression.
Immunocytochemistry (ICC) is another valuable form of immunostaining. While similar to IHC, in ICC, extracellular matrix material is stripped away, and the cell membrane is etched with alcohol to make it permeable to antibodies. This allows antibodies to pass through the cell membrane and bind to specific targets inside the cell. Organelles, cytoskeletal components, and other intracellular structures can be visualized in this way. While some ICC techniques use EIA, the enzyme can be replaced with a fluorescent molecule, making it a fluorescent immunoassay.
Exercise \(1\)
1. What is the difference between immunohistochemistry and immunocytochemistry?
2. What must be true of the product of the enzymatic reaction used in immunohistochemistry?
Enzyme-linked Immunosorbent Assays (ELISAs)
The enzyme-linked immunosorbent assays (ELISAs) are widely used EIAs. In the direct ELISA, antigens are immobilized in the well of a microtiter plate. An antibody that is specific for a particular antigen and is conjugated to an enzyme is added to each well. If the antigen is present, then the antibody will bind. After washing to remove any unbound antibodies, a colorless substrate (chromogen) is added. The presence of the enzyme converts the substrate into a colored end product (Figure \(1\)). While this technique is faster because it only requires the use of one antibody, it has the disadvantage that the signal from a direct ELISA is lower (lower sensitivity).
In a sandwich ELISA, the goal is to use antibodies to precisely quantify specific antigen present in a solution, such as antigen from a pathogen, a serum protein, or a hormone from the blood or urine to list just a few examples. The first step of a sandwich ELISA is to add the primary antibody to all the wells of a microtiter plate (Figure \(3\)). The antibody sticks to the plastic by hydrophobic interactions. After an appropriate incubation time, any unbound antibody is washed away. Comparable washes are used between each of the subsequent steps to ensure that only specifically bound molecules remain attached to the plate. A blocking protein is then added (e.g., albumin or the milk protein casein) to bind the remaining nonspecific protein-binding sites in the well. Some of the wells will receive known amounts of antigen to allow the construction of a standard curve, and unknown antigen solutions are added to the other wells. The primary antibody captures the antigen and, following a wash, the secondary antibody is added, which is a polyclonal antibody that is conjugated to an enzyme. After a final wash, a colorless substrate (chromogen) is added, and the enzyme converts it into a colored end product. The color intensity of the sample caused by the end product is measured with a spectrophotometer. The amount of color produced (measured as absorbance) is directly proportional to the amount of enzyme, which in turn is directly proportional to the captured antigen. ELISAs are extremely sensitive, allowing antigen to be quantified in the nanogram (10–9 g) per mL range.
In an indirect ELISA, we quantify antigen-specific antibody rather than antigen. We can use indirect ELISA to detect antibodies against many types of pathogens, including Borrelia burgdorferi (Lyme disease) and HIV. There are three important differences between indirect and direct ELISAs as shown in Figure \(4\). Rather than using antibody to capture antigen, the indirect ELISA starts with attaching known antigen (e.g., peptides from HIV) to the bottom of the microtiter plate wells. After blocking the unbound sites on the plate, patient serum is added; if antibodies are present (primary antibody), they will bind the antigen. After washing away any unbound proteins, the secondary antibody with its conjugated enzyme is directed against the primary antibody (e.g., antihuman immunoglobulin). The secondary antibodyallows us to quantify how much antigen-specific antibody is present in the patient’s serum by the intensity of the color produced from the conjugated enzyme-chromogen reaction.
As with several other tests for antibodies discussed in this chapter, there is always concern about cross-reactivity with antibodies directed against some other antigen, which can lead to false-positive results. Thus, we cannot definitively diagnose an HIV infection (or any other type of infection) based on a single indirect ELISA assay. We must confirm any suspected positive test, which is most often done using either an immunoblot that actually identifies the presence of specific peptides from the pathogen or a test to identify the nucleic acids associated with the pathogen, such as reverse transcriptase PCR (RT-PCR) or a nucleic acid antigen test.
Exercise \(2\)
1. What is the purpose of the secondary antibody in a direct ELISA?
2. What do the direct and indirect ELISAs quantify?
Clinical Focus: Part 2
Although contacting and testing the 1300 patients for HIV would be time consuming and expensive, administrators hoped to minimize the hospital’s liability by proactively seeking out and treating potential victims of the rogue employee’s crime. Early detection of HIV is important, and prompt treatment can slow the progression of the disease.
There are a variety of screening tests for HIV, but the most widely used is the indirect ELISA. As with other indirect ELISAs, the test works by attaching antigen (in this case, HIV peptides) to a well in a 96-well plate. If the patient is HIV positive, anti-HIV antibodies will bind to the antigen and be identified by the second antibody-enzyme conjugate.
Exercise \(3\)
1. How accurate is an indirect ELISA test for HIV, and what factors could impact the test’s accuracy?
2. Should the hospital use any other tests to confirm the results of the indirect ELISA?
Immunofiltration and Immunochromatographic Assays
For some situations, it may be necessary to detect or quantify antigens or antibodies that are present at very low concentration in solution. Immunofiltration techniques have been developed to make this possible. In immunofiltration, a large volume of fluid is passed through a porous membrane into an absorbent pad. An antigen attached to the porous membrane will capture antibody as it passes; alternatively, we can also attach an antibody to the membrane to capture antigen.
The method of immunofiltration has been adapted in the development of immunochromatographic assays, commonly known as lateral flow tests or strip tests. These tests are quick and easy to perform, making them popular for point-of-care use (i.e., in the doctor’s office) or in-home use. One example is the TORCH test that allows doctors to screen pregnant women or newborns for infection by an array of viruses and other pathogens (Toxoplasma, other viruses, rubella, cytomegalovirus, herpes simplex). In-home pregnancy tests are another widely used example of a lateral flow test (Figure \(5\)). Immunofiltration tests are also popular in developing countries, because they are inexpensive and do not require constant refrigeration of the dried reagents. However, the technology is also built into some sophisticated laboratory equipment.
In lateral flow tests (Figure \(6\)), fluids such as urine are applied to an absorbent pad on the test strip. The fluid flows by capillary action and moves through a stripe of beads with antibodies attached to their surfaces. The fluid in the sample actually hydrates the reagents, which are present in a dried state in the stripe. Antibody-coated beads made of latex or tiny gold particles will bind antigens in the test fluid. The antibody-antigen complexes then flow over a second stripe that has immobilized antibody against the antigen; this stripe will retain the beads that have bound antigen. A third control stripe binds any beads. A red color (from gold particles) or blue (from latex beads) developing at the test line indicates a positive test. If the color only develops at the control line, the test is negative.
Like ELISA techniques, lateral flow tests take advantage of antibody sandwiches, providing sensitivity and specificity. While not as quantitative as ELISA, these tests have the advantage of being fast, inexpensive, and not dependent on special equipment. Thus, they can be performed anywhere by anyone. There are some concerns about putting such powerful diagnostic tests into the hands of people who may not understand the tests’ limitations, such as the possibility of false-positive results. While home pregnancy tests have become widely accepted, at-home antibody-detection tests for diseases like HIV have raised some concerns in the medical community. Some have questioned whether self-administration of such tests should be allowed in the absence of medical personnel who can explain the test results and order appropriate confirmatory tests. However, with growing numbers of lateral flow tests becoming available, and the rapid development of lab-on-a-chip technology (Figure 20.1), home medical tests are likely to become even more commonplace in the future.
Exercise \(4\)
1. What physical process does the lateral flow method require to function?
2. Explain the purpose of the third strip in a lateral flow assay.
Table \(1\) compares some of the key mechanisms and examples of some of the EIAs discussed in this section as well as immunoblots, which were discussed in Detecting Antigen-Antibody Complexes.
Table \(1\): Immunoblots & Enzyme Immunoassays
Type of Assay Mechanism Specific Procedures Examples
Immunoblots Uses enzyme-antibody conjugates to identify specific proteins that have been transferred to an absorbent membrane Western blot: Detects the presence of a particular protein Detecting the presence of HIV peptides (or peptides from other infectious agents) in patient sera
Immunostaining Uses enzyme-antibody conjugates to stain specific molecules on or in cells Immunohistochemistry: Used to stain specific cells in a tissue Stain for presence of CD8 cells in host tissue
Enzyme-linked immunosorbent assay (ELISA) Uses enzyme-antibody conjugates to quantify target molecules Direct ELISA: Uses a single antibody to detect the presence of an antigen Detection of HIV antigen p24 up to one month after being infected
Indirect ELISA: Measures the amount of antibody produced against an antigen Detection of HIV antibodies in serum
Immunochromatographic (lateral flow) assays Techniques use the capture of flowing, color-labeled antigen-antibody complexes by fixed antibody for disease diagnosis Sandwich ELISA: Measures the amount of antigen bound by the antibody Detection of antibodies for various pathogens in patient sera (e.g., rapid strep, malaria dipstick)
Pregnancy test detecting human chorionic gonadotrophin in urine
Clinical Focus: Part 3
Although the indirect ELISA for HIV is a sensitive assay, there are several complicating considerations. First, if an infected person is tested too soon after becoming infected, the test can yield false-negative results. The seroconversion window is generally about three weeks, but in some cases, it can be more than two months.
In addition to false negatives, false positives can also occur, usually due to previous infections with other viruses that induce cross-reacting antibodies. The false-positive rate depends on the particular brand of test used, but 0.5% is not unusual.1 Because of the possibility of a false positive, all positive tests are followed up with a confirmatory test. This confirmatory test is often an immunoblot (western blot) in which HIV peptides from the patient’s blood are identified using an HIV-specific mAb-enzyme conjugate. A positive western blot would confirm an HIV infection and a negative blot would confirm the absence of HIV despite the positive ELISA.
Unfortunately, western blots for HIV antigens often yield indeterminant results, in which case, they neither confirm nor invalidate the results of the indirect ELISA. In fact, the rate of indeterminants can be 10–49% (which is why, combined with their cost, western blots are not used for screening). Similar to the indirect ELISA, an indeterminant western blot can occur because of cross-reactivity or previous viral infections, vaccinations, or autoimmune diseases.
Exercise \(5\)
1. Of the 1300 patients being tested, how many false-positive ELISA tests would be expected?
2. Of the false positives, how many indeterminant western blots could be expected?
3. How would the hospital address any cases in which a patient’s western blot was indeterminant?
Key Concepts and Summary
• Enzyme immunoassays (EIA) are used to visualize and quantify antigens. They use an antibody conjugated to an enzyme to bind the antigen, and the enzyme converts a substrate into an observable end product. The substrate may be either a chromogen or a fluorogen.
• Immunostaining is an EIA technique for visualizing cells in a tissue (immunohistochemistry) or examining intracellular structures (immunocytochemistry).
• Direct ELISA is used to quantify an antigen in solution. The primary antibody captures the antigen, and the secondary antibody delivers an enzyme. Production of end product from the chromogenic substrate is directly proportional to the amount of captured antigen.
• Indirect ELISA is used to detect antibodies in patient serum by attaching antigen to the well of a microtiter plate, allowing the patient (primary) antibody to bind the antigen and an enzyme-conjugated secondary antibody to detect the primary antibody.
• Immunofiltration and immunochromatographic assays are used in lateral flow tests, which can be used to diagnose pregnancy and various diseases by detecting color-labeled antigen-antibody complexes in urine or other fluid samples
Footnotes
1. 1 Thomas, Justin G., Victor Jaffe, Judith Shaffer, and Jose Abreu, “HIV Testing: US Recommendations 2014,” Osteopathic Family Physician 6, no. 6 (2014). | textbooks/bio/Microbiology/Microbiology_(OpenStax)/20%3A_Laboratory_Analysis_of_the_Immune_Response/20.04%3A_Enzyme_Immunoassays_%28EIA%29_and_Enzyme-Linked_Immunosorbent_Assays_%28ELISA%29.txt |
Learning Objectives
• Describe the benefits of immunofluorescent antibody assays in comparison to nonfluorescent assays
• Compare direct and indirect fluorescent antibody assays
• Explain how a flow cytometer can be used to quantify specific subsets of cells present in a complex mixture of cell types
• Explain how a fluorescence-activated cell sorter can be used to separate unique types of cells
Rapid visualization of bacteria from a clinical sample such as a throat swab or sputum can be achieved through fluorescent antibody (FA) techniques that attach a fluorescent marker (fluorogen) to the constant region of an antibody, resulting in a reporter molecule that is quick to use, easy to see or measure, and able to bind to target markers with high specificity. We can also label cells, allowing us to precisely quantify particular subsets of cells or even purify these subsets for further research.
As with the enzyme assays, FA methods may be direct, in which a labeled mAb binds an antigen, or indirect, in which secondary polyclonal antibodies bind patient antibodies that react to a prepared antigen. Applications of these two methods were demonstrated in Figure 2.3.8. FA methods are also used in automated cell counting and sorting systems to enumerate or segregate labeled subpopulations of cells in a sample.
Direct Fluorescent Antibody Techniques
Direct fluorescent antibody (DFA) tests use a fluorescently labeled mAb to bind and illuminate a target antigen. DFA tests are particularly useful for the rapid diagnosis of bacterial diseases. For example, fluorescence-labeled antibodies against Streptococcus pyogenes (group A strep) can be used to obtain a diagnosis of strep throat from a throat swab. The diagnosis is ready in a matter of minutes, and the patient can be started on antibiotics before even leaving the clinic. DFA techniques may also be used to diagnose pneumonia caused by Mycoplasma pneumoniae or Legionella pneumophila from sputum samples (Figure \(1\)). The fluorescent antibodies bind to the bacteria on a microscope slide, allowing ready detection of the bacteria using a fluorescence microscope. Thus, the DFA technique is valuable for visualizing certain bacteria that are difficult to isolate or culture from patient samples.
Exercise \(1\)
In a direct fluorescent antibody test, what does the fluorescent antibody bind to?
Indirect Fluorescent Antibody Techniques
Indirect fluorescent antibody (IFA) tests (Figure \(2\)) are used to look for antibodies in patient serum. For example, an IFA test for the diagnosis of syphilis uses T. pallidum cells isolated from a lab animal (the bacteria cannot be grown on lab media) and a smear prepared on a glass slide. Patient serum is spread over the smear and anti-treponemal antibodies, if present, are allowed to bind. The serum is washed off and a secondary antibody added. The secondary antibody is an antihuman immunoglobulin conjugated to a fluorogen. On examination, the T. pallidum bacteria will only be visible if they have been bound by the antibodies from the patient’s serum.
The IFA test for syphilis provides an important complement to the VDRL test discussed in Detecting Antigen-Antibody Complexes. The VDRL is more likely to generate false-positive reactions than the IFA test; however, the VDRL is a better test for determining whether an infection is currently active.
IFA tests are also useful for the diagnosis of autoimmune diseases. For example, systemic lupus erythematosus (SLE) (see Autoimmune Disorders) is characterized by elevated expression levels of antinuclear antibodies (ANA). These autoantibodies can be expressed against a variety of DNA-binding proteins and even against DNA itself. Because autoimmunity is often difficult to diagnose, especially early in disease progression, testing for ANA can be a valuable clue in making a diagnosis and starting appropriate treatment.
