Bacteria cause diseases by three main mechanisms: (a) inva- sion of tissues followed by inflammation, (b) toxin production, and (c) immunopathogenesis. Table 10-6 summarizes a list of bacteria with their virulence factors.
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Invasion of tissues followed by inflammation
Invasiveness refers to the ability of an organism to invade the host cells after establishing infection. “Invasion” is the term commonly used to describe the entry of bacteria into host cells, implying an active role for the organisms and a passive
role for the host cells. For many disease-causing bacteria, invasion of the host’s epithelium is central to the infectious process. Some bacteria (e.g., Salmonella spp.) invade tissues through the intracellular junctions in the cytoplasm. Some bacteria (e.g., Shigella spp.) multiply within host cells, whereas other bacteria do not.
Shigella spp. initiate infection process by adhering to host cells in the small intestine. There are multiple proteins, including the invasion plasmid antigens (IpA-D), that contribute to the process.
Once inside the cells, the shigellae either are lysed or escape from the phagocytic vesicle, where they multiply in the cytoplasm.
Other bacteria (e.g., Yersinia species, N. gonorrhoeae, Chlamydia trachomatis) invade specific types of the host’s epithelial cells and may subsequently enter the tissue. Once inside the host cell, the bacteria may remain enclosed in a vacuole composed of the host cell membrane, or the vacuole membrane may dis- solved and bacteria may disperse within the cell and from one cell to another.
Invasion of tissues followed by inflammation is enhanced by many factors, which include: (a) enzymes, (b) antiphagocytic factors, (c) biofilms, (d) inflammation, and (e) intracellular survival.
1. Enzymes: Invasion of bacteria is enhanced by many enzymes.
Many species of bacteria produce enzymes that are not intrin- sically toxic but do play important roles in the infectious process. Some of these enzymes are discussed below:
■ Hyaluronidases and collagenase: Hyaluronidases and col- lagenase are the enzymes that hydrolyze hyaluronic acid and degrade collagen, respectively; thereby allowing the bacteria to spread through subcutaneous tissues.
Hyaluronidases are produced by many bacteria (e.g., staphylococci, streptococci, and anaerobes) and aid in their spread through tissues. For example, hyaluroni- dase produced by S. pyogenes degrades hyaluronic acid in the subcutaneous tissue, thereby facilitating the organ- ism to spread rapidly.
Clostridium perfringens produces the proteolytic enzyme collagenase, which degrades collagen (the major protein of fi brous connective tissue), and promotes the spread of infection in tissue.
■ Coagulase: Staphylococcus aureus produces the enzyme coagulase, which in association with blood factors coagulates the plasma. Coagulase contributes to the formation of fibrin walls around staphylococcal lesions, which protects bacteria from phagocytosis by walling off the infected area. The enzyme also causes deposition of fibrin on the surfaces of individual staphylococci, which may help protect them from phagocytosis or from destruction within phagocytic cells.
■ Streptokinase ( fibrinolysin): Many hemolytic strepto- cocci produce enzyme streptokinase, which activates a proteolytic enzyme of plasma. This enzyme is then able to dissolve coagulated plasma and thereby possibly aids in the rapid spread of streptococci through tissues.
Streptokinase has been used in the treatment of acute myocardial infarction to dissolve fibrin clots.
Organism Virulence factors
Staphylococcus aureus Coagulase, protein A Streptococcus pyogenes M protein
Streptococcus pneumoniae Capsular polysaccharide Enterococcus faecalis Cytolysin, biofilm formation
Neisseria gonorrhoeae Pili, opacity-associated proteins (Opa), IgA proteases
Neisseria meningitidis Capsular polysaccharide
Bacillus anthracis Capsule, edema factor, lethal factor, protective antigen
Listeria monocytogenes Internalin
Escherichia coli Heat-labile and heat-stable enterotoxins, pili
Haemophilus influenzae Capsular polysaccharide
Vibrio cholerae Cholera toxin
Mycobacterium tuberculosis Mycolic acid cell wall TABLE 10-6 Bacterial virulence factors
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■ IgA1 proteases: Certain pathogenic bacteria produce enzymes IgA1 proteases that split IgA1 at specific pro- line–threonine or proline–serine bonds in the hinge region and inactivate its antibody activity. IgA1 protease is an important virulence factor of the pathogens, such as N. gonorrhoeae, Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae. Production of IgA1 protease allows the pathogens to inactivate the primary antibody found on mucosal surfaces and thereby facilitates the attachment of these bacteria to the mucous membrane.
2. Antiphagocytic factors: Many bacterial pathogens are rapidly killed once they are ingested by polymorphonuclear cells or macrophages. Some pathogens evade phagocyto- sis or leukocyte microbicidal mechanisms by several anti- phagocytic factors; the most important being (a) capsule, (b) cell wall proteins, (c) cytotoxins, and (d) surface antigens.
