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25 Microbial Infection and Pathogenesis I Human–Pathogen Interactions 851 II Enzymes and Toxins of Pathogenesis 858...

25 Microbial Infection and Pathogenesis I Human–Pathogen Interactions 851 II Enzymes and Toxins of Pathogenesis 858 MICROBIOLOGYNOW Killing Pathogens on Contact The adherence of pathogenic bacteria to surfaces plays a animal and plant tissue surfaces exhibit antibacterial proper- crucial role in their ability to colonize areas of the body and ties, including shark skin, insect wings, and lotus leaves. cause disease. In addition to natural tissue surfaces in the Such natural surfaces are composed of nanostructures that body, such as tooth enamel and mucous membranes, kill bacteria on contact, likely by disrupting the integrity of their pathogens can also adhere to artificial surfaces associated cytoplasmic membranes. Borrowing this concept, antibacte- with implanted medical devices, including catheters, pros- rial nanofabrication methods have centered on titanium thetic heart valves, and artificial joints. The adherence of dioxide (TiO2) as a potential substrate to mimic natural bacteria to these sites can lead to the formation of biofilms, contact killing. Although TiO2 has shown limited potential for which are notoriously difficult to treat using antibiotics. killing gram-positive cocci, such as Staphylococcus aureus Because of the tendency of pathogenic bacteria to (bottom photo), it has shown considerable promise in destroy- colonize artificial surfaces in the body, prosthetic joint infec- ing gram-negative bacteria on contact, including Escherichia tions (PJIs) are a constant threat in orthopedic medicine. coli, in which the spiky nanostructures cause indentation and Considering the ineffectiveness of antibiotics in treating eventual puncture of the cells (arrow, top photo). these infections, successful treatment of PJIs often requires If artificial joints and other medical implants can be man- physical removal of the colonized biofilm from the prosthesis ufactured with surfaces having antibacterial nanostructures, and surrounding tissues in a surgical procedure called PJIs and other biofilm-associated infections may become debridement. To avoid the complications of treating infection, much easier to prevent and control—no antibiotics can we find ways to prevent the formation of biofilms that necessary. lead to PJIs in the first place? Source: Jenkins, J., et al. 2018. Characterization of bactericidal tita- Taking a lesson from nature itself, researchers are turning nium surfaces using electron microscopy. Microsc. Anal. (Am. Ed.). to the field of biomimetics to address these issues. Several 32: 15. 850 Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 850 18/03/21 18:54  CHAPTER 25 Microbial Infection and Pathogenesis  851 I Human–Pathogen Staphylococcus aureus cell Interactions T o induce pathogenesis and begin to parasitize the host, pathogenic microbes must first adhere to host tissues and SdrG adhesin USDA Agricultural Research Service overcome immune defenses that seek to inhibit microbial N2 Fibrinogen colonization and invasion. coating Substrate N3 H umans are exposed to microorganisms of all sorts in their envi- ronment. Whether one is walking outdoors, sitting indoors, or participating in any type of physical activity, environmental microbes and humans interact. The human body is also a natural home to enormous numbers of microorganisms, as we saw in Chap- (a) (b) ter 24. Most of these are harmless, and in fact essential, with only a small percentage capable of causing disease. However, those that do, Figure 25.2 Bacterial adherence. A bacterial pathogen attaches specifically to host tissues by way of complementary receptors on the bacterial and host surfaces. along with pathogens that are not part of the normal human micro- (a) Cryogenic transmission electron micrograph of the gram-positive bacterium biota, possess specific traits that underlie their pathogenic lifestyles. Staphylococcus aureus adhering to an extracellular matrix of fibrinogen, which the A major focus of this chapter will be a consideration of these specific cells convert to a fibrin clot as a defense against host immune responses (see also traits and how they induce the diseased state. Figure 25.12b). The S. aureus cells are about 0.75 mm in diameter. (b) Expanded dia- gram of S. aureus binding fibrinogen using the N2 and N3 domains of its SdrG adhe- 25.1 Microbial Adherence sin protein. In the world of infectious diseases, the term infection is used to imply the growth of microorganisms on or in the host, whereas the sort or another. These include mucous membranes, the skin surface, term disease is reserved for actual tissue damage or injury that or under mucous membranes or the skin during penetration of these impairs host function. If a pathogen gains access to the specific tis- sites from puncture wounds, insect bites, cuts, or other abrasions. sues it infects, disease will occur only if it first adheres to those tis- The portal of entry may be critical for the establishment of an infec- sues, multiplies to yield many cells or viral particles, and then tion because a pathogen that gains access to incompatible tissues is proceeds to damage tissues (or the entire organism) by the release typically ineffective. For example, if cells of the bacterium Streptococ- of toxic or invasive substances (Figure 25.1). Adherence is the first cus pneumoniae are swallowed, they will be killed by the strong acid- Mastering Microbiology step, and although adherence is required to initiate disease, it is not ity of the stomach, whereas if the same cells reach the respiratory Art Activity: sufficient to initiate disease because the host has many innate tract, they could trigger a fatal case of pneumonia. Figure 25.1 Microbial defenses that can thwart infection; we consider these in Chapter 26. Receptor molecules coating the surfaces of both the pathogen and pathogenesis cells of its host are often critical for adhering the pathogen to host Adherence Molecules tissues. Specific receptors can be important for the binding of any Pathogens typically adhere to epithelial cells through specific inter- type of pathogenic microbe, including bacteria, viruses, and para- actions between molecules on the pathogen and molecules on the sites (Figure 25.2). Receptors on pathogens have evolved to bind spe- host tissues. In addition, pathogens may adhere to each other, form- cifically to complementary molecules in the host, and the ing biofilms (◀ Sections 4.9 and 20.4), with the biofilm itself adher- complementary nature of the receptors on pathogen and host cells ing to specific tissues. In medical microbiology, adherence is the alerts the pathogen that it has arrived on a suitable infection site. Receptors on the pathogen surface are called adhesins and are com- enhanced ability of a microorganism to attach to a cell or a surface. 