The IFA for ANA begins by fixing cells grown in culture to a glass slide and making them permeable to antibody. The slides are then incubated with serial dilutions of serum from the patient. After incubation, the slide is washed to remove unbound proteins, and the fluorescent antibody (antihuman IgG conjugated to a fluorogen) added. After an incubation and wash, the cells can be examined for fluorescence evident around the nucleus (Figure \(3\)). The titer of ANA in the serum is determined by the highest dilution showing fluorescence. Because many healthy people express ANA, the American College of Rheumatology recommends that the titer must be at least 1:40 in the presence of symptoms involving two or more organ systems to be considered indicative of SLE.1
Exercise \(2\)
1. In an indirect fluorescent antibody test, what does the fluorescent antibody bind to?
2. What is the ANA test looking for?
Flow Cytometry
Fluorescently labeled antibodies can be used to quantify cells of a specific type in a complex mixture using flow cytometry (Figure \(4\)), an automated, cell-counting system that detects fluorescing cells as they pass through a narrow tube one cell at a time. For example, in HIV infections, it is important to know the level of CD4 T cells in the patient’s blood; if the numbers fall below 500 per μL of blood, the patient becomes more likely to acquire opportunistic infections; below 200 per μL, the patient can no longer mount a useful adaptive immune response at all. The analysis begins by incubating a mixed-cell population (e.g., white blood cells from a donor) with a fluorescently labeled mAb specific for a subpopulation of cells (e.g., anti-CD4). Some experiments look at two cell markers simultaneously by adding a different fluorogen to the appropriate mAb. The cells are then introduced to the flow cytometer through a narrow capillary that forces the cells to pass in single file. A laser is used to activate the fluorogen. The fluorescent light radiates out in all directions, so the fluorescence detector can be positioned at an angle from the incident laser light.
Figure \(4\) shows the obscuration bar in front of the forward-scatter detector that prevents laser light from hitting the detector. As a cell passes through the laser bar, the forward-scatter detector detects light scattered around the obscuration bar. The scattered light is transformed into a voltage pulse, and the cytometer counts a cell. The fluorescence from a labeled cell is detected by the side-scatter detectors. The light passes through various dichroic mirrors such that the light emitted from the fluorophore is received by the correct detector.
Data are collected from both the forward- and side-scatter detectors. One way these data can be presented is in the form of a histogram. The forward scatter is placed on the y-axis (to represent the number of cells), and the side scatter is placed on the x-axis (to represent the fluoresence of each cell). The scaling for the x-axis is logarithmic, so fluorescence intensity increases by a factor of 10 with each unit increase along the axis. Figure \(5\) depicts an example in which a culture of cells is combined with an antibody attached to a fluorophore to detect CD8 cells and then analyzed by flow cytometry. The histogram has two peaks. The peak on the left has lower fluorescence readings, representing the subset of the cell population (approximately 30 cells) that does not fluoresce; hence, they are not bound by antibody and therefore do not express CD8. The peak on the right has higher fluorescence readings, representing the subset of the cell population (approximately 100 cells) that show fluorescence; hence, they are bound by the antibody and therefore do express CD8.
Exercise \(3\)
1. What is the purpose of the laser in a flow cytometer?
2. In the output from a flow cytometer, the area under the histogram is equivalent to what?
Clinical Focus: Resolution
After notifying all 1300 patients, the hospital begins scheduling HIV screening. Appointments were scheduled a minimum of 3 weeks after the patient’s last hospital visit to minimize the risk of false negatives. Because some false positives were anticipated, the public health physician set up a counseling protocol for any patient whose indirect ELISA came back positive.
Of the 1300 patients, eight tested positive using the ELISA. Five of these tests were invalidated by negative western blot tests, but one western blot came back positive, confirming that the patient had indeed contracted HIV. The two remaining western blots came back indeterminate. These individuals had to submit to a third test, a PCR, to confirm the presence or absence of HIV sequences. Luckily, both patients tested negative.
As for the lone patient confirmed to have HIV, the tests cannot prove or disprove any connection to the syringes compromised by the former hospital employee. Even so, the hospital’s insurance will fully cover the patient’s treatment, which began immediately.
Although we now have drugs that are typically effective at controlling the progression of HIV and AIDS, there is still no cure. If left untreated, or if the drug regimen fails, the patient will experience a gradual decline in the number of CD4 helper T cells, resulting in severe impairment of all adaptive immune functions. Even moderate declines of helper T cell numbers can result in immunodeficiency, leaving the patient susceptible to opportunistic infections. To monitor the status of the patient’s helper T cells, the hospital will use flow cytometry. This sensitive test allows physicians to precisely determine the number of helper T cells so they can adjust treatment if the number falls below 500 cells/µL.
Cell Sorting Using Immunofluorescence
The flow cytometer and immunofluorescence can also be modified to sort cells from a single sample into purified subpopulations of cells for research purposes. This modification of the flow cytometer is called a fluorescence-activated cell sorter (FACS). In a FACS, fluorescence by a cell induces the device to put a charge on a droplet of the transporting fluid containing that cell. The charge is specific to the wavelength of the fluorescent light, which allows for differential sorting by those different charges. The sorting is accomplished by an electrostatic deflector that moves the charged droplet containing the cell into one collecting vessel or another. The process results in highly purified subpopulations of cells.
One limitation of a FACS is that it only works on isolated cells. Thus, the method would work in sorting white blood cells, since they exist as isolated cells. But for cells in a tissue, flow cytometry can only be applied if we can excise the tissue and separate it into single cells (using proteases to cleave cell-cell adhesion molecules) without disrupting cell integrity. This method may be used on tumors, but more often, immunohistochemistry and immunocytochemistry are used to study cells in tissues.
Link to Learning
Watch videos to learn more about how flow cytometry and a FACS work.
Exercise \(4\)
In fluorescence activated cell sorting, what characteristic of the target cells allows them to be separated?
Table \(1\) compares the mechanisms of the fluorescent antibody techniques discussed in this section.
Table \(1\): Fluorescent Antibody Techniques
Type of Assay Mechanism Examples
Direct fluorescent antibody (DFA) Uses fluorogen-antibody conjugates to label bacteria from patient samples Visualizing Legionella pneumophila from a throat swab
Indirect fluorescent antibody (IFA) Detects disease-specific antibodies in patent serum Diagnosing syphilis; detecting antinuclear antibodies (ANA) for lupus and other autoimmune diseases
Flow cytometry Labels cell membranes with fluorogen-antibody conjugate markers excited by a laser; machine counts the cell and records the relative fluorescence Counting the number of fluorescently labeled CD4 or CD8 cells in a sample
Fluorescence activated cell sorter (FACS) Form of flow cytometry that both counts cells and physically separates them into pools of high and low fluorescence cells Sorting cancer cells
Key Concepts and Summary
• Immunofluorescence assays use antibody-fluorogen conjugates to illuminate antigens for easy, rapid detection.
• Direct immunofluorescence can be used to detect the presence of bacteria in clinical samples such as sputum.
• Indirect immunofluorescence detects the presence of antigen-specific antibodies in patient sera. The fluorescent antibody binds to the antigen-specific antibody rather than the antigen.
• The use of indirect immunofluorescence assays to detect antinuclear antibodies is an important tool in the diagnosis of several autoimmune diseases.
• Flow cytometry uses fluorescent mAbs against cell-membrane proteins to quantify specific subsets of cells in complex mixtures.
• Fluorescence-activated cell sorters are an extension of flow cytometry in which fluorescence intensity is used to physically separate cells into high and low fluorescence populations.
Footnotes
1. 1 Gill, James M., ANNA M. Quisel, PETER V. Rocca, and DENE T. Walters. “Diagnosis of systemic lupus erythematosus.” American family physician 68, no. 11 (2003): 2179-2186. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/20%3A_Laboratory_Analysis_of_the_Immune_Response/20.05%3A_Fluorescent_Auto-Antibody_Techniques.txt |
20.1: Practical Applications of Monoclonal and Polyclonal Antibodies
In addition to being crucial for our normal immune response, antibodies provide powerful tools for research and diagnostic purposes. The high specificity of antibodies makes them an excellent tool for detecting and quantifying a broad array of targets, from drugs to serum proteins to microorganisms. With in vitro assays, antibodies can be used to precipitate soluble antigens, agglutinate cells, and neutralize drugs, toxins, and viruses.
Multiple Choice
For many uses in the laboratory, polyclonal antibodies work well, but for some types of assays, they lack sufficient ________ because they cross-react with inappropriate antigens.
1. specificity
2. sensitivity
3. accuracy
4. reactivity
Answer
A
How are monoclonal antibodies produced?
1. Antibody-producing B cells from a mouse are fused with myeloma cells and then the cells are grown in tissue culture.
2. A mouse is injected with an antigen and then antibodies are harvested from its serum.
3. They are produced by the human immune system as a natural response to an infection.
4. They are produced by a mouse’s immune system as a natural response to an infection.
Answer
A
Fill in the Blank
When we inject an animal with the same antigen a second time a few weeks after the first, ________ takes place, which means the antibodies produced after the second injection will on average bind the antigen more tightly.
Answer
affinity maturation
When using mAbs to treat disease in humans, the mAbs must first be ________ by replacing the mouse constant region DNA with human constant region DNA.
Answer
humanized
If we used normal mouse mAbs to treat human disease, multiple doses would cause the patient to respond with ________ against the mouse antibodies.
Answer
neutralizing antibodies
A polyclonal response to an infection occurs because most antigens have multiple ________,
Answer
epitopes
Short Answer
Describe two reasons why polyclonal antibodies are more likely to exhibit cross-reactivity than monoclonal antibodies.
Critical Thinking
Suppose you were screening produce in a grocery store for the presence of E. coli contamination. Would it be better to use a polyclonal anti-E. coli antiserum or a mAb against an E. coli membrane protein? Explain.
20.2: Detecting Antigen-Antibody Complexes in vitro
Laboratory tests to detect antibodies and antigens outside of the body (e.g., in a test tube) are called in vitro assays. When both antibodies and their corresponding antigens are present in a solution, we can often observe a precipitation reaction in which large complexes (lattices) form and settle out of solution.
Multiple Choice
The formation of ________ is a positive result in the VDRL test.
1. flocculant
2. precipitin
3. coagulation
4. a bright pink color
Answer
A
The titer of a virus neutralization test is the highest dilution of patient serum
1. in which there is no detectable viral DNA.
2. in which there is no detectable viral protein.
3. that completely blocks plaque formation.
4. that reduces plaque formation by at least 50%.
Answer
D
In the Ouchterlony assay, we see a sharp precipitin arc form between antigen and antiserum. Why does this arc remain visible for a long time?
1. The antibody molecules are too large to diffuse through the agar.
2. The precipitin lattice is too large to diffuse through the agar.
3. Methanol, added once the arc forms, denatures the protein and blocks diffusion.
4. The antigen molecules are chemically coupled to the gel matrix.
Answer
B
Fill in the Blank
When slowly adding antigen to an antiserum, the amount of precipitin would gradually increase until reaching the ________; addition of more antigen after this point would actually decrease the amount of precipitin.
Answer
equivalence zone or zone of equivalence
The radial immunodiffusion test quantifies antigen by mixing ________ into a gel and then allowing antigen to diffuse out from a well cut in the gel.
Answer
antiserum
Short Answer
Explain why hemolysis in the complement fixation test is a negative test for infection.
What is meant by the term “neutralizing antibodies,” and how can we quantify this effect using the viral neutralization assay?
Critical Thinking
Both IgM and IgG antibodies can be used in precipitation reactions. However, one of these immunoglobulin classes will form precipitates at much lower concentrations than the other. Which class is this, and why is it so much more efficient in this regard?
20.3: Agglutination Assays
In addition to causing precipitation of soluble molecules and flocculation of molecules in suspension, antibodies can also clump together cells or particles (e.g., antigen-coated latex beads) in a process called agglutination. Agglutination can be used as an indicator of the presence of antibodies against bacteria or red blood cells. Agglutination assays are usually quick and easy to perform on a glass slide or microtiter plate.
Multiple Choice
We use antisera to distinguish between various ________ within a species of bacteria.
1. isotypes
2. serovars
3. subspecies
4. lines
Answer
B
When using antisera to characterize bacteria, we will often link the antibodies to ________ to better visualize the agglutination.
1. latex beads
2. red blood cells
3. other bacteria
4. white blood cells
Answer
A
The antibody screening test that is done along with pretransfusion blood typing is used to ensure that the recipient
1. does not have a previously undetected bacterial or viral infection.
2. is not immunocompromised.
3. actually does have the blood type stated in the online chart.
4. is not making antibodies against antigens outside the ABO or Rh systems.
Answer
D
The direct Coombs’ test is designed to detect when people have a disease that causes them to
1. have an excessively high fever.
2. quit making antibodies.
3. make too many red blood cells.
4. produce antibodies that bind to their own red blood cells.
Answer
D
Viral hemagglutination assays only work with certain types of viruses because
1. the virus must be able to cross-link red blood cells directly.
2. the virus must be able to lyse red blood cells.
3. the virus must not be able to lyse red blood cells.
4. other viruses are too dangerous to work with in a clinical lab setting.
Answer
A
Fill in the Blank
In the major cross-match, we mix ________ with the donor red blood cells and look for agglutination.
Answer
patient serum
Coombs’ reagent is an antiserum with antibodies that bind to human ________.
Answer
immunoglobulins/antibodies and/or complement
Short Answer
Explain why the titer of a direct hemagglutination assay is the highest dilution that still causes hemagglutination, whereas in the viral hemagglutination inhibition assay, the titer is the highest dilution at which hemagglutination is not observed.
Why would a doctor order a direct Coombs’ test when a baby is born with jaundice?
Critical Thinking
When shortages of donated blood occur, O-negative blood may be given to patients, even if they have a different blood type. Why is this the case? If O-negative blood supplies were depleted, what would be the next-best choice for a patient with a different blood type in critical need of a transfusion? Explain your answers.
20.4: Enzyme Immunoassays (EIA) and Enzyme-Linked Immunosorbent Assays (ELISA)
Enzyme immunoassays (EIA) are used to visualize and quantify antigens. They use an antibody conjugated to an enzyme to bind the antigen, and the enzyme converts a substrate into an observable end product. The substrate may be either a chromogen or a fluorogen. Immunostaining is an EIA technique for visualizing cells in a tissue (immunohistochemistry) or examining intracellular structures (immunocytochemistry). Direct ELISA is used to quantify an antigen in solution.
Multiple Choice
In an enzyme immunoassay, the enzyme
1. is bound by the antibody’s antigen-binding site.
2. is attached to the well of a microtiter plate.
3. is conjugated to the suspect antigen.
4. is bound to the constant region of the secondary antibody.
Answer
D
When using an EIA to study microtubules or other structures inside a cell, we first chemically fix the cell and then treat the cells with alcohol. What is the purpose of this alcohol treatment?