■ Capsule: The capsule surrounding bacteria, such as S. pneumoniae (Fig. 10-3) and N. meningitidis, is the most important antiphagocytic factor. It retards the phago- cytosis of bacteria by preventing the phagocytes from adhering to the bacteria.
■ Cell wall proteins: The cell wall proteins, such as the protein A and protein M, of S. aureus and S. pyogenes especially are antiphagocytic. For example, protein A of S. aureus binds to IgG and prevents the activation of complement. M protein of S. pyogenes is antiphagocytic.
■ Cytotoxins: Certain bacteria produce cytotoxins that interfere with chemotaxis or killing of phagocytes. For example, S. aureus produces hemolysins and leukocidins that lyse and damage RBCs and WBCs.
■ Surface antigens: Surface antigens of bacteria, such as Vi antigen of S. typhi and K antigen of E. coli make the bacteria resistant to phagocytosis and lytic activity of complement.
A list of intracellular pathogens is given in Table 10-7.
3. Biofi lms: The biofi lm is an aggregate of interactive bacteria attached to a solid surface or to each other and encased in an exopolysaccharide matrix. Biofi lms consist of single cells and microcolonies of bacteria, all found together in a highly hydrated, predominantly anionic exopolymer matrix. This is distinct from planktonic or free-living bacterial growth, in which interactions of the microorganisms do not occur.
Biofi lms form a slimy coat on solid surfaces and occur throughout the nature. A single species of bacteria may be involved, or more than one species may coaggregate to form a biofi lm. Fungi, including yeasts, are occasionally involved.
Biofi lms are important in human infections that are persis- tent and diffi cult to treat. A few such infections include:
(a) S. epidermidis and S. aureus infections of central venous catheters;
(b) Eye infections that occur with contact lenses and intraocular lenses;
(c) Infections in dental plaque; and
(d) Pseudomonas aeruginosa airway infections in cystic fi brosis patients.
Key Points
Biofi lm confers an inherent resistance to antimicrobial agents, whether these antimicrobial agents are antibiotics, disinfec- tants, or germicides. The mechanisms of resistance are:
■ Delayed penetration of antimicrobial agent through the biofilm matrix;
■ Altered growth rate of biofilm organisms; and
■ Other physiological changes due to biofilm mode of growth.
4. Infl ammation: Infl ammation is an important host defense induced by the presence of bacteria in the body. It is of two types: pyogenic and granulomatous. Pyogenic infl ammation is the host defense seen primarily against pyogenic or pus- producing bacteria, such as S. pyogenes. It typically consists of neutrophils and the production of specifi c antibodies and elevated level of complement. Granulomatous infl am- mation is the host defense seen primarily against intracel- lular granuloma-producing bacteria, such as Mycobacterium tuberculosis, Mycobacterium leprae, etc. The response consists of production of macrophages and CD4+ T cells.
Organism Examples
Bacteria Mycobacterium spp.
Listeria monocytogenes Brucella spp.
Legionella pneumophila Francisella spp.
Yersinia pestis Salmonella Typhi Shigella dysenteriae Rickettsia Chlamydia
Viruses All viruses
Parasites Leishmania spp.
Trypanosoma cruzi Plasmodium spp.
Babesia spp.
Toxoplasma gondii Cryptosporidium parvum Microsporidium spp.
Fungus Histoplasma capsulatum
TABLE 10-7 Intracellular pathogens
Inhibition of phagocytosis by capsule
Phagocyte Streptococcus
pneumoniae
FIG. 10-3. Schematic diagram showing inhibition of phagocytosis by the capsule of Streptococcus pneumoniae.
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5. Intracellular survival: A few mechanisms that are suggested for intracellular survival of bacteria include (a) inhibition of phagolysosome fusion, (b) resistance to action of lysosomal enzymes, and (c) adaptation to cyto- plasmic replication as follows:
■ Bacteria (such as Chlamydia, M. tuberculosis) that interfere with the formation of phagolysosomes in a phagocyte can survive intracellularly and evade host defense pro- cess. These bacteria live within cells and are protected from attack by macrophages and neutrophils. The bac- teria that do not interfere with the formation of phagoly- sosomes are otherwise killed inside the phagocytes.
■ Presence of capsular polysaccharide in Mycobacterium lepraemurium and mycoside in M. tuberculosis makes these bacteria resistant to action of lysosomal enzymes.
■ Certain bacteria, such as rickettsiae, escape from the phagosome into the cytoplasm of the host cell before the fusion of phagosome with lysosome takes place and hence continue to remain intracellular.