6 UNIT Pathogens gain access to host tissues by way of a portal of entry of one posed of glycoprotein or lipoprotein covalently bound to the outer The infection process The disease process TOXICITY EXPOSURE ADHERENCE INVASION MULTIPLICATION Toxin effects TISSUE OR to pathogens to skin or mucosa through epithelium Growth and production are local or SYSTEMIC DAMAGE of virulence factors systemic and toxins INVASIVENESS Further growth at original and distant sites Figure 25.1 Microbial pathogenesis. Following exposure to a pathogenic microbe, a series of events leads to infection, and a further series of events results in disease. Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 851 18/03/21 18:54  852   UNIT 6 MICROBE–HUMAN INTERACTIONS AND THE IMMUNE SYSTEM Capsule CDC/PHIL, M. Miller CDC/ Larry Stauffer, Oregon State PHL/PHIL (a) CDC/PHIL, Dr. Richard Facklam J.W. Costerton CDC/PHIL CDC/PHIL (b) (c) (a) (b) (c) Figure 25.4 Capsules and colonies of Streptococcus pneumoniae. (a) Figure 25.3 The bacterial capsule as a facilitator of pathogen attachment. Gram stain of S. pneumoniae cells; capsules are not visible. (b) S. pneumoniae (a) Bacillus anthracis growing on an agar plate. The mucoid colonies of encapsu- treated with anticapsular antibodies (Quellung reaction) that make the capsule lated cells are about 0.5 cm in diameter. (b) Light micrograph of cells of B. anthracis visible. (c) Colonies of encapsulated S. pneumoniae cells grown on blood agar growing in horse blood and stained to show the capsule (dark pink surrounding blue show a mucoid morphology with a sunken center. The colonies are about 2–3 cell). Cells are about 1 mm in diameter. (c) Cells of enteropathogenic Escherichia coli mm in diameter and a single cell of S. pneumoniae is about 0.8 mm in diameter. attached to the brush border of intestinal microvilli by way of a distinct capsule. The E. coli cells are about 0.5 mm in diameter. Many pathogens selectively adhere to particular types of cells through cell surface structures other than adhesins, capsules, or slime layer of the cell (Figure 25.2b). In addition to extracellular matrices, layers. For example, Neisseria gonorrhoeae, the pathogen that causes the such as the fibrinogen shown in Figure 25.2, host receptors for bac- sexually transmitted disease gonorrhea (▶ Section 31.13), adheres spe- terial adhesins include cell surface glycoproteins or complex mem- cifically to mucosal epithelial cells in the genitourinary tract, eye, rec- brane lipids, such as gangliosides or globosides (both are tum, and throat; by contrast, other tissues are not infected. sphingolipids containing sugars and other molecules). N. gonorrhoeae has a cell surface protein called Opa (opacity associated protein) that binds specifically to a host protein found only on the Adherence Structures: Capsules, Fimbriae, surface of epithelial cells of these body regions, allowing adherence of Pili, and Flagella the pathogen to host cells. Likewise, influenza virus targets upper respi- Some adhesins form part of an outer cell surface structure that may ratory tract mucosal cells and attaches specifically to these and later to or may not be covalently linked to components of the cell wall. For lung epithelial cells by way of the protein hemagglutinin present on example, some notable pathogenic bacteria form a capsule. In the virus surface (◀ Section 11.9 and ▶ Section 31.8). Bacillus anthracis (the bacterium that causes anthrax), the capsule is Fimbriae and pili are bacterial cell surface protein structures composed of polypeptide containing only the amino acid (◀ Section 2.6) that function in attachment (Figure 25.5). For instance, d-glutamate. The capsule of B. anthracis can be seen in cells by light along with Opa, the pili of Neisseria gonorrhoeae play a key role in microscopy, and the encapsulated cells form smooth, slimy colonies attachment to urogenital epithelia, and fimbriated strains of Esche- when grown on agar plates (Figure 25.3). The electron microscope richia coli are more frequent causes of urinary tract infections than can also clearly reveal bacterial capsules (Figure 25.3c). The capsule strains lacking fimbriae. Among the best-characterized fimbriae are 6 surface contains specific receptors that facilitate adherence to host the type I fimbriae of enteric bacteria (Escherichia, Klebsiella, Salmonella, UNIT tissues, but the inherently sticky nature of the capsule itself also and Shigella), which are uniformly distributed on the surface of cells assists in the overall attachment process. Although capsules are (Figure 25.5). Pili are typically longer and fewer in number than fim- important for adherence of some pathogens to host tissues, many briae, and in addition to attachment, some pili function in the bacte- important pathogens, such as Vibrio cholerae, the causative agent of rial genetic transfer process of conjugation (◀ Section 9.8). Both pili the disease cholera, lack them. and fimbriae function by specifically binding to host cell surface gly- Besides adherence, capsules are important for protecting patho- coproteins, thereby initiating adherence. Flagella may also facilitate genic bacteria from host defenses. For example, the only known adherence of bacterial cells to host cells (Figure 25.5), although their virulence factor for Streptococcus pneumoniae (bacterial pneumonia) role is thought to be less important than that of fimbriae and pili. is its polysaccharide capsule (Figure 25.4). Encapsulated strains of S. pneumoniae grow abundantly in the lungs where they initiate Check Your Understanding host responses that interfere with lung function, cause extensive Distinguish between infection and disease. host damage, and can cause death (▶ Section 31.2). By contrast, What event is required but not sufficient to cause an infectious nonencapsulated strains of S. pneumoniae are quickly and effi- disease? ciently ingested and destroyed by phagocytes, white blood cells Describe the molecules or structures that facilitate pathogen that ingest and kill bacteria by a process called phagocytosis (▶ Sec- adherence to host tissues. tions 26.5–26.7). Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 852 18/03/21 18:54  CHAPTER 25 Microbial Infection and Pathogenesis  853 been called the “intrabody phageome.” Although poorly under- Flagellum stood, the human phageome may play a role in modulating inflam- mation and other immune responses, which may have implications Fimbriae in controlling autoimmune diseases such as type 1 diabetes and Crohn’s disease (▶ Section 28.1). Growth of the Microbial Community: An Example from Human Dental Caries Infection requires growth of the pathogen after it has attached to and colonized a surface (Figures 25.1 and 25.6), and the actual dis- ease process may not be the result of a single type of microbe but of a community of interacting microorganisms. An example of this is found in the oral microbial disease dental caries (tooth decay), where attachment and infection have been well studied as models of these key events in the disease process. Even on a freshly cleaned tooth surface, acidic glycoproteins CDC/PHIL from the saliva form a thin organic film several micrometers thick; this film provides an attachment site for bacterial cells, and oral Figure 25.5 Fimbriae. Computer-generated image of a scanning electron micro- streptococci quickly colonize it. These include in particular the two graph of cells of Salmonella enterica (typhi ) showing the numerous thin fimbriae Streptococcus species most often implicated in tooth decay: and the much thicker peritrichously arranged flagella. A single cell is about 1 mm in S. sobrinus and S. mutans. Both of these organisms produce a cap- diameter. sule (Section 25.1). The S. sobrinus capsule contains adhesins spe- cific for host salivary glycoproteins ( Figure 25.7a, b), whereas S. mutans resides in crevices and small fissures where it relies on 25.2 Colonization and Invasion dextran—a strongly adhesive exopolysaccharide—that it produces If a single pathogenic virus or cell attaches to its specific host tissue, to secure cells to the tooth and gum surface (Figure 25.7c, d). Both it alone is insufficient to cause disease; the pathogen must establish S. sobrinus and S. mutans are lactic acid bacteria (◀ Section 16.6) that residence there and multiply. Colonization, the growth of a micro- ferment glucose to lactic acid, the agent that destroys tooth enamel. organism after it has gained access to host tissues, begins at birth as However, the trigger for decay activities is sucrose (table sugar), a newborn child is naturally exposed to a suite of harmless (and in since it is sucrose that allows these species to produce the dextran many cases necessary) bacteria and viruses that will be the infant’s exopolysaccharide and capsules necessary for attachment and initial normal microbiota (Chapter 24). colonization. The human body is rich in organic nutrients and provides condi- Extensive bacterial growth of these oral streptococci results in a tions of controlled pH, osmotic pressure, and temperature that are thick biofilm called dental plaque (Figure 25.7). Using phyloge- favorable for the growth of microorganisms. However, each body netic probes, it has been possible to more thoroughly explore the region, such as the skin, respiratory, gastrointestinal, and genitouri- microbial diversity of dental plaque, and it is clear that the two nary tracts, differs chemically and physically from others, and thus Streptococcus species are not the entire story. Many other gram- provides a selective environment for the growth of certain microbes positive and gram-negative Bacteria are present in plaque, including and not others. The result is that pathogens show rather rigid tissue species of Corynebacterium, Porphyromonas, Leptotrichia, Neisseria, the specificities (▶ Table 26.1), and this reality is often helpful in the 6 UNIT diagnosis of microbial infections. Colonization typically begins at sites in the mucous membranes Exposure Adhesion and Biofilm colonization formation (Figure 25.6). Mucous membranes are composed of epithelial cells, tightly packed cells that interface with the external environment. Microbial They are found throughout the body, lining the urogenital, respira- cells tory, and gastrointestinal tracts. The epithelial cells in mucous mem- Mucus branes secrete mucus, a thick liquid secretion that contains water-soluble proteins and glycoproteins. Mucus retains moisture Epithelial cell and naturally inhibits microbial attachment because most microbes are swept away by physical processes, such as swallowing or sneez- ing. Nevertheless, some microbes—both pathogens and nonpatho- gens—adhere to the epithelial surface and colonize. If these attached Time microbes are pathogens, it sets the stage for infection, invasion, and (a) (b) (c) disease (Figure 25.1). Nonpathogenic microbes that penetrate Figure 25.6 Bacterial interactions with mucous membranes. (a) Loose association mucous membranes include large numbers—potentially many bil- upon initial exposure. (b) Penetration of mucus leading to adhesion and colonization. lions—of innocuous bacteriophages that contribute to what has (c) Further colonization leading to biofilm formation. Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 853 18/03/21 18:54  854   UNIT 6 MICROBE–HUMAN INTERACTIONS AND THE IMMUNE SYSTEM dental caries and related, often more serious, oral pathologies, including gingivitis and periodontitis, bacterial diseases that erode the tooth-supporting gum and bone tissues. Invasion and Systemic Infection C. Lai, M.A. Listgarten, and B. Rosan In the case of dental caries, the bacterial infection primarily resides on the tooth and gum surfaces. By contrast, in most infectious dis- eases, the pathogen must invade past the tissue surface in order to promote disease. Invasion is the ability of a pathogen to enter into host cells or tissues, spread, and cause disease. Some pathogens remain localized after initial entry, multiplying and invading at a (a) (b) single focus of infection, such as the boil that may arise from Staphy- lococcus skin infections (▶ Section 31.9). However, sometimes the Dextran pathogens enter the bloodstream, from where they can travel to dis- tant parts of the body. Depending on the pathogen and the condi- tion of an individual and that individual’s immune system, the presence of bacteria in the blood can have mild or highly severe consequences. The mere presence of bacteria in the blood is called bacteremia; this condition is typically self-limiting and asymptomatic, as the I.L. Shechmeister and J. Bozzola CDC/PHIL, Dr. Richard Facklam Cells bacterial