1. It makes holes in the cell membrane large enough for antibodies to pass.
2. It makes the membrane sticky so antibodies will bind and be taken up by receptor-mediated endocytosis.
3. It removes negative charges from the membrane, which would otherwise repulse the antibodies.
4. It prevents nonspecific binding of the antibodies to the cell membrane.
Answer
A
In a lateral-flow pregnancy test, you see a blue band form on the control line and no band form on the test line. This is probably a ________ test for pregnancy.
1. positive
2. false-positive
3. false-negative
4. negative
Answer
D
When performing an FEIA, the fluorogen replaces the ________ that is used in an EIA.
1. antigen
2. chromogenic substrate
3. enzyme
4. secondary antibody
Answer
B
Fill in the Blank
To detect antibodies against bacteria in the bloodstream using an EIA, we would run a(n) ________, which we would start by attaching antigen from the bacteria to the wells of a microtiter plate.
Answer
indirect ELISA
Short Answer
Why is it important in a sandwich ELISA that the antigen has multiple epitopes? And why might it be advantageous to use polyclonal antisera rather than mAb in this assay?
The pregnancy test strip detects the presence of human chorionic gonadotrophin in urine. This hormone is initially produced by the fetus and later by the placenta. Why is the test strip preferred for this test rather than using either a direct or indirect ELISA with their more quantifiable results?
Critical Thinking
Label the primary and secondary antibodies, and discuss why the production of end product will be proportional to the amount of antigen.
20.5: Fluorescent Auto-Antibody Techniques
Rapid visualization of bacteria from a clinical sample such as a throat swab or sputum can be achieved through fluorescent antibody (FA) techniques that attach a fluorescent marker (fluorogen) to the constant region of an antibody, resulting in a reporter molecule that is quick to use, easy to see or measure, and able to bind to target markers with high specificity. We can also label cells, allowing us to precisely quantify particular subsets of cells or even purify them for further research.
Multiple Choice
Suppose you need to quantify the level of CD8 T cells in the blood of a patient recovering from influenza. You treat a sample of the patient’s white blood cells using a fluorescent mAb against CD8, pass the cells through a flow cytometer, and produce the histogram shown below. The area under the peak to the left (blue) is three times greater than the area of the peak on the right (red). What can you determine from these data?
1. There are no detectable CD8 cells.
2. There are three times as many CD4 cells than CD8 cells.
3. There are three times as many CD8 cells than CD4 cells.
4. CD8 cells make up about one-fourth of the total number of cells.
Answer
D
In the data described in the previous question, the average fluorescence intensity of cells in the second (red) peak is about ________ that in the first (blue) peak.
1. three times
2. 100 times
3. one-third
4. 1000 times
Answer
B
In a direct fluorescent antibody test, which of the following would we most likely be looking for using a fluorescently-labeled mAb?
1. bacteria in a patient sample
2. bacteria isolated from a patient and grown on agar plates
3. antiserum from a patient smeared onto a glass slide
4. antiserum from a patient that had bound to antigen-coated beads
Answer
A
Fill in the Blank
In flow cytometry, cell subsets are labeled using a fluorescent antibody to a membrane protein. The fluorogen is activated by a(n) ________ as the cells pass by the detectors.
Answer
laser
Fluorescence in a flow cytometer is measured by a detector set at an angle to the light source. There is also an in-line detector that can detect cell clumps or ________.
Answer
fragments
Critical Thinking
A patient suspected of having syphilis is tested using both the VDRL test and IFA. The IFA test comes back positive, but the VDRL test is negative. What is the most likely reason for these results?
A clinician suspects that a patient with pneumonia may be infected by Legionella pneumophila. Briefly describe two reasons why a DFA test might be better for detecting this pathogen than standard bacteriology techniques. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/20%3A_Laboratory_Analysis_of_the_Immune_Response/20.E%3A_Laboratory_Analysis_of_the_Immune_Response_%28Exercises%29.txt |
The human body is covered in skin, and like most coverings, skin is designed to protect what is underneath. One of its primary purposes is to prevent microbes in the surrounding environment from invading underlying tissues and organs. But in spite of its role as a protective covering, skin is not itself immune from infection. Certain pathogens and toxins can cause severe infections or reactions when they come in contact with the skin. Other pathogens are opportunistic, breaching the skin’s natural defenses through cuts, wounds, or a disruption of normal microbiota resulting in an infection in the surrounding skin and tissue. Still other pathogens enter the body via different routes—through the respiratory or digestive systems, for example—but cause reactions that manifest as skin rashes or lesions.
Nearly all humans experience skin infections to some degree. Many of these conditions are, as the name suggests, “skin deep,” with symptoms that are local and non-life-threatening. At some point, almost everyone must endure conditions like acne, athlete’s foot, and minor infections of cuts and abrasions, all of which result from infections of the skin. But not all skin infections are quite so innocuous. Some can become invasive, leading to systemic infection or spreading over large areas of skin, potentially becoming life-threatening.
• 21.1: Anatomy and Normal Microbiota of the Skin and Eyes
Human skin consists of two main layers, the epidermis and dermis, which are situated on top of the hypodermis, a layer of connective tissue. The skin is an effective physical barrier against microbial invasion. The skin’s relatively dry environment and normal microbiota discourage colonization by transient microbes. The skin’s normal microbiota varies from one region of the body to another. The conjunctiva of the eye is a frequent site for microbial infection; deeper infections are less common.
• 21.2: Bacterial Infections of the Skin and Eyes
Staphylococcus and Streptococcus cause many different types of skin infections, many of which occur when bacteria breach the skin barrier through a cut or wound. S. aureus are frequently associated with purulent skin infections that manifest as folliculitis, furuncles, or carbuncles. S. aureus is also a leading cause of staphylococcal scalded skin syndrome (SSSS). S. aureus is generally drug resistant and current MRSA strains are resistant to a wide range of antibiotics.
• 21.3: Viral Infections of the Skin and Eyes
Papillomas (warts) are caused by human papillomaviruses. Herpes simplex virus (especially HSV-1) mainly causes oral herpes, but lesions can appear on other areas of the skin and mucous membranes. Roseola and fifth disease are common viral illnesses that cause skin rashes; roseola is caused by HHV-6 and HHV-7 while fifth disease is caused by parvovirus 19. Viral conjunctivitis is often caused by adenoviruses and may be associated with the common cold. Herpes keratitis is caused by herpesviruses.
• 21.4: Mycoses of the Skin and Eyes
Mycoses can be cutaneous, subcutaneous, or systemic. Common cutaneous mycoses include tineas caused by dermatophytes of the genera Trichophyton, Epidermophyton, and Microsporum. Tinea corporis is called ringworm. Tineas on other parts of the body have names associated with the affected body part. Aspergillosis is a fungal disease caused by molds of the genus Aspergillus. Primary cutaneous aspergillosis enters through a break in the skin, such as the site of an injury or a surgical wound.
• 21.5: Protozoan and Helminthic Infections of the Eyes
The protozoan Acanthamoeba and the helminth Loa loa are two parasites that can breach the skin barrier, causing infections of the skin and eyes. Acanthamoeba keratitis is a parasitic infection of the eye that often results from improper disinfection of contact lenses or swimming while wearing contact lenses. Loiasis, or eye worm, is a disease endemic to Africa that is caused by parasitic worms that infect the subcutaneous tissue of the skin and eyes. It is transmitted by deerfly vectors.
• 21.E: Skin and Eye Infections (Exercises)
These are exercises for Chapter 21 "Skin and Eye Infections" in OpenStax's Microbiology Textmap.
Thumbnail: Multiple plantar warts have grown on this toe.
21: Skin and Eye Infections
Learning Objectives
• Describe the major anatomical features of the skin and eyes
• Compare and contrast the microbiomes of various body sites, such as the hands, back, feet, and eyes
• Explain how microorganisms overcome defenses of skin and eyes in order to cause infection
• Describe general signs and symptoms of disease associated with infections of the skin and eyes
Clinical Focus: Part 1
Sam, a college freshman with a bad habit of oversleeping, nicked himself shaving in a rush to get to class on time. At the time, he didn’t think twice about it. But two days later, he noticed the cut was surrounded by a reddish area of skin that was warm to the touch. When the wound started oozing pus, he decided he had better stop by the university’s clinic. The doctor took a sample from the lesion and then cleaned the area.
Exercise \(1\)
What type of microbe could be responsible for Sam’s infection?
Human skin is an important part of the innate immune system. In addition to serving a wide range of other functions, the skin serves as an important barrier to microbial invasion. Not only is it a physical barrier to penetration of deeper tissues by potential pathogens, but it also provides an inhospitable environment for the growth of many pathogens. In this section, we will provide a brief overview of the anatomy and normal microbiota of the skin and eyes, along with general symptoms associated with skin and eye infections.
Layers of the Skin
Human skin is made up of several layers and sublayers. The two main layers are the epidermis and the dermis. These layers cover a third layer of tissue called the hypodermis, which consists of fibrous and adipose connective tissue (Figure \(1\)).
The epidermis is the outermost layer of the skin, and it is relatively thin. The exterior surface of the epidermis, called the stratum corneum, primarily consists of dead skin cells. This layer of dead cells limits direct contact between the outside world and live cells. The stratum corneum is rich in keratin, a tough, fibrous protein that is also found in hair and nails. Keratin helps make the outer surface of the skin relatively tough and waterproof. It also helps to keep the surface of the skin dry, which reduces microbial growth. However, some microbes are still able to live on the surface of the skin, and some of these can be shed with dead skin cells in the process of desquamation, which is the shedding and peeling of skin that occurs as a normal process but that may be accelerated when infection is present.
Beneath the epidermis lies a thicker skin layer called the dermis. The dermis contains connective tissue and embedded structures such as blood vessels, nerves, and muscles. Structures called hair follicles (from which hair grows) are located within the dermis, even though much of their structure consists of epidermal tissue. The dermis also contains the two major types of glands found in human skin: sweat glands (tubular glands that produce sweat) and sebaceous glands (which are associated with hair follicles and produce sebum, a lipid-rich substance containing proteins and minerals).
Perspiration (sweat) provides some moisture to the epidermis, which can increase the potential for microbial growth. For this reason, more microbes are found on the regions of the skin that produce the most sweat, such as the skin of the underarms and groin. However, in addition to water, sweat also contains substances that inhibit microbial growth, such as salts, lysozyme, and antimicrobial peptides. Sebum also serves to protect the skin and reduce water loss. Although some of the lipids and fatty acids in sebum inhibit microbial growth, sebum contains compounds that provide nutrition for certain microbes.
Exercise \(2\)
How does desquamation help with preventing infections?
Normal Microbiota of the Skin
The skin is home to a wide variety of normal microbiota, consisting of commensal organisms that derive nutrition from skin cells and secretions such as sweat and sebum. The normal microbiota of skin tends to inhibit transient-microbe colonization by producing antimicrobial substances and outcompeting other microbes that land on the surface of the skin. This helps to protect the skin from pathogenic infection.
The skin’s properties differ from one region of the body to another, as does the composition of the skin’s microbiota. The availability of nutrients and moisture partly dictates which microorganisms will thrive in a particular region of the skin. Relatively moist skin, such as that of the nares (nostrils) and underarms, has a much different microbiota than the dryer skin on the arms, legs, hands, and top of the feet. Some areas of the skin have higher densities of sebaceous glands. These sebum-rich areas, which include the back, the folds at the side of the nose, and the back of the neck, harbor distinct microbial communities that are less diverse than those found on other parts of the body.
Different types of bacteria dominate the dry, moist, and sebum-rich regions of the skin. The most abundant microbes typically found in the dry and sebaceous regions are Betaproteobacteria and Propionibacteria, respectively. In the moist regions, Corynebacterium and Staphylococcus are most commonly found (Figure \(2\)). Viruses and fungi are also found on the skin, with Malassezia being the most common type of fungus found as part of the normal microbiota. The role and populations of viruses in the microbiota, known as viromes, are still not well understood, and there are limitations to the techniques used to identify them. However, Circoviridae, Papillomaviridae, and Polyomaviridaeappear to be the most common residents in the healthy skin virome.123
Exercise \(3\)
What are the four most common bacteria that are part of the normal skin microbiota?
Infections of the Skin
While the microbiota of the skin can play a protective role, it can also cause harm in certain cases. Often, an opportunistic pathogen residing in the skin microbiota of one individual may be transmitted to another individual more susceptible to an infection. For example, methicillin-resistant Staphylococcus aureus (MRSA) can often take up residence in the nares of health care workers and hospital patients; though harmless on intact, healthy skin, MRSA can cause infections if introduced into other parts of the body, as might occur during surgery or via a post-surgical incision or wound. This is one reason why clean surgical sites are so important.
Injury or damage to the skin can allow microbes to enter deeper tissues, where nutrients are more abundant and the environment is more conducive to bacterial growth. Wound infections are common after a puncture or laceration that damages the physical barrier of the skin. Microbes may infect structures in the dermis, such as hair follicles and glands, causing a localized infection, or they may reach the bloodstream, which can lead to a systemic infection.
In some cases, infectious microbes can cause a variety of rashes or lesions that differ in their physical characteristics. These rashes can be the result of inflammation reactions or direct responses to toxins produced by the microbes. Table \(1\) lists some of the medical terminology used to describe skin lesions and rashes based on their characteristics; Figure \(3\) and Figure \(4\) illustrate some of the various types of skin lesions. It is important to note that many different diseases can lead to skin conditions of very similar appearance; thus the terms used in the table are generally not exclusive to a particular type of infection or disease.
Table \(1\): Some Medical Terms Associated with Skin Lesions and Rashes
Term Definition
abscess localized collection of pus
bulla (pl., bullae) fluid-filled blister no more than 5 mm in diameter
carbuncle deep, pus-filled abscess generally formed from multiple furuncles
crust dried fluids from a lesion on the surface of the skin
cyst encapsulated sac filled with fluid, semi-solid matter, or gas, typically located just below the upper layers of skin
folliculitis a localized rash due to inflammation of hair follicles
furuncle (boil) pus-filled abscess due to infection of a hair follicle
macules smooth spots of discoloration on the skin
papules small raised bumps on the skin
pseudocyst lesion that resembles a cyst but with a less defined boundary
purulent pus-producing; suppurative
pustules fluid- or pus-filled bumps on the skin
pyoderma any suppurative (pus-producing) infection of the skin
suppurative producing pus; purulent
ulcer break in the skin; open sore
vesicle small, fluid-filled lesion
wheal swollen, inflamed skin that itches or burns, such as from an insect bite
Exercise \(4\)
How can asymptomatic health care workers transmit bacteria such as MRSA to patients?