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Toxin production
Toxins produced by bacteria are generally classified into two groups: exotoxins and endotoxins.
1. Exotoxins: Exotoxins are heat-labile proteins that are pro- duced by several Gram-positive and Gram-negative bacteria.
These are bacterial products, which are secreted into tissues and directly harm tissues or trigger destructive biological activities (Fig. 10-4). The genes coding for these proteins are frequently encoded on plasmid or on bacteriophage DNA.
Some important toxins encoded by plasmids are tetanus toxin of C. tetani and heat-labile and heat-stable toxins of enterotoxigenic E. coli. Toxins encoded by bacteriophage DNA are cholera toxins, diphtheria toxins, and botulinum toxin.
Superantigens: Superantigens are special group of toxins.
These molecules activate T-cell nonspecifi cally by binding simultaneously to a T-cell receptor and major histocom- patibility complex class II (MHC II) molecules on another cell, without requiring antigen. Nonspecifi c activation of T cells results in a life-threatening autoimmune-like response by producing a large amount of interleukins, such as IL-1 and IL-2. Furthermore, stimulation of T cells by superantigen can also lead to the death of activated T cell, resulting in loss of specifi c T-cell clones and that of their immune response. Staphylococcal enterotoxin (toxic shock syndrome toxin) of S. aureus and erythrogenic toxin of type A or C of S. pyogenes are examples of superantigens.
2. Endotoxins: The term endotoxin was coined in 1893 by Pfeiffer to distinguish the class of toxic substances released after lysis of bacteria from the toxic substances (exotoxins) secreted by bacteria.
Exotoxin Cell wall
Endotoxin
FIG. 10-4. Schematic diagram showing release of exotoxin and endotoxin.
Key Points
Exotoxins show the following features:
■ Exotoxins are good antigens; they induce the synthesis of pro- tective antibody called antitoxins. Some of these antitoxins are useful in the treatment of botulism, tetanus, and other dis- eases. Exotoxins treated with formaldehyde or acid or heat can be converted into toxoid. The toxoids lack toxicity but retain antigenicity. Hence, these are used in protective vaccines.
■ Exotoxins are some of the most toxic substances known.
They are highly potent even in minute amounts. Botulinum toxin is the most potent one, and it has been estimated that 3 kg of botulinum toxin can kill all persons in the world.
Similarly, the fatal dose of tetanus toxin for a human is esti- mated to be less than 1 g.
■ Many toxins have a dimeric A–B subunit structure.
Diphtheria toxin, tetanus toxin, cholera toxins, and the enterotoxin of E. coli are some of the examples of exotoxins that have an A–B subunit structure. A is the active subunit that possesses the toxic activity, and B is the binding subunit that is responsible for the binding of exotoxin to specific receptors of the membrane of human cell (Fig. 10-5).
■ These toxins are very specific in their mechanism of action and act at specific sites of a tissue. The biochemical targets
of A–B toxin include ribosomes, transport mechanisms, and intracellular signaling (cyclic adenosine monophos- phate, CAMP; G protein production); all these cause diar- rhea, loss of neuronal functions, or even death.
■ The exotoxins have specific pharmacological activities and do not produce fever, unlike endotoxins.
Key Points
Endotoxins show the following properties:
■ They are produced by Gram-negative bacteria, but not by Gram-positive bacteria.
■ They are lipopolysaccharide (LPS) components of the outer membrane of Gram-negative bacteria. These form an inte- gral part of the cell wall unlike exotoxins, which are actively released from the cells.
■ The genes that encode the enzymes that produce the LPS are present on the bacterial chromosome, but not on plasmids or bacteriophage DNA, which usually encodes the exotoxins.
■ They are heat stable, and they are released from the bacte- rial cell surface by disintegration of the cell wall.
■ They are weakly antigenic and do not induce, or poorly induce, protective antibodies. Hence, their action is not neutralized by the protective antibodies.
■ They cannot be toxoided.
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Biological activity of endotoxin: Gram-negative bacteria produce endotoxin during infection. The toxicity of endo- toxin is low in comparison with exotoxins. All endotoxins usually produce the same generalized effect of fever and shock. The lipid A protein of LPS is responsible for endo- toxin activities (Table 10-8). The endotoxin binds to specifi c receptors, such as CD14 and TLR4, present on macro- phages, B cells, and other cells. Endotoxin exerts profound biological effects on the host and may be lethal. Biological activities of the endotoxins include the following:
■ Mitogenic effects on B lymphocytes that increase resis- tance to viral and bacterial infections.
■ Production of gamma interferon by T lymphocytes, which may enhance the antiviral state, promote the rejection of tumor cells, and activates the macrophages and natural killer cells.