cells do not grow in the bloodstream, and the immune system quickly removes them. By contrast, in septicemia bacteria multiply in the bloodstream, and the organism spreads systemically (c) (d) Figure 25.7 Cariogenic Streptococcus spp. and dental plaque. (a) The low-magnification micrograph shows predominantly streptococcal cell morphol- ogy embedded in dental plaque. The species Streptococcus sobrinus (arrows) appears darker from a specific staining technique. (b) Higher-magnification ­micrograph showing a region in the plaque with S. sobrinus cells (dark, arrow). Note the extensive capsule (lighter grey area) surrounding the S. sobrinus cells. (c) Light micrograph of a Streptococcus mutans culture showing the characteristic cell chains of streptococci. (d) Scanning electron micrograph of the sticky dextran material that holds cells together in filaments. Individual cells of both S. sobrinus and S. mutans are about 1 mm in diameter. Tooth surface filamentous anaerobe Fusobacterium, and many others (Figure 25.8). Jessica Mark Welch and Gary Borisy Moreover, the different species likely play specific structural and 6 functional roles in mature dental plaque. This can be seen in FISH- UNIT stained (◀ Section 19.5) sections of plaque, where filamentous streamers of cells of Corynebacterium attached to a thin biofilm on the tooth surface anchor cells of Streptococcus and other bacteria a short distance away from the tooth surface (Figure 25.8). Such an arrangement probably allows Streptococcus cells to extend out from Growth of biofilm the tooth surface into regions of the oral cavity where saliva, sugars, Gram-positive Gram-negative and other nutrients are more abundant. Corynebacterium Fusobacterium Dental plaque is thus a complex mixed-culture biofilm composed Streptococcus Porphyromonas of several different genera of Bacteria and their accumulated prod- ucts. A few Archaea are also present in dental plaque, primarily Figure 25.8 Bacterial diversity of dental plaque. Confocal micrograph of a FISH- methanogenic species such as Methanobrevibacter oralis. As dental stained (◀ Section 19.5) section through human dental plaque using a suite of phy­ plaque accumulates, the microbiota secrete locally high concentra- logenetic probes containing different fluorescent tags. Color matching to specific tions of lactic acid that decalcifies tooth enamel, resulting in dental groups is shown below the photo. The tooth surface would be on the left with the caries. Tooth enamel is strongly calcified tissue, and the ability of filamentous Corynebacterium species (purple) containing attached streptococci microbes to invade this tissue plays a major role in the extent of (green) flaring out from an attachment site on the tooth surface. Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 854 18/03/21 18:54  CHAPTER 25 Microbial Infection and Pathogenesis  855 from an initial focus and produces toxins or other poisonous sub- and discovered that strains of S. pneumoniae that contained a cap- stances. Septicemia usually begins as an infection in a specific organ sule (“smooth” strains because they formed smooth colonies on such as the intestine, kidney, or lung, and then spreads rapidly plates) were highly virulent for mice, whereas mutant derivatives throughout the body from there. Septicemia is typically associated lacking a capsule (“rough” strains) were not. The S. pneumoniae with major symptoms and may lead to massive inflammation, cul- capsule is the primary virulence factor of this bacterium because it minating in septic shock (sepsis) and death. Viremia is the term used helps the bacterium evade immune surveillance. Griffith’s key dis- to describe viruses present in the bloodstream, and measles, a highly covery was that the smooth phenotype could be transferred to infectious disease in those not vaccinated (▶ Section 31.6), is a good rough cells by treating rough cells with an extract from smooth example of a systemic viremia. cells (◀ Figure 1.38). This was the first experimental example of A pathogen that causes disease in a given host can trigger mild or transformation, a bacterial genetic transfer process (◀ Section 9.6), severe outcomes depending on its inherent capacity to elicit disease. and the transforming constituent in the extract was later shown (by We consider the principles that govern these capacities now with a other scientists) to be DNA. focus on the important infectious disease concept called virulence. Griffith’s choice of experimental organism was fortuitous because S. pneumoniae is both readily transformable and highly virulent for Check Your Understanding mice. Only a few cells of an encapsulated strain of S. pneumoniae can At what body sites do pathogens typically attach and colonize? establish a fatal infection and kill all mice in a test population. As a How does the capsule of Streptococcus mutans assist in the result, the LD50 for S. pneumoniae in mice is not proportional to the formation of dental caries? number of cells delivered (Figure 25.9). By contrast, the number of Mastering Microbiology cells of a less virulent pathogen, such as the gram-negative enteric Which is the more serious condition, bacteremia or septicemia, Art Activity: and why? bacterium Salmonella enterica (typhimurium), necessary to kill all of Figure 25.9 Microbial the mice in the test population is about 10,000-fold greater than the virulence highly virulent S. pneumoniae cells, and the LD50 is proportionally related to the number of cells of the pathogen injected into the mice 25.3 Pathogenicity, Virulence, (Figure 25.9). and Virulence Attenuation There are many examples of highly virulent human pathogens, Unique properties of each pathogen contribute to its pathogenicity, especially among viruses. For example, some strains of the influenza the overall ability to cause disease. The measure of pathogenicity is virus are so highly virulent that only a few virions can initiate called virulence, the relative ability of a pathogen to cause disease. Pathogenicity and virulence are not uniform properties of a given pathogen and can differ dramatically between different strains of CDC/Janice Haney Carr CDC/Janice Haney Carr the same bacterial species or virus. Highly virulent strains of a given pathogen tend to emerge every so often and when they do, they often trigger a particularly severe course of disease that is rapid and widespread, potentially resulting in an epidemic or pandemic (Chap- ter 30). The virulence of a given pathogen depends on a number of factors including its relative abilities to adhere, colonize, and invade Highly virulent Moderately virulent (Figure 25.1), as well as its arsenal of virulence factors. organism organism 100 (Streptococcus [Salmonella enterica pneumoniae) (typhimurium)] Virulence Percentage of mice killed Virulence is the net outcome of host–pathogen interactions, a 80 dynamic relationship between the two organisms influenced by 6 UNIT ever-changing conditions in the pathogen, the host, and the environ- 60 ment. Host damage in an infectious disease is mediated by v­ irulence factors, toxic or destructive substances produced by the pathogen that directly or indirectly enhance invasiveness and host damage by 40 facilitating and promoting infection. The second part of this chapter is devoted to major virulence factors. 