Anatomy and Microbiota of the Eye
Although the eye and skin have distinct anatomy, they are both in direct contact with the external environment. An important component of the eye is the nasolacrimal drainage system, which serves as a conduit for the fluid of the eye, called tears. Tears flow from the external eye to the nasal cavity by the lacrimal apparatus, which is composed of the structures involved in tear production (Figure \(5\)). The lacrimal gland, above the eye, secretes tears to keep the eye moist. There are two small openings, one on the inside edge of the upper eyelid and one on the inside edge of the lower eyelid, near the nose. Each of these openings is called a lacrimal punctum. Together, these lacrimal puncta collect tears from the eye that are then conveyed through lacrimal ducts to a reservoir for tears called the lacrimal sac, also known as the dacrocyst or tear sac.
From the sac, tear fluid flows via a nasolacrimal duct to the inner nose. Each nasolacrimal duct is located underneath the skin and passes through the bones of the face into the nose. Chemicals in tears, such as defensins, lactoferrin, and lysozyme, help to prevent colonization by pathogens. In addition, mucins facilitate removal of microbes from the surface of the eye.
The surfaces of the eyeball and inner eyelid are mucous membranes called conjunctiva. The normal conjunctival microbiota has not been well characterized, but does exist. One small study (part of the Ocular Microbiome project) found twelve genera that were consistently present in the conjunctiva.4 These microbes are thought to help defend the membranes against pathogens. However, it is still unclear which microbes may be transient and which may form a stable microbiota.5
Use of contact lenses can cause changes in the normal microbiota of the conjunctiva by introducing another surface into the natural anatomy of the eye. Research is currently underway to better understand how contact lenses may impact the normal microbiota and contribute to eye disease.
The watery material inside of the eyeball is called the vitreous humor. Unlike the conjunctiva, it is protected from contact with the environment and is almost always sterile, with no normal microbiota (Figure \(6\)).
Infections of the Eye
The conjunctiva is a frequent site of infection of the eye; like other mucous membranes, it is also a common portal of entry for pathogens. Inflammation of the conjunctiva is called conjunctivitis, although it is commonly known as pinkeyebecause of the pink appearance in the eye. Infections of deeper structures, beneath the cornea, are less common (Figure \(7\)). Conjunctivitis occurs in multiple forms. It may be acute or chronic. Acute purulent conjunctivitis is associated with pus formation, while acute hemorrhagic conjunctivitis is associated with bleeding in the conjunctiva. The term blepharitis refers to an inflammation of the eyelids, while keratitis refers to an inflammation of the cornea (Figure \(7\)); keratoconjunctivitis is an inflammation of both the cornea and the conjunctiva, and dacryocystitis is an inflammation of the lacrimal sac that can often occur when a nasolacrimal duct is blocked.
Infections leading to conjunctivitis, blepharitis, keratoconjunctivitis, or dacryocystitis may be caused by bacteria or viruses, but allergens, pollutants, or chemicals can also irritate the eye and cause inflammation of various structures. Viral infection is a more likely cause of conjunctivitis in cases with symptoms such as fever and watery discharge that occurs with upper respiratory infection and itchy eyes. Table \(2\) summarizes some common forms of conjunctivitis and blepharitis.
Table \(2\): Types of Conjunctivities and Blepharitis
Condition Description Causative Agent(s)
Acute purulent conjunctivitis Conjunctivitis with purulent discharge Bacterial (Haemophilus, Staphylococcus)
Acute hemorrhagic conjunctivitis Involves subconjunctival hemorrhages Viral (Picornaviradae)
Acute ulcerative blepharitis Infection involving eyelids; pustules and ulcers may develop Bacterial (Staphylococcal) or viral (herpes simplex, varicella-zoster, etc.)
Follicular conjunctivitis Inflammation of the conjunctiva with nodules (dome-shaped structures that are red at the base and pale on top) Viral (adenovirus and others); environmental irritants
Dacryocystitis Inflammation of the lacrimal sac often associated with a plugged nasolacrimal duct Bacterial (Haemophilus, Staphylococcus, Streptococcus)
Keratitis Inflammation of cornea Bacterial, viral, or protozoal; environmental irritants
Keratoconjunctivitis Inflammation of cornea and conjunctiva Bacterial, viral (adenoviruses), or other causes (including dryness of the eye)
Nonulcerative blepharitis Inflammation, irritation, redness of the eyelids without ulceration Environmental irritants; allergens
Papillary conjunctivitis Inflammation of the conjunctiva; nodules and papillae with red tops develop Environmental irritants; allergens
Exercise \(5\)
How does the lacrimal apparatus help to prevent eye infections?
Key Concepts and Summary
• Human skin consists of two main layers, the epidermis and dermis, which are situated on top of the hypodermis, a layer of connective tissue.
• The skin is an effective physical barrier against microbial invasion.
• The skin’s relatively dry environment and normal microbiota discourage colonization by transient microbes.
• The skin’s normal microbiota varies from one region of the body to another.
• The conjunctiva of the eye is a frequent site for microbial infection, but deeper eye infections are less common; multiple types of conjunctivitis exist.
Footnotes
1. 1 Belkaid, Y., and J.A. Segre. “Dialogue Between Skin Microbiota and Immunity,” Science 346 (2014) 6212:954–959.
2. 2 Foulongne, Vincent, et al. “Human Skin Microbiota: High Diversity of DNA Viruses Identified on the Human Skin by High Throughput Sequencing.” PLoS ONE (2012) 7(6): e38499. doi: 10.1371/journal.pone.0038499.
3. 3 Robinson, C.M., and J.K. Pfeiffer. “Viruses and the Microbiota.” Annual Review of Virology (2014) 1:55–59. doi: 10.1146/annurev-virology-031413-085550.
4. 4 Abelson, M.B., Lane, K., and Slocum, C.. “The Secrets of Ocular Microbiomes.” Review of Ophthalmology June 8, 2015. www.reviewofophthalmology.com...isease/c/55178. Accessed Sept 14, 2016.
5. 5 Shaikh-Lesko, R. “Visualizing the Ocular Microbiome.” The Scientist May 12, 2014. http://www.the-scientist.com/?articl...lar-Microbiome. Accessed Sept 14, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/21%3A_Skin_and_Eye_Infections/21.01%3A_Anatomy_and_Normal_Microbiota_of_the_Skin_and_Eyes.txt |
Learning Objectives
• Identify the most common bacterial pathogens that cause infections of the skin and eyes
• Compare the major characteristics of specific bacterial diseases affecting the skin and eyes
Despite the skin’s protective functions, infections are common. Gram-positive Staphylococcus spp. and Streptococcus spp. are responsible for many of the most common skin infections. However, many skin conditions are not strictly associated with a single pathogen. Opportunistic pathogens of many types may infect skin wounds, and individual cases with identical symptoms may result from different pathogens or combinations of pathogens.
In this section, we will examine some of the most important bacterial infections of the skin and eyes and discuss how biofilms can contribute to and exacerbate such infections. Key features of bacterial skin and eye infections are also summarized in the Disease Profile boxes throughout this section.
Staphylococcal Infections of the Skin
Staphylococcus species are commonly found on the skin, with S. epidermidis and S. hominis being prevalent in the normal microbiota. S. aureus is also commonly found in the nasal passages and on healthy skin, but pathogenic strains are often the cause of a broad range of infections of the skin and other body systems.
S. aureus is quite contagious. It is spread easily through skin-to-skin contact, and because many people are chronic nasal carriers (asymptomatic individuals who carry S. aureus in their nares), the bacteria can easily be transferred from the nose to the hands and then to fomites or other individuals. Because it is so contagious, S. aureus is prevalent in most community settings. This prevalence is particularly problematic in hospitals, where antibiotic-resistant strains of the bacteria may be present, and where immunocompromised patients may be more susceptible to infection. Resistant strains include methicillin-resistant S. aureus (MRSA), which can be acquired through health-care settings (hospital-acquired MRSA, or HA-MRSA) or in the community (community-acquired MRSA, or CA-MRSA). Hospital patients often arrive at health-care facilities already colonized with antibiotic-resistant strains of S. aureus that can be transferred to health-care providers and other patients. Some hospitals have attempted to detect these individuals in order to institute prophylactic measures, but they have had mixed success (see Eye on Ethics: Screening Patients for MRSA).
When a staphylococcal infection develops, choice of medication is important. As discussed above, many staphylococci (such as MRSA) are resistant to some or many antibiotics. Thus, antibiotic sensitivity is measured to identify the most suitable antibiotic. However, even before receiving the results of sensitivity analysis, suspected S. aureus infections are often initially treated with drugs known to be effective against MRSA, such as trimethoprim-sulfamethoxazole(TMP/SMZ), clindamycin, a tetracycline (doxycycline or minocycline), or linezolid.
The pathogenicity of staphylococcal infections is often enhanced by characteristic chemicals secreted by some strains. Staphylococcal virulence factors include hemolysins called staphylolysins, which are cytotoxic for many types of cells, including skin cells and white blood cells. Virulent strains of S. aureus are also coagulase-positive, meaning they produce coagulase, a plasma-clotting protein that is involved in abscess formation. They may also produce leukocidins, which kill white blood cells and can contribute to the production of pus and Protein A, which inhibits phagocytosis by binding to the constant region of antibodies. Some virulent strains of S. aureus also produce other toxins, such as toxic shock syndrome toxin-1 (see Virulence Factors of Bacterial and Viral Pathogens).
To confirm the causative agent of a suspected staphylococcal skin infection, samples from the wound are cultured. Under the microscope, gram-positive Staphylococcus species have cellular arrangements that form grapelike clusters; when grown on blood agar, colonies have a unique pigmentation ranging from opaque white to cream. A catalase testis used to distinguish Staphylococcus from Streptococcus, which is also a genus of gram-positive cocci and a common cause of skin infections. Staphylococcus species are catalase-positive while Streptococcus species are catalase-negative.
Other tests are performed on samples from the wound in order to distinguish coagulase-positive species of Staphylococcus (CoPS) such as S. aureus from common coagulase-negative species (CoNS) such as S. epidermidis. Although CoNS are less likely than CoPS to cause human disease, they can cause infections when they enter the body, as can sometimes occur via catheters, indwelling medical devices, and wounds. Passive agglutination testing can be used to distinguish CoPS from CoNS. If the sample is coagulase-positive, the sample is generally presumed to contain S. aureus. Additional genetic testing would be necessary to identify the particular strain of S. aureus.
Another way to distinguish CoPS from CoNS is by culturing the sample on mannitol salt agar (MSA). Staphylococcus species readily grow on this medium because they are tolerant of the high concentration of sodium chloride (7.5% NaCl). However, CoPS such as S. aureus ferment mannitol (which will be evident on a MSA plate), whereas CoNS such as S. epidermidis do not ferment mannitol but can be distinguished by the fermentation of other sugars such as lactose, malonate, and raffinose (Figure \(1\)).
Screening Patients for MRSA
According to the CDC, 86% of invasive MRSA infections are associated in some way with healthcare, as opposed to being community-acquired. In hospitals and clinics, asymptomatic patients who harbor MRSA may spread the bacteria to individuals who are more susceptible to serious illness.
In an attempt to control the spread of MRSA, hospitals have tried screening patients for MRSA. If patients test positive following a nasal swab test, they can undergo decolonization using chlorhexidine washes or intranasal mupirocin. Some studies have reported substantial reductions in MRSA disease following implementation of these protocols, while others have not. This is partly because there is no standard protocol for these procedures. Several different MRSA identification tests may be used, some involving slower culturing techniques and others rapid testing. Other factors, such as the effectiveness of general hand-washing protocols, may also play a role in helping to prevent MRSA transmission. There are still other questions that need to be addressed: How frequently should patients be screened? Which individuals should be tested? From where on the body should samples be collected? Will increased resistance develop from the decolonization procedures?
Even if identification and decolonization procedures are perfected, ethical questions will remain. Should patients have the right to decline testing? Should a patient who tests positive for MRSA have the right to decline the decolonization procedure, and if so, should hospitals have the right to refuse treatment to the patient? How do we balance the individual’s right to receive care with the rights of other patients who could be exposed to disease as a result?
Superficial Staphylococcal Infections
S. aureus is often associated with pyoderma, skin infections that are purulent. Pus formation occurs because many strains of S. aureus produce leukocidins, which kill white blood cells. These purulent skin infections may initially manifest as folliculitis, but can lead to furuncles or deeper abscesses called carbuncles.
Folliculitis generally presents as bumps and pimples that may be itchy, red, and/or pus-filled. In some cases, folliculitis is self-limiting, but if it continues for more than a few days, worsens, or returns repeatedly, it may require medical treatment. Sweat, skin injuries, ingrown hairs, tight clothing, irritation from shaving, and skin conditions can all contribute to folliculitis. Avoidance of tight clothing and skin irritation can help to prevent infection, but topical antibiotics (and sometimes other treatments) may also help. Folliculitis can be identified by skin inspection; treatment is generally started without first culturing and identifying the causative agent.
In contrast, furuncles (boils) are deeper infections (Figure \(2\)). They are most common in those individuals (especially young adults and teenagers) who play contact sports, share athletic equipment, have poor nutrition, live in close quarters, or have weakened immune systems. Good hygiene and skin care can often help to prevent furuncles from becoming more infective, and they generally resolve on their own. However, if furuncles spread, increase in number or size, or lead to systemic symptoms such as fever and chills, then medical care is needed. They may sometimes need to be drained (at which time the pathogens can be cultured) and treated with antibiotics.
When multiple boils develop into a deeper lesion, it is called a carbuncle (Figure \(2\)). Because carbuncles are deeper, they are more commonly associated with systemic symptoms and a general feeling of illness. Larger, recurrent, or worsening carbuncles require medical treatment, as do those associated with signs of illness such as fever. Carbuncles generally need to be drained and treated with antibiotics. While carbuncles are relatively easy to identify visually, culturing and laboratory analysis of the wound may be recommended for some infections because antibiotic resistance is relatively common.
Proper hygiene is important to prevent these types of skin infections or to prevent the progression of existing infections.
Staphylococcal scalded skin syndrome (SSSS) is another superficial infection caused by S. aureus that is most commonly seen in young children, especially infants. Bacterial exotoxins first produce erythema (redness of the skin) and then severe peeling of the skin, as might occur after scalding (Figure \(3\)). SSSS is diagnosed by examining characteristics of the skin (which may rub off easily), using blood tests to check for elevated white blood cell counts, culturing, and other methods. Intravenous antibiotics and fluid therapy are used as treatment.
Impetigo
The skin infection impetigo causes the formation of vesicles, pustules, and possibly bullae, often around the nose and mouth. Bullae are large, fluid-filled blisters that measure at least 5 mm in diameter. Impetigo can be diagnosed as either nonbullous or bullous. In nonbullous impetigo, vesicles and pustules rupture and become encrusted sores. Typically the crust is yellowish, often with exudate draining from the base of the lesion. In bullous impetigo, the bullae fill and rupture, resulting in larger, draining, encrusted lesions (Figure \(4\)).
Especially common in children, impetigo is particularly concerning because it is highly contagious. Impetigo can be caused by S. aureus alone, by Streptococcus pyogenes alone, or by coinfection of S. aureus and S. pyogenes. Impetigo is often diagnosed through observation of its characteristic appearance, although culture and susceptibility testing may also be used.