■ Activation of the complement cascade with the forma- tion of C3a and C5a.
■ Production and release of acute-phase cytokines, such as IL-1, TNF-␣ (tumor necrosis factor-alpha), IL-6, and prostaglandins.
Endotoxic shock: Endotoxins at low concentration induce a protective response, such as fever, vasodilation, and
activation of immunity and infl ammatory response.
However, endotoxins at very high concentration, as seen in blood of patients with Gram-negative bacterial sepsis, cause a syndrome of endotoxic shock. Endotoxic shock is characterized by fever, leukopenia, thrombocytope- nia, sudden fall of blood pressure, circulatory collapse, and sudden death. This is because high concentration of endotoxin can activate the alternative pathway of complement and cause vasodilatation and capillary leak- age, resulting in high fever, hypertension, and shock.
It also causes activation of blood coagulation pathway, leading to disseminated intravascular coagulation.
Endotoxins are not destroyed by autoclaving; hence infu- sion of sterile solution containing endotoxins can cause serious illness.
Detection of endotoxins in medical solutions: Endotoxins are omnipresent in the environment. Solutions for human use (such as intravenous fl uids) are prepared under carefully controlled conditions to ensure sterility and to remove endotoxin. Representative samples of every manufacturing batch are checked for endotoxins by one of two procedures:
(a) limulus lysate test or (b) rabbit pyrogenicity test.
■ Limulus lysate test: The test depends on the ability of endotoxin to induce gelation of lysates of amoebocyte cells from the horseshoe crab Limulus polyphemus. Test kits are commercially available. It is simple, fast, and sensitive to detect endotoxin at a level of 1 ng/mL.
■ Rabbit pyrogenicity test: The test depends on the exquisite sensitivity of rabbits to the pyrogenic effects of endo- toxin. In this test, a sample of the solution to be tested is injected intravenously into the ear veins of adult rab- bits, while the rectal temperature of the animal is moni- tored. Careful monitoring of the temperature responses provides a sensitive and reliable indicator of the pres- ence of endotoxin in the solution.
Table 10-9 summarizes differences between exotoxins and endotoxins.
DNA
Exotoxin coding mRNA
Nucleus
Exotoxin
Exotoxin is released from bacteria
Altered exotoxin
1
Exotoxin enters cell by binding to host cell receptor through binding
component (B) 2
Active component (A) of exotoxin alters cell function by inhibiting protein synthesis
3
FIG. 10-5. Schematic diagram showing mode of action of exotoxin.
Clinical features Mechanism
Fever Interleukin-1
Inflammation Activation of alternative pathway of complement (C3a, C5a)
Disseminated intravascular coagulation (DIC)
Activation of Hageman factor Shock (hypotension) Bradykinin, nitric oxide Leukopenia, thrombocytopenia,
decreased peripheral circulation, and perfusion to organs
Secondary to DIC TABLE 10-8
Mechanisms of endotoxin-mediated toxicity
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Feature Endotoxin Exotoxin
Nature Lipopolysaccharide Protein (polypeptide)
Source Gram-negative bacterial cell wall Gram-positive bacteria and some Gram-negative bacteria
Location of genes Chromosome Plasmid or bacteriophage
Nature of secretion Not secreted by the bacterial cell Actively secreted by the bacteria
Release of toxin Cell lysis Filtration of bacterial cultures
Heat stability Highly stable (withstand even 100°C for an hour) Heat-labile, destroyed mostly at 60°C Mode of action Mediated by interleukins (IL-1) and tumor necrosis factor Mostly enzyme-like action
Effect Nonspecific (fever, shock, etc.) Specific pharmacological effect
Tissue affinity No Specific affinity for certain tissues
Diseases Gram-negative bacterial sepsis, meningococcemia Botulism, diphtheria, and staphylococcal toxic shock syndrome
Fatal dose Only large doses are fatal Small doses (even a few micrograms) are fatal
Antigenicity Poorly antigenic Highly antigenic
Neutralization by antibodies Ineffective Neutralized by specific antibodies
Vaccine No effective vaccine Specific toxoids are available
TABLE 10-9 Differences between endotoxins and exotoxins
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Immunopathogenesis
In certain diseases, the symptoms are caused not by the organism itself, but due to immune response to the presence of organisms. For example, immune complexes deposited in the glomerulus of the kidney cause poststreptococcal glo- merulonephritis. Antibodies that are produced against the M proteins of S. pyogenes cross-react with joint, heart, and brain tissues producing disease manifestations of rheumatic fever.
Similarly, the host immune response is an important cause of disease symptoms in patients suffering from syphilis caused by T. pallidum, Lyme disease caused by Borrelia, and other diseases.