20 Virulence is a quantifiable entity, especially if a pathogen is lethal and an experimental animal model is available. For example, the LD50 (LD stands for “lethal dose”) is defined as the number of cells 101 102 103 104 105 106 107 of a pathogen (or virions, for a viral pathogen) that kills 50% of the Number of cells injected per mouse population of the host organism in a test group. Highly virulent pathogens frequently show little difference in the number of cells Figure 25.9 Microbial virulence. Differences in microbial virulence demon­ required to kill 100% of a test group as compared with the LD50. To strated by the number of cells of Streptococcus pneumoniae (red bars) and Salmo- illustrate this, recall the foundational work of the British microbi- nella enterica (typhimurium) (green bars) required to kill mice. A colorized scanning ologist Frederick Griffith (◀ Section 1.14 and Figure 1.38). Griffith electron micrograph of each bacterium is shown above its respective graph. worked with the gram-positive bacterium Streptococcus pneumoniae Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 855 18/03/21 18:54  856   UNIT 6 MICROBE–HUMAN INTERACTIONS AND THE IMMUNE SYSTEM disease even though mortality rates are typically low. Ebola virus is rubella, chicken pox/shingles, and yellow fever (Figure 25.10). also highly virulent, and the tiny inoculum necessary to initiate dis- Although attenuated viruses are “live” in the sense that, unlike ease often results in a fatal infection. By contrast, the bacterial patho- “killed” strains, they are capable of replication and could in prin- gen Vibrio cholerae (which causes cholera) is not especially virulent, ciple become virulent once again, properly attenuated virus vac- as a large inoculum of this intestinal pathogen is necessary to initiate cines (those free of any unattenuated virions) typically show disease (▶ Section 33.3). greater efficacy and generate an overall stronger immune response than do killed virus vaccines. Attenuation The virulence of a pathogen can change. Attenuation is the decrease Check Your Understanding or loss of virulence of a pathogen (Figure 25.10). When pathogens What are virulence factors? How can the LD50 test be used to de­ are kept in laboratory culture rather than isolated from diseased fine virulence of a pathogen? animals, their virulence often decreases, or may be completely What circumstances can contribute to attenuation of a pathogen? lost. Strains that have either a reduced virulence or are no longer virulent are said to be attenuated. Attenuation is thought to occur because nonvirulent or weakly virulent mutants grow faster than 25.4 Genetics of Virulence and virulent strains in laboratory media where virulence has no selec- tive advantage. After successive transfers in fresh media, such the Compromised Host mutants are therefore selectively favored. However, if an attenu- The virulence of a bacterial pathogen and the eventual outcome of ated culture is reinoculated into an animal, the organism may an infectious disease are the net result of genetic and physiological regain its original virulence, especially with continued in vivo pas- features of both the pathogen and the host. In the case of the host, a sage as more-virulent strains are naturally selected. In some cases, pathogen may infect a healthy, well-rested young adult or an indi- though, the loss of virulence is permanent. For example, if a dele- vidual compromised by an ailment, such as a physiological condi- tion mutation led to a major modification of a required receptor tion (old age, hospitalization, immune suppression), an ongoing molecule (Figure 25.2b) or to the inability to produce a key viru- infectious disease (for example, acquired immunodeficiency syn- lence factor, such as the production of a toxin or invasive enzyme, drome [AIDS] caused by the human immunodeficiency virus [HIV]), then the mutant strain would be permanently attenuated. or a genetic disease (for example, cystic fibrosis). The outcome of Attenuated strains of various pathogens are valuable to clinical infection—health or disease—may be very different in these different medicine because they are often used for the production of vac- individuals, even if they are infected by the same strain of a viral or cines, especially viral vaccines. This principle was first demon- bacterial pathogen. strated in the 1880s by Louis Pasteur with his remarkable development of the first rabies vaccine (◀ Section 1.11 and Figure 1.32a), and since that time, attenuated viral strains have also been Final preparation of attenuated vaccine in employed in the production of vaccines for measles, mumps, fertilized chicken eggs Isolate yellow fever virus from diseased patient Multiple passages of Multiple passages Multiple passages virus in Rhesus macaques of virus in mouse of virus in chicken embryos embryos 6 UNIT Administration of vaccine to susceptible individual to elicit immune response without disease symptoms Attenuation of virulence Figure 25.10 Attenuation of virulence in vaccine production. To produce the yellow fever vaccine, the isolated virus is attenuated by doz- ens of passages in hosts that are increasingly dissimilar to the original human host, with the final preparation occurring in embryonated chicken eggs. With each successive passage, the virus gradually becomes more adapted to its new host, while simultaneously becoming less virulent to the original human host. When introduced to a susceptible individual, the weakened viral strain elicits a protective immune response to yellow fever without causing symptoms of the disease itself. Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 856 18/03/21 18:54  CHAPTER 25 Microbial Infection and Pathogenesis  857 The virulence of a pathogen may be encoded by firmly entrenched Several genes that encode these virulence factors in Salmonella and chromosomal genes or by highly mobile genetic elements. For exam- related gram-negative pathogens, such as pathogenic strains of Esch- ple, the gram-negative bacterium Bordetella pertussis, the causative agent erichia coli (▶ Section 33.11), are found clustered together on the of whooping cough (pertussis, ▶ Section 31.3), makes several toxins chromosome as pathogenicity islands. Salmonella pathogenicity island 1 including pertussis toxin, a potent AB-type exotoxin (Section 25.6); (SPI1) is a cluster of genes that encodes over 10 distinct proteins collectively, these toxins trigger the symptoms of whooping cough. that promote virulence and invasion. One of these is invH, a gene Other species of Bordetella do not make pertussis toxin, and the chro- encoding a surface adhesion protein (Section 25.1). Several inv mosomal gene encoding pertussis toxin does not readily move from genes encode proteins important for trafficking of virulence factors. B. pertussis to other species. But in contrast to B. pertussis, some bacterial For example, the InvJ regulator protein controls assembly of struc- pathogens routinely exchange genes encoding virulence factors with tural proteins InvG, PrgH, PrgI, PrgJ, and PrgK, which form a type III different bacterial species or even genera, and thus highly related ver- secretion system called the injectisome, an organelle in the bacterial sions of their most potent weapons may appear in several different envelope that allows for the direct transfer of virulence proteins into pathogens. Salmonella is a well-studied example of the genetic transfer host cells through a needle-like assembly (◀ Section 6.13 and of virulence factors, and we focus on this bacterium now. Figures 6.43 and 6.44). A second Salmonella pathogenicity island, SPI2, contains genes Virulence in Salmonella: Pathogenicity that are responsible for causing more systemic than localized disease Islands and Plasmids and resistance to host defenses. In addition, several plasmid-encoded Salmonella species infect humans, leading to various gastrointestinal virulence factors, such as antibiotic resistance genes encoded on illnesses (▶ Section 33.10), and they encode a large number of viru- R plasmids (◀ Section 6.2), can spread between Salmonella species lence factors that are important in disease. These include type I fim- and other genera of enteric bacteria. Pathogenicity islands and briae (Section 25.1) to facilitate attachment of cells to gastrointestinal R plasmids allow for the facile and rapid transfer of virulence factors. tissues; several different classes of exotoxins (Section 25.6); antiphago- It is thus not uncommon for genes encoding factors in one pathogen cytic proteins that block engulfment of bacterial cells by host phago- to be similar, if not identical, to those in another because of transfer cytes; proteins that promote survival if the bacterium does get of parts or all of the islands or plasmids between species by horizon- phagocytosed; siderophores, organic molecules that bind iron tightly tal gene exchange (Chapter 9). and, in pathogenic bacteria, allow the bacteria to outcompete host In the well-known opportunistic pathogen (see next subsection) sequestration systems for iron; and endotoxin (Section 25.8). With Pseudomonas aeruginosa, pathogenicity islands can also contain genes the exception of endotoxin, many of these virulence factors are encoding antibiotic resistance. As for Salmonella, many cases of mul- encoded by genes present on mobile DNA rather than on the cell’s tiple antibiotic resistance in P. aeruginosa are linked to plasmids. chromosome (Figure 25.11). However, in some strains of P. aeruginosa, genomic islands contain- ing transposable elements (◀ Section 9.11) are present. By transposi- Mastering Microbiology tion these islands have “captured” multiple antibiotic resistance Art Activity: Enterotoxin genes and can then disseminate them to other organisms; the Figure 25.10 (diarrhea) Siderophores Virulence genomic islands are present in P. aeruginosa in place of or in addi- factors in Injectisome (iron uptake) Salmonella (inv and prg tion to resistance plasmids. Regardless of how they are encoded, products form Type I fimbriae these strains have become resistant to virtually all of the clinically complex) (adherence) useful antibiotics that have traditionally been used to control P. aeruginosa infections. This is a particularly serious problem for the Endotoxin in LPS layer Virulence compromised host and in the hospital environment, and we con- (fever) SPI2 plasmid sider these issues now. SPI1 6 UNIT Anti- The Compromised Host phagocytic proteins Some individuals are simply more susceptible to infection than induced Cytotoxin others for reasons that have little to do with the virulence of the by oxyR (inhibits host cell protein synthesis; Ca2+ efflux pathogen. These so-called compromised hosts are individuals in from host cell; adherence) which one or more mechanisms of resistance to disease are inac- O antigen (inhibits tive and in whom the probability of infection is therefore Vi capsule antigen; phagocyte inhibits complement binding increased. Many hospital patients with noninfectious diseases (for killing) Pathogenicity example, cancer and heart disease) acquire microbial infections islands on Flagellum (motility) chromosome more readily because they are compromised hosts. Such healthcare- H antigen (adherence; inhibits phagocyte killing) associated infections (also called nosocomial infections; ▶ Sec­t ion 29.2) affect up to 2 million individuals each year in the United Figure 25.11 Virulence factors in Salmonella. Factors important for virulence States, with a nearly 5% mortality rate. Invasive medical proce- and the development of pathogenesis in this gram-negative enteric pathogen are dures such as catheterization, hypodermic injection, spinal shown. Genes encoding many of the factors reside on the pathogenicity islands ­puncture, biopsy, and surgery may unintentionally introduce or plasmids. Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 857 18/03/21 18:54  858   UNIT 6 MICROBE–HUMAN INTERACTIONS AND THE IMMUNE SYSTEM microorganisms into the patient. The stress of surgery and the anti-inflammatory drugs given to reduce pain and swelling can II Enzymes and Toxins also reduce host resistance. Some factors can compromise host resistance outside the hospi- of Pathogenesis tal, including lifestyle choices that affect major organs of the body, such as intravenous drug usage, tobacco use, and excessive con- M any pathogenic microbes increase their competitiveness by synthesizing potent products—enzymes and toxins— that allow them to access nutrients in the host. These virulence sumption of alcohol, or genetic diseases that eliminate parts of the immune system (▶ Section 28.2). People that are physically com- factors may help the pathogen in initial invasion, be the final re­ promised for any of a number of reasons may be more susceptible sult of successful colonization, or be secreted into food or other to infections, not only because they are physically weakened but nonliving substances later ingested by the host. also because their living conditions and lifestyle choices may put them in more continual contact with infectious agents. For exam- ple, infection with the human immunodeficiency virus (HIV) pre- B acterial pathogens damage host tissues (or the entire host) in two major ways: (1) by secreting tissue-destroying enzymes and (2) by secreting or shedding toxins that target specific host tissues disposes an individual to infections from opportunistic pathogens, microbes that cause disease only in the absence of or the entire host. In contrast to bacterial pathogens, most viral normal host resistance. HIV causes AIDS by destroying a specific pathogens damage host tissues by lysing cells directly, although class of immune cell, the CD4 T lymphocytes (▶ Section 31.15), some viruses are nonlytic and instead introduce genes into host cells which are key to an effective immune response. The reduction in that may eventually harm the host (◀ Section 5.7). CD4 T cells reduces immunity, and an opportunistic pathogen— We turn our focus now to the enzymes and toxins of well-studied one that does not cause disease in a healthy, uninfected host—can pathogenic bacteria, contrasting their potency and modes of action. then cause serious disease or even death. Individuals with immu- Some of these virulence factors cause only minor disease symptoms, nodeficiencies from underlying genetic causes rather than infec- whereas others are among the most poisonous substances known. tion are also more susceptible to opportunistic infections because part of their immune system is either nonfunctional or 25.5 Enzymes as Virulence Factors suboptimal. Following adherence, colonization, and infection by a pathogen, The outcome of an infectious disease thus depends on several invasiveness requires the breakdown of host tissues and access to factors, both host and pathogen related. Two individuals exposed to nutrients released from host cells. In many classical bacterial patho- the same pathogen in the same way may well show different out- gens, this is accomplished through the activity of enzymes that attack comes. But once an infection has proceeded to the actual stage of and destroy cells in one type of tissue or another (Table 25.1). disease, the symptoms that appear are due to harmful products of the pathogens, and we turn our attention to these now. Tissue-Destroying Enzymes Many virulence factors are enzymes. For example, streptococci, Check Your Understanding staphylococci, and certain clostridia produce hyaluronidase What major virulence factors are produced by Salmonella? (Table 25.1), an enzyme that promotes spreading of organisms in What is an opportunistic pathogen? What steps can a per­ tissues by breaking down the polysaccharide hyaluronic acid. son take to help avoid opportunistic infections? Among other functions, hyaluronic acid is a component of the extra- What is a nosocomial infection? cellular matrix and functions as a type of “intercellular cement” in animal connective tissues, helping to maintain the organization of 6 Enzyme virulence factors of some well-known gram-positive bacterial pathogens UNIT TABLE 25.1 Organism Disease Enzymea Enzyme activity Staphylococcus aureus Pus-forming infections Coagulase Induces fibrin clotting; allows bacterial cells to remain at site of infection (prevents access to pathogens by cells of the immune response) Nuclease; lipase Break down nucleic acids or lipids Streptococcus pyogenes Pus-forming infections; scarlet fever; Hyaluronidase Dissolves hyaluronic acid in connective tissues; allows bacte- strep throat rial cells to spread (enhances pathogen invasion) Streptokinase Dissolves fibrin clots; allows bacterial cells to spread Clostridium perfringens Gas gangrene; food poisoning Collagenase Breaks down collagen (a protein), allowing the bacterium to spread to other tissues Protease Breaks down proteins a The activities of coagulase, hyaluronidase, and streptokinase are depicted in Figure 25.12. Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 858 18/03/21 18:54  CHAPTER 25 Microbial Infection and Pathogenesis  859 individual cells into tissues. The activity of hyaluronidase causes streptokinase to dissolve fibrin clots and make further invasion pos- host cells to slough apart, allowing pathogens at an initial coloniza- sible (Table 25.1, Figure 25.12b). Streptokinase specifically activates tion site to spread between host cells to attack subsurface tissues the host to produce plasmin, an enzyme that degrades fibrin blood (Figure 25.12a). Similarly, the clostridia that cause gas gangrene pro- clots. Because of this powerful activity, streptokinase also has a med- Mastering Microbiology duce collagenase, an enzyme that destroys collagen (a major protein ically beneficial function. The protein is marketed as a pharmaceuti- Art Activity: of connective tissues in muscle and other body tissues); collagenase cal and administered intravenously to dissolve clots for conditions Figure 25.11a Activity of some enables these bacteria to gain access to deeper host tissues and in which normal blood flow is blocked by blood clots, such as from enzyme virulence spread through the body. Recall that clostridia are anaerobes, and heart attacks, deep vein thromboses, or embolisms. factors colonizing deeper tissues allows them to reach less oxic conditions In contrast to the fibrin-destroying activity of streptokinase, some and provides a ready source of nutrients from destroyed tissues (gan- pathogens produce enzymes that actually promote the formation of grene clostridia are typically proteolytic species, ◀ Section 16.8 and fibrin clots. These clots protect the pathogen from host responses. ▶ Section 32.9). Many pathogenic streptococci and staphylococci For example, coagulase (Table 25.1), produced by virulent Staphylo- also