Topical or oral antibiotic treatment is typically effective in treating most cases of impetigo. However, cases caused by S. pyogenes can lead to serious sequelae (pathological conditions resulting from infection, disease, injury, therapy, or other trauma) such as acute glomerulonephritis (AGN), which is severe inflammation in the kidneys.
Nosocomial S. epidermidis Infections
Though not as virulent as S. aureus, the staphylococcus S. epidermidis can cause serious opportunistic infections. Such infections usually occur only in hospital settings. S. epidermidis is usually a harmless resident of the normal skin microbiota. However, health-care workers can inadvertently transfer S. epidermidis to medical devices that are inserted into the body, such as catheters, prostheses, and indwelling medical devices. Once it has bypassed the skin barrier, S. epidermidis can cause infections inside the body that can be difficult to treat. Like S. aureus, S. epidermidis is resistant to many antibiotics, and localized infections can become systemic if not treated quickly. To reduce the risk of nosocomial (hospital-acquired) S. epidermidis, health-care workers must follow strict procedures for handling and sterilizing medical devices before and during surgical procedures.
Exercise \(1\)
Why are Staphylococcus aureus infections often purulent?
Streptococcal Infections of the Skin
Streptococcus are gram-positive cocci with a microscopic morphology that resembles chains of bacteria. Colonies are typically small (1–2 mm in diameter), translucent, entire edge, with a slightly raised elevation that can be either nonhemolytic, alpha-hemolytic, or beta-hemolytic when grown on blood agar (Figure \(5\)). Additionally, they are facultative anaerobes that are catalase-negative.
The genus Streptococcus includes important pathogens that are categorized in serological Lancefield groups based on the distinguishing characteristics of their surface carbohydrates. The most clinically important streptococcal species in humans is S. pyogenes, also known as group A streptococcus (GAS). S. pyogenes produces a variety of extracellular enzymes, including streptolysins O and S, hyaluronidase, and streptokinase. These enzymes can aid in transmission and contribute to the inflammatory response.1 S. pyogenes also produces a capsule and M protein, a streptococcal cell wall protein. These virulence factors help the bacteria to avoid phagocytosis while provoking a substantial immune response that contributes to symptoms associated with streptococcal infections.
S. pyogenes causes a wide variety of diseases not only in the skin, but in other organ systems as well. Examples of diseases elsewhere in the body include pharyngitis and scarlet fever, which will be covered in later chapters.
Cellulitis, Erysipelas, and Erythema Nosodum
Common streptococcal conditions of the skin include cellulitis, erysipelas, and erythema nodosum. An infection that develops in the dermis or hypodermis can cause cellulitis, which presents as a reddened area of the skin that is warm to the touch and painful. The causative agent is often S. pyogenes, which may breach the epidermis through a cut or abrasion, although cellulitis may also be caused by staphylococci. S. pyogenes can also cause erysipelas, a condition that presents as a large, intensely inflamed patch of skin involving the dermis (often on the legs or face). These infections can be suppurative, which results in a bullous form of erysipelas. Streptococcal and other pathogens may also cause a condition called erythema nodosum, characterized by inflammation in the subcutaneous fat cells of the hypodermis. It sometimes results from a streptococcal infection, though other pathogens can also cause the condition. It is not suppurative, but leads to red nodules on the skin, most frequently on the shins (Figure \(6\)).
In general, streptococcal infections are best treated through identification of the specific pathogen followed by treatment based upon that particular pathogen’s susceptibility to different antibiotics. Many immunological tests, including agglutination reactions and ELISAs, can be used to detect streptococci. Penicillin is commonly prescribed for treatment of cellulitis and erysipelas because resistance is not widespread in streptococci at this time. In most patients, erythema nodosum is self-limiting and is not treated with antimicrobial drugs. Recommended treatments may include nonsteroidal anti-inflammatory drugs (NSAIDs), cool wet compresses, elevation, and bed rest.
Necrotizing Fasciitis
Streptococcal infections that start in the skin can sometimes spread elsewhere, resulting in a rare but potentially life-threatening condition called necrotizing fasciitis, sometimes referred to as flesh-eating bacterial syndrome. S. pyogenes is one of several species that can cause this rare but potentially-fatal condition; others include Klebsiella, Clostridium, Escherichia coli, S. aureus, and Aeromonas hydrophila.
Necrotizing fasciitis occurs when the fascia, a thin layer of connective tissue between the skin and muscle, becomes infected. Severe invasive necrotizing fasciitis due to Streptococcus pyogenes occurs when virulence factors that are responsible for adhesion and invasion overcome host defenses. S. pyogenes invasins allow bacterial cells to adhere to tissues and establish infection. Bacterial proteases unique to S. pyogenes aggressively infiltrate and destroy host tissues, inactivate complement, and prevent neutrophil migration to the site of infection. The infection and resulting tissue death can spread very rapidly, as large areas of skin become detached and die. Treatment generally requires debridement (surgical removal of dead or infected tissue) or amputation of infected limbs to stop the spread of the infection; surgical treatment is supplemented with intravenous antibiotics and other therapies (Figure \(7\)).
Necrotizing fasciitis does not always originate from a skin infection; in some cases there is no known portal of entry. Some studies have suggested that experiencing a blunt force trauma can increase the risk of developing streptococcal necrotizing fasciitis.2
Exercise \(2\)
How do staphylococcal infections differ in general presentation from streptococcal infections?
Clinical Focus: Part 2
Observing that Sam’s wound is purulent, the doctor tells him that he probably has a bacterial infection. She takes a sample from the lesion to send for laboratory analysis, but because it is Friday, she does not expect to receive the results until the following Monday. In the meantime, she prescribes an over-the-counter topical antibiotic ointment. She tells Sam to keep the wound clean and apply a new bandage with the ointment at least twice per day.
Exercise \(3\)
1. How would the lab technician determine if the infection is staphylococcal or streptococcal? Suggest several specific methods.
2. What tests might the lab perform to determine the best course of antibiotic treatment?
Pseudomonas Infections of the Skin
Another important skin pathogen is Pseudomonas aeruginosa, a gram-negative, oxidase-positive, aerobic bacillus that is commonly found in water and soil as well as on human skin. P. aeruginosa is a common cause of opportunistic infections of wounds and burns. It can also cause hot tub rash, a condition characterized by folliculitis that frequently afflicts users of pools and hot tubs (recall the Clinical Focus case in Microbial Biochemistry). P. aeruginosa is also the cause of otitis externa (swimmer’s ear), an infection of the ear canal that causes itching, redness, and discomfort, and can progress to fever, pain, and swelling (Figure \(8\)).
Wounds infected with P. aeruginosa have a distinctive odor resembling grape soda or fresh corn tortillas. This odor is caused by the 2-aminoacetophenone that is used by P. aeruginosa in quorum sensing and contributes to its pathogenicity. Wounds infected with certain strains of P. aeruginosa also produce a blue-green pus due to the pigments pyocyanin and pyoverdin, which also contribute to its virulence. Pyocyanin and pyoverdin are siderophores that help P. aeruginosa survive in low-iron environments by enhancing iron uptake. P. aeruginosa also produces several other virulence factors, including phospholipase C (a hemolysin capable of breaking down red blood cells), exoenzyme S(involved in adherence to epithelial cells), and exotoxin A (capable of causing tissue necrosis). Other virulence factors include a slime that allows the bacterium to avoid being phagocytized, fimbriae for adherence, and proteases that cause tissue damage. P. aeruginosa can be detected through the use of cetrimide agar, which is selective for Pseudomonas species (Figure \(9\)).
Pseudomonas spp. tend to be resistant to most antibiotics. They often produce β-lactamases, may have mutations affecting porins (small cell wall channels) that affect antibiotic uptake, and may pump some antibiotics out of the cell, contributing to this resistance. Polymyxin B and gentamicin are effective, as are some fluoroquinolones. Otitis externa is typically treated with ear drops containing acetic acid, antibacterials, and/or steroids to reduce inflammation; ear drops may also include antifungals because fungi can sometimes cause or contribute to otitis externa. Wound infections caused by Pseudomonas spp. may be treated with topical antibiofilm agents that disrupt the formation of biofilms.
Exercise \(4\)
Name at least two types of skin infections commonly caused by Pseudomonas spp.
Acne
One of the most ubiquitous skin conditions is acne. Acne afflicts nearly 80% of teenagers and young adults, but it can be found in individuals of all ages. Higher incidence among adolescents is due to hormonal changes that can result in overproduction of sebum.
Acne occurs when hair follicles become clogged by shed skin cells and sebum, causing non-inflammatory lesions called comedones. Comedones (singular “comedo”) can take the form of whitehead and blackhead pimples. Whiteheads are covered by skin, whereas blackhead pimples are not; the black color occurs when lipids in the clogged follicle become exposed to the air and oxidize (Figure \(10\)).
Often comedones lead to infection by Propionibacterium acnes, a gram-positive, non-spore-forming, aerotolerant anaerobic bacillus found on skin that consumes components of sebum. P. acnes secretes enzymes that damage the hair follicle, causing inflammatory lesions that may include papules, pustules, nodules, or pseudocysts, depending on their size and severity.
Treatment of acne depends on the severity of the case. There are multiple ways to grade acne severity, but three levels are usually considered based on the number of comedones, the number of inflammatory lesions, and the types of lesions. Mild acne is treated with topical agents that may include salicylic acid (which helps to remove old skin cells) or retinoids (which have multiple mechanisms, including the reduction of inflammation). Moderate acne may be treated with antibiotics (erythromycin, clindamycin), acne creams (e.g., benzoyl peroxide), and hormones. Severe acne may require treatment using strong medications such as isotretinoin (a retinoid that reduces oil buildup, among other effects, but that also has serious side effects such as photosensitivity). Other treatments, such as phototherapy and laser therapy to kill bacteria and possibly reduce oil production, are also sometimes used.
Exercise \(5\)
What is the role of Propionibacterium acnes in causing acne?
Clinical Focus: Resolution
Sam uses the topical antibiotic over the weekend to treat his wound, but he does not see any improvement. On Monday, the doctor calls to inform him that the results from his laboratory tests are in. The tests show evidence of both Staphylococcus and Streptococcus in his wound. The bacterial species were confirmed using several tests. A passive agglutination test confirmed the presence of S. aureus. In this type of test, latex beads with antibodies cause agglutination when S. aureus is present. Streptococcus pyogenes was confirmed in the wound based on bacitracin (0.04 units) susceptibility as well as latex agglutination tests specific for S. pyogenes.
Because many strains of S. aureus are resistant to antibiotics, the doctor had also requested an antimicrobial susceptibility test (AST) at the same time the specimen was submitted for identification. The results of the AST indicated no drug resistance for the Streptococcus spp.; the Staphylococcus spp. showed resistance to several common antibiotics, but were susceptible to cefoxitin and oxacillin. Once Sam began to use these new antibiotics, the infection resolved within a week and the lesion healed.
Anthrax
The zoonotic disease anthrax is caused by Bacillus anthracis, a gram-positive, endospore-forming, facultative anaerobe. Anthrax mainly affects animals such as sheep, goats, cattle, and deer, but can be found in humans as well. Sometimes called wool sorter’s disease, it is often transmitted to humans through contact with infected animals or animal products, such as wool or hides. However, exposure to B. anthracis can occur by other means, as the endospores are widespread in soils and can survive for long periods of time, sometimes for hundreds of years.
The vast majority of anthrax cases (95–99%) occur when anthrax endospores enter the body through abrasions of the skin.3 This form of the disease is called cutaneous anthrax. It is characterized by the formation of a nodule on the skin; the cells within the nodule die, forming a black eschar, a mass of dead skin tissue (Figure \(11\)). The localized infection can eventually lead to bacteremia and septicemia. If untreated, cutaneous anthrax can cause death in 20% of patients.4 Once in the skin tissues, B. anthracis endospores germinate and produce a capsule, which prevents the bacteria from being phagocytized, and two binary exotoxins that cause edema and tissue damage. The first of the two exotoxins consists of a combination of protective antigen (PA) and an enzymatic lethal factor (LF), forming lethal toxin (LeTX). The second consists of protective antigen (PA) and an edema factor (EF), forming edema toxin (EdTX).
Less commonly, anthrax infections can be initiated through other portals of entry such as the digestive tract (gastrointestinal anthrax) or respiratory tract (pulmonary anthrax or inhalation anthrax). Typically, cases of noncutaneous anthrax are more difficult to treat than the cutaneous form. The mortality rate for gastrointestinal anthrax can be up to 40%, even with treatment. Inhalation anthrax, which occurs when anthrax spores are inhaled, initially causes influenza-like symptoms, but mortality rates are approximately 45% in treated individuals and 85% in those not treated. A relatively new form of the disease, injection anthrax, has been reported in Europe in intravenous drug users; it occurs when drugs are contaminated with B. anthracis. Patients with injection anthrax show signs and symptoms of severe soft tissue infection that differ clinically from cutaneous anthrax. This often delays diagnosis and treatment, and leads to a high mortality rate.5
B. anthracis colonies on blood agar have a rough texture and serrated edges that eventually form an undulating band (Figure \(11\)). Broad spectrum antibiotics such as penicillin, erythromycin, and tetracycline are often effective treatments.
Unfortunately, B. anthracis has been used as a biological weapon and remains on the United Nations’ list of potential agents of bioterrorism.6 Over a period of several months in 2001, a number of letters were mailed to members of the news media and the United States Congress. As a result, 11 individuals developed cutaneous anthrax and another 11 developed inhalation anthrax. Those infected included recipients of the letters, postal workers, and two other individuals. Five of those infected with pulmonary anthrax died. The anthrax spores had been carefully prepared to aerosolize, showing that the perpetrator had a high level of expertise in microbiology.7
A vaccine is available to protect individuals from anthrax. However, unlike most routine vaccines, the current anthrax vaccine is unique in both its formulation and the protocols dictating who receives it.8 The vaccine is administered through five intramuscular injections over a period of 18 months, followed by annual boosters. The US Food and Drug Administration (FDA) has only approved administration of the vaccine prior to exposure for at-risk adults, such as individuals who work with anthrax in a laboratory, some individuals who handle animals or animal products (e.g., some veterinarians), and some members of the United States military. The vaccine protects against cutaneous and inhalation anthrax using cell-free filtrates of microaerophilic cultures of an avirulent, nonencapsulated strain of B. anthracis.9 The FDA has not approved the vaccine for routine use after exposure to anthrax, but if there were ever an anthrax emergency in the United States, patients could be given anthrax vaccine after exposure to help prevent disease.
Exercise \(6\)
What is the characteristic feature of a cutaneous anthrax infection?
Bacterial Infections of the Skin
Bacterial infections of the skin can cause a wide range of symptoms and syndromes, ranging from the superficial and relatively harmless to the severe and even fatal. Most bacterial skin infections can be diagnosed by culturing the bacteria and treated with antibiotics. Antimicrobial susceptibility testing is also often necessary because many strains of bacteria have developed antibiotic resistance. Figure \(12\) summarizes the characteristics of some common bacterial skin infections.
Bacterial Conjunctivitis
Like the skin, the surface of the eye comes in contact with the outside world and is somewhat prone to infection by bacteria in the environment. Bacterial conjunctivitis (pinkeye) is a condition characterized by inflammation of the conjunctiva, often accompanied by a discharge of sticky fluid (described as acute purulent conjunctivitis) (Figure \(13\)). Conjunctivitis can affect one eye or both, and it usually does not affect vision permanently. Bacterial conjunctivitis is most commonly caused by Haemophilus influenzae, but can also be caused by other species such as Moraxella catarrhalis, S. pneumoniae, and S. aureus. The causative agent may be identified using bacterial cultures, Gram stain, and diagnostic biochemical, antigenic, or nucleic acid profile tests of the isolated pathogen. Bacterial conjunctivitis is very contagious, being transmitted via secretions from infected individuals, but it is also self-limiting. Bacterial conjunctivitis usually resolves in a few days, but topical antibiotics are sometimes prescribed. Because this condition is so contagious, medical attention is recommended whenever it is suspected. Individuals who use contact lenses should discontinue their use when conjunctivitis is suspected. Certain symptoms, such as blurred vision, eye pain, and light sensitivity, can be associated with serious conditions and require medical attention.
Neonatal Conjunctivitis
Newborns whose mothers have certain sexually transmitted infections are at risk of contracting ophthalmia neonatorum or inclusion conjunctivitis, which are two forms of neonatal conjunctivitis contracted through exposure to pathogens during passage through the birth canal. Gonococcal ophthalmia neonatorum is caused by Neisseria gonorrhoeae, the bacterium that causes the STD gonorrhea (Figure \(14\)). Inclusion (chlamydial) conjunctivitis is caused by Chlamydia trachomatis, the anaerobic, obligate, intracellular parasite that causes the STD chlamydia.
To prevent gonoccocal ophthalmia neonatorum, silver nitrate ointments were once routinely applied to all infants’ eyes shortly after birth; however, it is now more common to apply antibacterial creams or drops, such as erythromycin. Most hospitals are required by law to provide this preventative treatment to all infants, because conjunctivitis caused by N. gonorrhoeae, C. trachomatis, or other bacteria acquired during a vaginal delivery can have serious complications. If untreated, the infection can spread to the cornea, resulting in ulceration or perforation that can cause vision loss or even permanent blindness. As such, neonatal conjunctivitis is treated aggressively with oral or intravenous antibiotics to stop the spread of the infection. Causative agents of inclusion conjunctivitis may be identified using bacterial cultures, Gram stain, and diagnostic biochemical, antigenic, or nucleic acid profile tests.
Exercise \(7\)
Compare and contrast bacterial conjunctivitis with neonatal conjunctivitis.
Trachoma
Trachoma, or granular conjunctivitis, is a common cause of preventable blindness that is rare in the United States but widespread in developing countries, especially in Africa and Asia. The condition is caused by the same species that causes neonatal inclusion conjunctivitis in infants, Chlamydia trachomatis. C. trachomatis can be transmitted easily through fomites such as contaminated towels, bed linens, and clothing and also by direct contact with infected individuals. C. trachomatis can also be spread by flies that transfer infected mucous containing C. trachomatis from one human to another.
Infection by C. trachomatis causes chronic conjunctivitis, which leads to the formation of necrotic follicles and scarring in the upper eyelid. The scars turn the eyelashes inward (a condition known as trichiasis) and mechanical abrasion of the cornea leads to blindness (Figure \(15\)). Antibiotics such as azithromycin are effective in treating trachoma, and outcomes are good when the disease is treated promptly. In areas where this disease is common, large public health efforts are focused on reducing transmission by teaching people how to avoid the risks of the infection.
Exercise \(8\)
Why is trachoma rare in the United States?
SAFE Eradication of Trachoma
Though uncommon in the United States and other developed nations, trachoma is the leading cause of preventable blindness worldwide, with more than 4 million people at immediate risk of blindness from trichiasis. The vast majority of those affected by trachoma live in Africa and the Middle East in isolated rural or desert communities with limited access to clean water and sanitation. These conditions provide an environment conducive to the growth and spread of Chlamydia trachomatis, the bacterium that causes trachoma, via wastewater and eye-seeking flies.
In response to this crisis, recent years have seen major public health efforts aimed at treating and preventing trachoma. The Alliance for Global Elimination of Trachoma by 2020 (GET 2020), coordinated by the World Health Organization (WHO), promotes an initiative dubbed “SAFE,” which stands for “Surgery, Antibiotics, Facial cleanliness, and Environmental improvement.” The Carter Center, a charitable, nongovernment organization led by former US President Jimmy Carter, has partnered with the WHO to promote the SAFE initiative in six of the most critically impacted nations in Africa. Through its Trachoma Control Program, the Carter Center trains and equips local surgeons to correct trichiasis and distributes antibiotics to treat trachoma. The program also promotes better personal hygiene through health education and improves sanitation by funding the construction of household latrines. This reduces the prevalence of open sewage, which provides breeding grounds for the flies that spread trachoma.
Bacterial Keratitis
Keratitis can have many causes, but bacterial keratitis is most frequently caused by Staphylococcus epidermidisand/or Pseudomonas aeruginosa. Contact lens users are particularly at risk for such an infection because S. epidermidis and P. aeruginosa both adhere well to the surface of the lenses. Risk of infection can be greatly reduced by proper care of contact lenses and avoiding wearing lenses overnight. Because the infection can quickly lead to blindness, prompt and aggressive treatment with antibiotics is important. The causative agent may be identified using bacterial cultures, Gram stain, and diagnostic biochemical, antigenic, or nucleic acid profile tests of the isolated pathogen.
Exercise \(9\)
Why are contact lens wearers at greater risk for developing keratitis?
Biofilms and Infections of the Skin and Eyes
When treating bacterial infections of the skin and eyes, it is important to consider that few such infections can be attributed to a single pathogen. While biofilms may develop in other parts of the body, they are especially relevant to skin infections (such as those caused by S. aureus or P. aeruginosa) because of their prevalence in chronic skin wounds. Biofilms develop when bacteria (and sometimes fungi) attach to a surface and produce extracellular polymeric substances (EPS) in which cells of multiple organisms may be embedded. When a biofilm develops on a wound, it may interfere with the natural healing process as well as diagnosis and treatment.
Because biofilms vary in composition and are difficult to replicate in the lab, they are still not thoroughly understood. The extracellular matrix of a biofilm consists of polymers such as polysaccharides, extracellular DNA, proteins, and lipids, but the exact makeup varies. The organisms living within the extracellular matrix may include familiar pathogens as well as other bacteria that do not grow well in cultures (such as numerous obligate anaerobes). This presents challenges when culturing samples from infections that involve a biofilm. Because only some species grow in vitro, the culture may contain only a subset of the bacterial species involved in the infection.
Biofilms confer many advantages to the resident bacteria. For example, biofilms can facilitate attachment to surfaces on or in the host organism (such as wounds), inhibit phagocytosis, prevent the invasion of neutrophils, and sequester host antibodies. Additionally, biofilms can provide a level of antibiotic resistance not found in the isolated cells and colonies that are typical of laboratory cultures. The extracellular matrix provides a physical barrier to antibiotics, shielding the target cells from exposure. Moreover, cells within a biofilm may differentiate to create subpopulations of dormant cells called persister cells. Nutrient limitations deep within a biofilm add another level of resistance, as stress responses can slow metabolism and increase drug resistance.
Bacterial Infections of the Eyes
A number of bacteria are able to cause infection when introduced to the mucosa of the eye. In general, bacterial eye infections can lead to inflammation, irritation, and discharge, but they vary in severity. Some are typically short-lived, and others can become chronic and lead to permanent eye damage. Prevention requires limiting exposure to contagious pathogens. When infections do occur, prompt treatment with antibiotics can often limit or prevent permanent damage. Figure \(16\)summarizes the characteristics of some common bacterial infections of the eyes.
Key Concepts and Summary
• Staphylococcus and Streptococcus cause many different types of skin infections, many of which occur when bacteria breach the skin barrier through a cut or wound.
• S. aureus are frequently associated with purulent skin infections that manifest as folliculitis, furuncles, or carbuncles. S. aureus is also a leading cause of staphylococcal scalded skin syndrome (SSSS).
• S. aureus is generally drug resistant and current MRSA strains are resistant to a wide range of antibiotics.
• Community-acquired and hospital-acquired staphyloccocal infections are an ongoing problem because many people are asymptomatic carriers.
• Group A streptococci (GAS), S. pyogenes, is often responsible for cases of cellulitis, erysipelas, and erythema nosodum. GAS are also one of many possible causes of necrotizing fasciitis.
• P. aeruginosa is often responsible for infections of the skin and eyes, including wound and burn infections, hot tub rash, otitis externa, and bacterial keratitis.
• Acne is a common skin condition that can become more inflammatory when Propionibacterium acnes infects hair follicles and pores clogged with dead skin cells and sebum.
• Cutaneous anthrax occurs when Bacillus anthracis breaches the skin barrier. The infection results in a localized black eschar on skin. Anthrax can be fatal if B. anthracis spreads to the bloodstream.
• Common bacterial conjunctivitis is often caused by Haemophilus influenzae and usually resolves on its own in a few days. More serious forms of conjunctivitis include gonococcal ophthalmia neonatorum, inclusion conjunctivitis (chlamydial), and trachoma, all of which can lead to blindness if untreated.
• Keratitis is frequently caused by Staphylococcus epidermidis and/or Pseudomonas aeruginosa, especially among contact lens users, and can lead to blindness.
• Biofilms complicate the treatment of wound and eye infections because pathogens living in biofilms can be difficult to treat and eliminate.
Footnotes
1. 1 Starr, C.R. and Engelberg N.C. “Role of Hyaluronidase in Subcutaneous Spread and Growth of Group A Streptococcus.” Infection and Immunity 2006(7:1): 40–48. doi: 10.1128/IAI.74.1.40-48.2006.
2. 2 Nuwayhid, Z.B., Aronoff, D.M., and Mulla, Z.D.. “Blunt Trauma as a Risk Factor for Group A Streptococcal Necrotizing Fasciitis.” Annals of Epidemiology (2007) 17:878–881.
3. 3 Shadomy, S.V., Traxler, R.M., and Marston, C.K. “Infectious Diseases Related to Travel: Anthrax” 2015. Centers for Disease Control and Prevention. wwwnc.cdc.gov/travel/yellowbo...travel/anthrax. Accessed Sept 14, 2016.
4. 4 US FDA. “Anthrax.” 2015. www.fda.gov/BiologicsBloodVac.../ucm061751.htm. Accessed Sept 14, 2016.
5. 5 Berger, T., Kassirer, M., and Aran, A.A.. “Injectional Anthrax—New Presentation of an Old Disease.” Euro Surveillance 19 (2014) 32. http://www.ncbi.nlm.nih.gov/pubmed/25139073. Accessed Sept 14, 2016.
6. 6 United Nations Office at Geneva. “What Are Biological and Toxin Weapons?” http://www.unog.ch/80256EE600585943/...2571860035A6DB?. Accessed Sept 14, 2016.
7. 7 Federal Bureau of Investigation. “Famous Cases and Criminals: Amerithrax or Anthrax Investigation.” www.fbi.gov/history/famous-c...-investigation. Accessed Sept 14, 2016.
8. 8 Centers for Disease Control and Prevention. “Anthrax: Medical Care: Prevention: Antibiotics.” www.cdc.gov/anthrax/medical-c...revention.html. Accessed Sept 14, 2016.
9. 9 Emergent Biosolutions. AVA (BioThrax) vaccine package insert (Draft). Nov 2015. www.fda.gov/downloads/biologi...ductsblas/ucm0 | textbooks/bio/Microbiology/Microbiology_(OpenStax)/21%3A_Skin_and_Eye_Infections/21.02%3A_Bacterial_Infections_of_the_Skin_and_Eyes.txt |
Learning Objectives
• Identify the most common viruses associated with infections of the skin and eyes
• Compare the major characteristics of specific viral diseases affecting the skin and eyes
Until recently, it was thought that the normal microbiota of the body consisted primarily of bacteria and some fungi. However, in addition to bacteria, the skin is colonized by viruses, and recent studies suggest that Papillomaviridae, Polyomaviridae and Circoviridae also contribute to the normal skin microbiota. However, some viruses associated with skin are pathogenic, and these viruses can cause diseases with a wide variety of presentations.
Numerous types of viral infections cause rashes or lesions on the skin; however, in many cases these skin conditions result from infections that originate in other body systems. In this chapter, we will limit the discussion to viral skin infections that use the skin as a portal of entry. Later chapters will discuss viral infections such as chickenpox, measles, and rubella—diseases that cause skin rashes but invade the body through portals of entry other than the skin.
Papillomas
Papillomas (warts) are the expression of common skin infections by human papillomavirus (HPV) and are transmitted by direct contact. There are many types of HPV, and they lead to a variety of different presentations, such as common warts, plantar warts, flat warts, and filiform warts. HPV can also cause sexually-transmitted genital warts, which will be discussed in Urogenital System Infections. Vaccination is available for some strains of HPV.
Common warts tend to develop on fingers, the backs of hands, and around nails in areas with broken skin. In contrast, plantar warts (also called foot warts) develop on the sole of the foot and can grow inwards, causing pain and pressure during walking. Flat warts can develop anywhere on the body, are often numerous, and are relatively smooth and small compared with other wart types. Filiform warts are long, threadlike warts that grow quickly.
In some cases, the immune system may be strong enough to prevent warts from forming or to eradicate established warts. However, treatment of established warts is typically required. There are many available treatments for warts, and their effectiveness varies. Common warts can be frozen off with liquid nitrogen. Topical applications of salicylic acidmay also be effective. Other options are electrosurgery (burning), curettage (cutting), excision, painting with cantharidin (which causes the wart to die so it can more easily be removed), laser treatments, treatment with bleomycin, chemical peels, and immunotherapy (Figure \(1\)).
Oral Herpes
Another common skin virus is herpes simplex virus (HSV). HSV has historically been divided into two types, HSV-1 and HSV-2. HSV-1 is typically transmitted by direct oral contact between individuals, and is usually associated with oral herpes. HSV-2 is usually transmitted sexually and is typically associated with genital herpes. However, both HSV-1 and HSV-2 are capable of infecting any mucous membrane, and the incidence of genital HSV-1 and oral HSV-2 infections has been increasing in recent years. In this chapter, we will limit our discussion to infections caused by HSV-1; HSV-2 and genital herpes will be discussed in Urogenital System Infections.
Infection by HSV-1 commonly manifests as cold sores or fever blisters, usually on or around the lips (Figure \(2\)). HSV-1 is highly contagious, with some studies suggesting that up to 65% of the US population is infected; however, many infected individuals are asymptomatic.1 Moreover, the virus can be latent for long periods, residing in the trigeminal nerve ganglia between recurring bouts of symptoms. Recurrence can be triggered by stress or environmental conditions (systemic or affecting the skin). When lesions are present, they may blister, break open, and crust. The virus can be spread through direct contact, even when a patient is asymptomatic.