produce proteases, nucleases, and lipases that degrade host pro- coccus aureus, converts fibrinogen to fibrin, resulting in the clotting teins, nucleic acids, and lipids, respectively (Table 25.1). of blood and the formation of fibrin surrounding the S. aureus cells; Two virulence factors are enzymes that affect fibrin, the insoluble this blanket of fibrin protects the S. aureus cells from attack by cells blood protein that triggers blood clots, but the activities of the of the host’s immune system (Figure 25.12b). The fibrin matrix pro- enzymes yield opposing results (Figure 25.12b). Blood clotting is duced as a result of coagulase activity may also account for the local- triggered by tissue injury and functions not only to stop blood loss ized nature of many staphylococcal infections, as is typically seen in but also, in the case of a bacterial infection, to isolate the pathogen, boils and pimples (▶ Section 31.9). Coagulase-positive S. aureus limiting the infection to a local region. Some pathogens counter this strains are typically more virulent than coagulase-negative strains, a host protective mechanism by producing fibrinolytic enzymes, such likely reflection of the former’s ability to evade innate immune as streptokinase produced by Streptococcus pyogenes. This bacterium responses such as phagocytosis (Chapter 26) and continue growth is often associated with pus-forming wounds and secretes and tissue destruction for a longer period. Epithelial Streptococcus Hyaluronic Subepithelial Enzyme Activities at the Host’s Mucosal Surface cell pyogenes acid tissue Host mucosal surfaces are bathed in immune substances including enzymes such as lysozyme, an enzyme that cleaves the peptidoglycan of bacterial cells and promotes their osmotic lysis (◀ Section 2.3). Virulence factors produced by the gram-positive bacterium Enterococcus faecalis—a major cause of bacteremia, surgical wound infections, and urinary tract infections—subvert the protective role of lysozyme by altering the structure of the bacterium’s peptidogly- can such that lysozyme can no longer recognize its substrate. Antibodies are also present on mucosal surfaces, in particular a Hyaluronidase Production of Pathogen invades class of antibody called IgA (▶ Section 27.3). These “secretory anti- producer attaches hyaluronidase ( ) deeper tissues. to epithelia. bodies,” as they are called, help prevent pathogen adherence to host tissues (Section 25.1). However, certain pathogenic bacteria counter (a) Hyaluronidase this protective role by producing enzymes that specifically cleave IgA (IgAases), rendering this host defense useless; Neisseria species such Blood Staphylococcus Clot Streptococcus as N. gonorrhoeae (gonorrhea) and N. meningitidis (meningitis) are vessel aureus pyogenes particularly notorious in this regard. 6 UNIT We thus see that pathogens can produce enzymes both as offen- sive weapons—to destroy host tissues—and as defensive weapons— to destroy or inactivate defensive weapons of the host. Both strategies accomplish a similar objective: The invasiveness of the pathogen is increased, and this allows it to ultimately extract more resources Staphylococci enter Clot walls off Streptokinase ( ) from its host. in cut, produce pathogen, blocking dissolves clot, coagulase ( ). access to immune releasing pathogen Check Your Understanding system cells ( ). to bloodstream and deeper tissues. Identify host factors that limit or accelerate infection of a microorganism at selected local sites. (b) Coagulase and streptokinase How do streptokinase and coagulase promote bacterial Figure 25.12 Activity of some enzyme virulence factors. (a) Hyaluronidase. infection and invasion? (b) Coagulase (left) and streptokinase (right). Along with some other bacterial What is an IgAase, and why would a bacterial pathogen pathogens, virulent strains of Streptococcus pyogenes typically produce hyalu­ produce one? ronidase and streptokinase, and virulent strains of Staphylococcus aureus typi­ cally produce coagulase. Download more at Learnclax.com M25_MADI4790_16_GE_C25.indd 859 18/03/21 18:54  860   UNIT 6 MICROBE–HUMAN INTERACTIONS AND THE IMMUNE SYSTEM 25.6 AB-Type Exotoxins inhibits protein synthesis in eukaryotic cells. Rats and mice are rela- tively resistant to diphtheria toxin, whereas humans are very suscep- Toxicity is the ability of an organism to cause disease by means of tible, with only a single molecule of toxin sufficient to kill a cell. a toxin that inhibits host cell function or kills host cells (or the host Diphtheria has a significant mortality rate, especially in the young, itself). Exotoxins are toxic proteins secreted by the pathogen as it and death ensues from tissue destruction in vital organs such as the grows. These toxins travel from a site of infection and cause damage heart and liver as diphtheria toxin blocks protein synthesis. at distant sites. Some exotoxins are enterotoxins, toxic proteins Cells of C. diphtheriae secrete diphtheria toxin as a single polypep- whose site of action is the small intestine, generally causing secre- tide. One component of the toxin, subunit B, specifically binds to a tion of fluid into the intestinal lumen, resulting in vomiting and host cell receptor protein on eukaryotic cells, the heparin-binding Mastering diarrhea. Exotoxins fall into three categories in terms of their mecha- Microbiology epidermal growth factor (Figure 25.13). After binding, proteolytic nism: AB toxins, cytolytic toxins, and superantigen toxins. In this section Art Activity: cleavage between subunit B and the remaining portion of the pro- Figure 25.12 we consider the AB toxins, and in Section 25.7 we focus on cytolytic The activity of tein, subunit A, allows subunit A to move across the host cytoplas- diphtheria toxin and superantigen toxins. mic membrane into the cytoplasm. Here subunit A disrupts protein As the name implies, AB toxins consist of two subunits, A and B. synthesis by blocking transfer of an amino acid from tRNA to grow- The B component binds to a host cell surface molecule, facilitating ing polypeptide chains. Diphtheria toxin specifically inactivates the transfer of the A subunit across the cytoplasmic membrane, elongation factor 2 (EF-2), a protein that functions in growth of the where it damages the cell. Some of the best-known and most potent polypeptide chain, by catalyzing the attachment of adenosine exotoxins are AB toxins, including those expressed in diphtheria, diphosphate (ADP) ribose from NAD+. Following ADP-ribosylation, tetanus, botulism, and cholera (T