While the lips, mouth, and face are the most common sites for HSV-1 infections, lesions can spread to other areas of the body. Wrestlers and other athletes involved in contact sports may develop lesions on the neck, shoulders, and trunk. This condition is often called herpes gladiatorum. Herpes lesions that develop on the fingers are often called herpetic whitlow.
HSV-1 infections are commonly diagnosed from their appearance, although laboratory testing can confirm the diagnosis. There is no cure, but antiviral medications such as acyclovir, penciclovir, famciclovir, and valacyclovir are used to reduce symptoms and risk of transmission. Topical medications, such as creams with n-docosanol and penciclovir, can also be used to reduce symptoms such as itching, burning, and tingling.
Exercise \(1\)
What are the most common sites for the appearance of herpetic lesions?
Roseola and Fifth Disease
The viral diseases roseola and fifth disease are somewhat similar in terms of their presentation, but they are caused by different viruses. Roseola, sometimes called roseola infantum or exanthem subitum (“sudden rash”), is a mild viral infection usually caused by human herpesvirus-6 (HHV-6) and occasionally by HHV-7. It is spread via direct contact with the saliva or respiratory secretions of an infected individual, often through droplet aerosols. Roseola is very common in children, with symptoms including a runny nose, a sore throat, and a cough, along with (or followed by) a high fever (39.4 ºC). About three to five days after the fever subsides, a rash may begin to appear on the chest and abdomen. The rash, which does not cause discomfort, initially forms characteristic macules that are flat or papules that are firm and slightly raised; some macules or papules may be surrounded by a white ring. The rash may eventually spread to the neck and arms, and sometimes continues to spread to the face and legs. The diagnosis is generally made based upon observation of the symptoms. However, it is possible to perform serological tests to confirm the diagnosis. While treatment may be recommended to control the fever, the disease usually resolves without treatment within a week after the fever develops. For individuals at particular risk, such as those who are immunocompromised, the antiviral medication ganciclovir may be used.
Fifth disease (also known as erythema infectiosum) is another common, highly contagious illness that causes a distinct rash that is critical to diagnosis. Fifth disease is caused by parvovirus B19, and is transmitted by contact with respiratory secretions from an infected individual. Infection is more common in children than adults. While approximately 20% of individuals will be asymptomatic during infection,2 others will exhibit cold-like symptoms (headache, fever, and upset stomach) during the early stages when the illness is most infectious. Several days later, a distinct red facial rash appears, often called “slapped cheek” rash (Figure \(3\)). Within a few days, a second rash may appear on the arms, legs, chest, back, or buttocks. The rash may come and go for several weeks, but usually disappears within seven to twenty-one days, gradually becoming lacy in appearance as it recedes.
In children, the disease usually resolves on its own without medical treatment beyond symptom relief as needed. Adults may experience different and possibly more serious symptoms. Many adults with fifth disease do not develop any rash, but may experience joint pain and swelling that lasts several weeks or months. Immunocompromised individuals can develop severe anemia and may need blood transfusions or immune globulin injections. While the rash is the most important component of diagnosis (especially in children), the symptoms of fifth disease are not always consistent. Serological testing can be conducted for confirmation.
Exercise \(2\)
Identify at least one similarity and one difference between roseola and fifth disease.
Viral Conjunctivitis
Like bacterial conjunctivitis viral infections of the eye can cause inflammation of the conjunctiva and discharge from the eye. However, viral conjunctivitis tends to produce a discharge that is more watery than the thick discharge associated with bacterial conjunctivitis. The infection is contagious and can easily spread from one eye to the other or to other individuals through contact with eye discharge.
Viral conjunctivitis is commonly associated with colds caused by adenoviruses; however, other viruses can also cause conjunctivitis. If the causative agent is uncertain, eye discharge can be tested to aid in diagnosis. Antibiotic treatment of viral conjunctivitis is ineffective, and symptoms usually resolve without treatment within a week or two.
Herpes Keratitis
Herpes infections caused by HSV-1 can sometimes spread to the eye from other areas of the body, which may result in keratoconjunctivitis. This condition, generally called herpes keratitis or herpetic keratitis, affects the conjunctiva and cornea, causing irritation, excess tears, and sensitivity to light. Deep lesions in the cornea may eventually form, leading to blindness. Because keratitis can have numerous causes, laboratory testing is necessary to confirm the diagnosis when HSV-1 is suspected; once confirmed, antiviral medications may be prescribed.
Viral Infections of the Skin and Eyes
A number of viruses can cause infections via direct contact with skin and eyes, causing signs and symptoms ranging from rashes and lesions to warts and conjunctivitis. All of these viral diseases are contagious, and while some are more common in children (fifth disease and roseola), others are prevalent in people of all ages (oral herpes, viral conjunctivitis, papillomas). In general, the best means of prevention is avoiding contact with infected individuals. Treatment may require antiviral medications; however, several of these conditions are mild and typically resolve without treatment. Figure \(4\) summarizes the characteristics of some common viral infections of the skin and eyes.
Key Concepts and Summary
• Papillomas (warts) are caused by human papillomaviruses.
• Herpes simplex virus (especially HSV-1) mainly causes oral herpes, but lesions can appear on other areas of the skin and mucous membranes.
• Roseola and fifth disease are common viral illnesses that cause skin rashes; roseola is caused by HHV-6 and HHV-7 while fifth disease is caused by parvovirus 19.
• Viral conjunctivitis is often caused by adenoviruses and may be associated with the common cold. Herpes keratitis is caused by herpesviruses that spread to the eye.
Footnotes
1. 1 Wald, A., and Corey, L. “Persistence in the Population: Epidemiology, Transmission.” In: A. Arvin, G. Campadelli-Fiume, E. Mocarski et al. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press, 2007. www.ncbi.nlm.nih.gov/books/NBK47447/. Accessed Sept 14, 2016.
2. 2 Centers for Disease Control and Prevention. “Fifth Disease.” http://www.cdc.gov/parvovirusb19/fifth-disease.html. Accessed Sept 14, 2016. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/21%3A_Skin_and_Eye_Infections/21.03%3A_Viral_Infections_of_the_Skin_and_Eyes.txt |
Learning Objectives
• Identify the most common fungal pathogens associated with cutaneous and subcutaneous mycoses
• Compare the major characteristics of specific fungal diseases affecting the skin
Many fungal infections of the skin involve fungi that are found in the normal skin microbiota. Some of these fungi can cause infection when they gain entry through a wound; others mainly cause opportunistic infections in immunocompromised patients. Other fungal pathogens primarily cause infection in unusually moist environments that promote fungal growth; for example, sweaty shoes, communal showers, and locker rooms provide excellent breeding grounds that promote the growth and transmission of fungal pathogens.
Fungal infections, also called mycoses, can be divided into classes based on their invasiveness. Mycoses that cause superficial infections of the epidermis, hair, and nails, are called cutaneous mycoses. Mycoses that penetrate the epidermis and the dermis to infect deeper tissues are called subcutaneous mycoses. Mycoses that spread throughout the body are called systemic mycoses.
Tineas
A group of cutaneous mycoses called tineas are caused by dermatophytes, fungal molds that require keratin, a protein found in skin, hair, and nails, for growth. There are three genera of dermatophytes, all of which can cause cutaneous mycoses: Trichophyton, Epidermophyton, and Microsporum. Tineas on most areas of the body are generally called ringworm, but tineas in specific locations may have distinctive names and symptoms (see Table \(1\) and Figure \(1\)). Keep in mind that these names—even though they are Latinized—refer to locations on the body, not causative organisms. Tineas can be caused by different dermatophytes in most areas of the body.
Table \(1\): Tineas and locations
Some Common Tineas and Location on the Body
Tinea corporis (ringworm) Body
Tinea capitis (ringworm) Scalp
Tinea pedis (athlete’s foot) Feet
Tinea barbae (barber’s itch) Beard
Tinea cruris (jock itch) Groin
Tinea unguium (onychomycosis) Toenails, fingernails
Dermatophytes are commonly found in the environment and in soils and are frequently transferred to the skin via contact with other humans and animals. Fungal spores can also spread on hair. Many dermatophytes grow well in moist, dark environments. For example, tinea pedis (athlete’s foot) commonly spreads in public showers, and the causative fungi grow well in the dark, moist confines of sweaty shoes and socks. Likewise, tinea cruris (jock itch) often spreads in communal living environments and thrives in warm, moist undergarments.
Tineas on the body (tinea corporis) often produce lesions that grow radially and heal towards the center. This causes the formation of a red ring, leading to the misleading name of ringworm recall the Clinical Focus case in The Eukaryotes of Microbiology.
Several approaches may be used to diagnose tineas. A Wood’s lamp (also called a black lamp) with a wavelength of 365 nm is often used. When directed on a tinea, the ultraviolet light emitted from the Wood’s lamp causes the fungal elements (spores and hyphae) to fluoresce. Direct microscopic evaluation of specimens from skin scrapings, hair, or nails can also be used to detect fungi. Generally, these specimens are prepared in a wet mount using a potassium hydroxide solution (10%–20% aqueous KOH), which dissolves the keratin in hair, nails, and skin cells to allow for visualization of the hyphae and fungal spores. The specimens may be grown on Sabouraud dextrose CC (chloramphenicol/cyclohexamide), a selective agar that supports dermatophyte growth while inhibiting the growth of bacteria and saprophytic fungi (Figure \(2\)). Macroscopic colony morphology is often used to initially identify the genus of the dermatophyte; identification can be further confirmed by visualizing the microscopic morphology using either a slide culture or a sticky tape prep stained with lactophenol cotton blue.
Various antifungal treatments can be effective against tineas. Allylamine ointments that include terbinafine are commonly used; miconazole and clotrimazole are also available for topical treatment, and griseofulvin is used orally.
Exercise \(1\)
Why are tineas, caused by fungal molds, often called ringworm?
Cutaneous Aspergillosis
Another cause of cutaneous mycoses is Aspergillus, a genus consisting of molds of many different species, some of which cause a condition called aspergillosis. Primary cutaneous aspergillosis, in which the infection begins in the skin, is rare but does occur. More common is secondary cutaneous aspergillosis, in which the infection begins in the respiratory system and disseminates systemically. Both primary and secondary cutaneous aspergillosis result in distinctive eschars that form at the site or sites of infection (Figure \(3\)). Pulmonary aspergillosis will be discussed more thoroughly in Respiratory Mycoses).
Primary cutaneous aspergillosis usually occurs at the site of an injury and is most often caused by Aspergillus fumigatus or Aspergillus flavus. It is usually reported in patients who have had an injury while working in an agricultural or outdoor environment. However, opportunistic infections can also occur in health-care settings, often at the site of intravenous catheters, venipuncture wounds, or in association with burns, surgical wounds, or occlusive dressing. After candidiasis, aspergillosis is the second most common hospital-acquired fungal infection and often occurs in immunocompromised patients, who are more vulnerable to opportunistic infections.
Cutaneous aspergillosis is diagnosed using patient history, culturing, histopathology using a skin biopsy. Treatment involves the use of antifungal medications such as voriconazole (preferred for invasive aspergillosis), itraconazole, and amphotericin B if itraconazole is not effective. For immunosuppressed individuals or burn patients, medication may be used and surgical or immunotherapy treatments may be needed.
Exercise \(2\)
Identify the sources of infection for primary and secondary cutaneous aspergillosis.
Candidiasis of the Skin and Nails
Candida albicans and other yeasts in the genus Candida can cause skin infections referred to as cutaneous candidiasis. Candida spp. are sometimes responsible for intertrigo, a general term for a rash that occurs in a skin fold, or other localized rashes on the skin. Candida can also infect the nails, causing them to become yellow and harden (Figure \(4\)).
Candidiasis of the skin and nails is diagnosed through clinical observation and through culture, Gram stain, and KOH wet mounts. Susceptibility testing for anti-fungal agents can also be done. Cutaneous candidiasis can be treated with topical or systemic azole antifungal medications. Because candidiasis can become invasive, patients suffering from HIV/AIDS, cancer, or other conditions that compromise the immune system may benefit from preventive treatment. Azoles, such as clotrimazole, econazole, fluconazole, ketoconazole, and miconazole; nystatin; terbinafine; and naftifine may be used for treatment. Long-term treatment with medications such as itraconazole or ketoconazole may be used for chronic infections. Repeat infections often occur, but this risk can be reduced by carefully following treatment recommendations, avoiding excessive moisture, maintaining good health, practicing good hygiene, and having appropriate clothing (including footwear).
Candida also causes infections in other parts of the body besides the skin. These include vaginal yeast infections (see Fungal Infections of the Reproductive System) and oral thrush (see Microbial Diseases of the Mouth and Oral Cavity).
Exercise \(3\)
What are the signs and symptoms of candidiasis of the skin and nails?
Sporotrichosis
Whereas cutaneous mycoses are superficial, subcutaneous mycoses can spread from the skin to deeper tissues. In temperate regions, the most common subcutaneous mycosis is a condition called sporotrichosis, caused by the fungus Sporothrix schenkii and commonly known as rose gardener’s disease or rose thorn disease (recall Case in Point: Every Rose Has Its Thorn). Sporotrichosis is often contracted after working with soil, plants, or timber, as the fungus can gain entry through a small wound such as a thorn-prick or splinter. Sporotrichosis can generally be avoided by wearing gloves and protective clothing while gardening and promptly cleaning and disinfecting any wounds sustained during outdoor activities.
Sporothrix infections initially present as small ulcers in the skin, but the fungus can spread to the lymphatic system and sometimes beyond. When the infection spreads, nodules appear, become necrotic, and may ulcerate. As more lymph nodes become affected, abscesses and ulceration may develop over a larger area (often on one arm or hand). In severe cases, the infection may spread more widely throughout the body, although this is relatively uncommon.
Sporothrix infection can be diagnosed based upon histologic examination of the affected tissue. Its macroscopic morphology can be observed by culturing the mold on potato dextrose agar, and its microscopic morphology can be observed by staining a slide culture with lactophenol cotton blue. Treatment with itraconazole is generally recommended.
Exercise \(4\)
Describe the progression of a Sporothrix schenkii infection.
Mycoses of the Skin
Cutaneous mycoses are typically opportunistic, only able to cause infection when the skin barrier is breached through a wound. Tineas are the exception, as the dermatophytes responsible for tineas are able to grow on skin, hair, and nails, especially in moist conditions. Most mycoses of the skin can be avoided through good hygiene and proper wound care. Treatment requires antifungal medications. Figure \(5\) summarizes the characteristics of some common fungal infections of the skin.
Key Concepts and Summary
• Mycoses can be cutaneous, subcutaneous, or systemic.
• Common cutaneous mycoses include tineas caused by dermatophytes of the genera Trichophyton, Epidermophyton, and Microsporum. Tinea corporis is called ringworm. Tineas on other parts of the body have names associated with the affected body part.
• Aspergillosis is a fungal disease caused by molds of the genus Aspergillus. Primary cutaneous aspergillosis enters through a break in the skin, such as the site of an injury or a surgical wound; it is a common hospital-acquired infection. In secondary cutaneous aspergillosis, the fungus enters via the respiratory system and disseminates systemically, manifesting in lesions on the skin.
• The most common subcutaneous mycosis is sporotrichosis (rose gardener’s disease), caused by Sporothrix schenkii.
• Yeasts of the genus Candida can cause opportunistic infections of the skin called candidiasis, producing intertrigo, localized rashes, or yellowing of the nails. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/21%3A_Skin_and_Eye_Infections/21.04%3A_Mycoses_of_the_Skin_and_Eyes.txt |
Learning Objectives
• Identify two parasites that commonly cause infections of the skin and eyes
• Identify the major characteristics of specific parasitic diseases affecting the skin and eyes
Many parasitic protozoans and helminths use the skin or eyes as a portal of entry. Some may physically burrow into the skin or the mucosa of the eye; others breach the skin barrier by means of an insect bite. Still others take advantage of a wound to bypass the skin barrier and enter the body, much like other opportunistic pathogens. Although many parasites enter the body through the skin, in this chapter we will limit our discussion to those for which the skin or eyes are the primary site of infection. Parasites that enter through the skin but travel to a different site of infection will be covered in other chapters. In addition, we will limit our discussion to microscopic parasitic infections of the skin and eyes. Macroscopic parasites such as lice, scabies, mites, and ticks are beyond the scope of this text.
Acanthamoeba Infections
Acanthamoeba is a genus of free-living protozoan amoebae that are common in soils and unchlorinated bodies of fresh water. (This is one reason why some swimming pools are treated with chlorine.) The genus contains a few parasitic species, some of which can cause infections of the eyes, skin, and nervous system. Such infections can sometimes travel and affect other body systems. Skin infections may manifest as abscesses, ulcers, and nodules. When acanthamoebae infect the eye, causing inflammation of the cornea, the condition is called Acanthamoeba keratitis. Figure \(1\) illustrates the Acanthamoeba life cycle and various modes of infection.
While Acanthamoeba keratitis is initially mild, it can lead to severe corneal damage, vision impairment, or even blindness if left untreated. Similar to eye infections involving P. aeruginosa, Acanthamoeba poses a much greater risk to wearers of contact lenses because the amoeba can thrive in the space between contact lenses and the cornea. Prevention through proper contact lens care is important. Lenses should always be properly disinfected prior to use, and should never be worn while swimming or using a hot tub.
Acanthamoeba can also enter the body through other pathways, including skin wounds and the respiratory tract. It usually does not cause disease except in immunocompromised individuals; however, in rare cases, the infection can spread to the nervous system, resulting in a usually fatal condition called granulomatous amoebic encephalitis (GAE) (see Fungal and Parasitic Diseases of the Nervous System). Disseminated infections, lesions, and Acanthamoeba keratitis can be diagnosed by observing symptoms and examining patient samples under the microscope to view the parasite. Skin biopsies may be used.
Acanthamoeba keratitis is difficult to treat, and prompt treatment is necessary to prevent the condition from progressing. The condition generally requires three to four weeks of intensive treatment to resolve. Common treatments include topical antiseptics (e.g., polyhexamethylene biguanide, chlorhexidine, or both), sometimes with painkillers or corticosteroids (although the latter are controversial because they suppress the immune system, which can worsen the infection). Azoles are sometimes prescribed as well. Advanced cases of keratitis may require a corneal transplant to prevent blindness.
Exercise \(1\)
How are Acanthamoeba infections acquired?
Loiasis
The helminth Loa loa, also known as the African eye worm, is a nematode that can cause loiasis, a disease endemic to West and Central Africa (Figure \(3\)). The disease does not occur outside that region except when carried by travelers. There is evidence that individual genetic differences affect susceptibility to developing loiasis after infection by the Loa loa worm. Even in areas in which Loa loa worms are common, the disease is generally found in less than 30% of the population.1 It has been suggested that travelers who spend time in the region may be somewhat more susceptible to developing symptoms than the native population, and the presentation of infection may differ.2
The parasite is spread by deerflies (genus Chrysops), which can ingest the larvae from an infected human via a blood meal (Figure \(3\)). When the deerfly bites other humans, it deposits the larvae into their bloodstreams. After about five months in the human body, some larvae develop into adult worms, which can grow to several centimeters in length and live for years in the subcutaneous tissue of the host.
The name “eye worm” alludes to the visible migration of worms across the conjunctiva of the eye. Adult worms live in the subcutaneous tissues and can travel at about 1 cm per hour. They can often be observed when migrating through the eye, and sometimes under the skin; in fact, this is generally how the disease is diagnosed. It is also possible to test for antibodies, but the presence of antibodies does not necessarily indicate a current infection; it only means that the individual was exposed at some time. Some patients are asymptomatic, but in others the migrating worms can cause fever and areas of allergic inflammation known as Calabar swellings. Worms migrating through the conjunctiva can cause temporary eye pain and itching, but generally there is no lasting damage to the eye. Some patients experience a range of other symptoms, such as widespread itching, hives, and joint and muscle pain.
Worms can be surgically removed from the eye or the skin, but this treatment only relieves discomfort; it does not cure the infection, which involves many worms. The preferred treatment is diethylcarbamazine, but this medication produces severe side effects in some individuals, such as brain inflammation and possible death in patients with heavy infections. Albendazole is also sometimes used if diethylcarbamazine is not appropriate or not successful. If left untreated for many years, loiasis can damage the kidneys, heart, and lungs, though these symptoms are rare.
Exercise \(2\)
Describe the most common way to diagnose loiasis.
Link to Learning
See a video of a live Loa loa microfilaria under the microscope.
Parasitic Skin and Eye Infections
The protozoan Acanthamoeba and the helminth Loa loa are two parasites capable of causing infections of the skin and eyes. Figure \(4\) summarizes the characteristics of some common fungal infections of the skin.
Key Concepts and Summary
The protozoan Acanthamoeba and the helminth Loa loa are two parasites that can breach the skin barrier, causing infections of the skin and eyes. Acanthamoeba keratitis is a parasitic infection of the eye that often results from improper disinfection of contact lenses or swimming while wearing contact lenses. Loiasis, or eye worm, is a disease endemic to Africa that is caused by parasitic worms that infect the subcutaneous tissue of the skin and eyes. It is transmitted by deerfly vectors.
Footnotes
1. 1 Garcia, A.. et al. “Genetic Epidemiology of Host Predisposition Microfilaraemia in Human Loiasis.” Tropical Medicine and International Health 4 (1999) 8:565–74. http://www.ncbi.nlm.nih.gov/pubmed/10499080. Accessed Sept 14, 2016.
2. 2 Spinello, A., et al. “Imported Loa loa Filariasis: Three Cases and a Review of Cases Reported in Non-Endemic Countries in the Past 25 Years.” International Journal of Infectious Disease 16 (2012) 9: e649–e662. DOI: http://dx.doi.org/10.1016/j.ijid.2012.05.1023. | textbooks/bio/Microbiology/Microbiology_(OpenStax)/21%3A_Skin_and_Eye_Infections/21.05%3A_Protozoan_and_Helminthic_Infections_of_the_Eyes.txt |
21.1: Anatomy and Normal Microbiota of the Skin and Eyes
Human skin consists of two main layers, the epidermis and dermis, which are situated on top of the hypodermis, a layer of connective tissue. The skin is an effective physical barrier against microbial invasion. The skin’s relatively dry environment and normal microbiota discourage colonization by transient microbes. The skin’s normal microbiota varies from one region of the body to another. The conjunctiva of the eye is a frequent site for microbial infection; deeper infections are less common.
Multiple Choice
_____________ glands produce a lipid-rich substance that contains proteins and minerals and protects the skin.
1. Sweat
2. Mammary
3. Sebaceous
4. Endocrine
Answer
C
Which layer of skin contains living cells, is vascularized, and lies directly above the hypodermis?
1. the stratum corneum
2. the dermis
3. the epidermis
4. the conjunctiva
Answer
B
Fill in the Blank
The ________ is the outermost layer of the epidermis.
Answer
stratum corneum
The mucous membrane that covers the surface of the eyeball and inner eyelid is called the ________.
Answer
conjunctiva
Short Answer
What is the role of keratin in the skin?
What are two ways in which tears help to prevent microbial colonization?
Which label indicates a sweat gland?
(credit: modification of work by National Cancer Institute)
Critical Thinking
Explain why it is important to understand the normal microbiota of the skin.
Besides the presence or absence of ulceration, how do acute ulcerative and nonulcerative blepharitis differ?
21.2: Bacterial Infections of the Skin and Eyes
Staphylococcus and Streptococcus cause many different types of skin infections, many of which occur when bacteria breach the skin barrier through a cut or wound. S. aureus are frequently associated with purulent skin infections that manifest as folliculitis, furuncles, or carbuncles. S. aureus is also a leading cause of staphylococcal scalded skin syndrome (SSSS). S. aureus is generally drug resistant and current MRSA strains are resistant to a wide range of antibiotics.
Multiple Choice
Staphylococcus aureus is most often associated with being
1. coagulase-positive.
2. coagulase-negative.
3. catalase-negative.
4. gram-negative
Answer
A
M protein is produced by
1. Pseudomonas aeruginosa
2. Staphylococcus aureus
3. Propionibacterium acnes
4. Streptococcus pyogenes
Answer
D
___________ is a major cause of preventable blindness that can be reduced through improved sanitation.
1. Ophthalmia neonatorum
2. Keratitis
3. Trachoma
4. Cutaneous anthrax
Answer
C
Which species is frequently associated with nosocomial infections transmitted via medical devices inserted into the body?
1. Staphylococcus epidermidis
2. Streptococcus pyogenes
3. Proproniobacterium acnes
4. Bacillus anthracis
Answer
A
Fill in the Blank
A purulent wound produces ________.
Answer
pus
Short Answer
How are leukocidins associated with pus production?
What is a good first test to distinguish streptococcal infections from staphylococcal infections?
21.3: Viral Infections of the Skin and Eyes
Papillomas (warts) are caused by human papillomaviruses. Herpes simplex virus (especially HSV-1) mainly causes oral herpes, but lesions can appear on other areas of the skin and mucous membranes. Roseola and fifth disease are common viral illnesses that cause skin rashes; roseola is caused by HHV-6 and HHV-7 while fifth disease is caused by parvovirus 19. Viral conjunctivitis is often caused by adenoviruses and may be associated with the common cold. Herpes keratitis is caused by herpesviruses.
Multiple Choice
Warts are caused by
1. human papillomavirus.
2. herpes simplex virus.
3. adenoviruses.
4. parvovirus B19.
Answer
A
Which of these viruses can spread to the eye to cause a form of keratitis?
1. human papillomavirus
2. herpes simplex virus 1
3. parvovirus 19
4. circoviruses
Answer
B
Cold sores are associated with:
1. human papillomavirus
2. roseola
3. herpes simplex viruses
4. human herpesvirus 6
Answer
C
Which disease is usually self-limiting but is most commonly treated with ganciclovir if medical treatment is needed?
1. roseola
2. oral herpes
3. papillomas
4. viral conjunctivitis
Answer
A
Adenoviruses can cause:
1. viral conjunctivitis
2. herpetic conjunctivitis
3. papillomas
4. oral herpes
Answer
A
Fill in the Blank
Human herpesvirus 6 is the causative agent of ________.
Answer
roseola
Short Answer
Compare and contrast bacterial and viral conjunctivitis.
21.4: Mycoses of the Skin and Eyes
Mycoses can be cutaneous, subcutaneous, or systemic. Common cutaneous mycoses include tineas caused by dermatophytes of the genera Trichophyton, Epidermophyton, and Microsporum. Tinea corporis is called ringworm. Tineas on other parts of the body have names associated with the affected body part. Aspergillosis is a fungal disease caused by molds of the genus Aspergillus. Primary cutaneous aspergillosis enters through a break in the skin, such as the site of an injury or a surgical wound.
Multiple Choice
___________ is a superficial fungal infection found on the head.
1. Tinea cruris
2. Tinea capitis
3. Tinea pedis
4. Tinea corporis
Answer
B
For what purpose would a health-care professional use a Wood’s lamp for a suspected case of ringworm?
1. to prevent the rash from spreading
2. to kill the fungus
3. to visualize the fungus
4. to examine the fungus microscopically
Answer
C
Sabouraud dextrose agar CC is selective for:
1. all fungi
2. non-saprophytic fungi
3. bacteria
4. viruses
Answer
B
The first-line recommended treatment for sporotrichosis is:
1. itraconazole
2. clindamycin
3. amphotericin
4. nystatin
Answer
A
Fill in the Blank
The most common subcutaneous mycosis in temperate regions is ________.
Answer
sporotrichosis
Short Answer
What yeasts commonly cause opportunistic infections?
Critical Thinking
What steps might you recommend to a patient for reducing the risk of developing a fungal infection of the toenails?
21.5: Protozoan and Helminthic Infections of the Eyes
The protozoan Acanthamoeba and the helminth Loa loa are two parasites that can breach the skin barrier, causing infections of the skin and eyes. Acanthamoeba keratitis is a parasitic infection of the eye that often results from improper disinfection of contact lenses or swimming while wearing contact lenses. Loiasis, or eye worm, is a disease endemic to Africa that is caused by parasitic worms that infect the subcutaneous tissue of the skin and eyes. It is transmitted by deerfly vectors.
Multiple Choice
Which of the following is most likely to cause an Acanthamoeba infection?
1. swimming in a lake while wearing contact lenses
2. being bitten by deerflies in Central Africa
3. living environments in a college dormitory with communal showers
4. participating in a contact sport such as wrestling
Answer
A
The parasitic Loa loa worm can cause great pain when it:
1. moves through the bloodstream
2. exits through the skin of the foot
3. travels through the conjunctiva
4. enters the digestive tract
Answer
C
A patient tests positive for Loa loa antibodies. What does this test indicate?
1. The individual was exposed to Loa loa at some point.
2. The individual is currently suffering from loiasis.
3. The individual has never been exposed to Loa loa.
4. The individual is immunosuppressed.
Answer
A
________ is commonly treated with a combination of chlorhexidine and polyhexamethylene biguanide.
1. Acanthamoeba keratitis
2. Sporotrichosis
3. Candidiasis
4. Loiasis
Answer
A
Fill in the Blank
Eye worm is another name for ________.
Answer
loiasis
The ________ is the part of the eye that is damaged due to Acanthamoeba keratitis.
Answer
cornea
Critical Thinking
Why might a traveler to a region with Loa loa worm have a greater risk of serious infection compared with people who live in the region?
What preventative actions might you recommend to a patient traveling to a region where loiasis is endemic? | textbooks/bio/Microbiology/Microbiology_(OpenStax)/21%3A_Skin_and_Eye_Infections/21.E%3A_Skin_and_Eye_Infections_%28Exercises%29.txt |
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