The successful pathogen must survive and multiply in the face of these formidable host defenses. Microbial virulence factors that permit the establishment of the pathogen in the hostile host environment are essential. If these factors are lost, the capacity to infect the host or become transmitted successfully goes with them. Not surprisingly, these virulence factors are also the target(s) in the design of vaccines. The following sections provide a general overview of the classes of bacterial virulence factors that permit them to over-come host defenses. No pathogen possesses all of the classes of virulence factors, nor are all virulence factors absolutely essential for a pathogen to reach its goal of sufficient mul-tiplication to establish itself in the host or to be transmitted to a new susceptible host.
A number of microorganisms synthesize protein molecules that are toxic to their hosts and are secreted into their environment or are found associated with the microbial surface. These exotoxins usually possess some degree of host cell specificity, which is dictated by the nature of the binding of one or more toxin components to a specific host cell receptor. The distribution of host cell receptors often dictates the degree and the breadth of the toxicity. Bacterial exotoxins, whether synthesized by Gram-positive or Gram-negative bacteria, fall into two broad classes, each of which represents a general pathogenic theme common to many bacterial species.
The best known pathogenic exotoxin theme is represented by the A – B exotoxins. These toxins are divisible into two general domains. One, the B subunit, is associated with the binding specificity of the molecule to the host cell. Generally speaking, the B region binds to a specific host cell surface glycoprotein or glycolipid. The other, subunit A, is the catalytic domain, which enzymatically attacks a susceptible host function or structure. The actual biochemical structure of exotoxins varies. In some cases (diphtheria toxin), the single B subunit of the toxin is linked through a disulfide bond to the A subunit. In other cases (pertussis toxin), multiple B subunits may join with a single A enzymatic subunit. In any event, following attachment of the B domain to the host cell surface, the A domain is transported by direct fusion or by endocytosis into the host cell. Many of the most po-tent A – B bacterial toxins are ADP-ribosylating enzymes. Some of these affect the pro-tein-synthesizing apparatus of the cell (diphtheria toxin, Pseudomonas exotoxin); others affect the cytoskeleton (Clostridium botulinum toxin C2) or the normal signal transduc-tion activities of the host (Bordetella pertussis and Vibrio cholerae). It is notable that the major natural substrates of the toxin ADP-ribosyltransferases are guanine nucleotide-binding proteins (G proteins), which are involved in signal transduction in eukaryotic cells. In a very simplistic way, one can think that the ADP-ribosylating toxins are all geared to interrupt the biochemical lines of communication within and between host cells. An understanding of bacterial toxins, therefore, sheds as much light on the intimate details of normal animal cell regulation as it does on bacterial pathogenicity.
Several bacterial toxins have been examined in exquisite detail at the biochemical level. The crystal structures of several have been “solved.” In many cases, the precise amino acids making up the catalytic site of the toxin are so well known that a single amino acid substitution can be made that is sufficient to detoxify the molecule. These toxoids are the basis for new generations of vaccines. Given this level of biochemical so-phistication, it is somewhat disconcerting to realize that the actual role of bacterial toxins in microbial pathogenicity has not been clarified. A number of the most fearsome human diseases are the result of intoxication by secreted bacterial toxins. Human disease as a consequence of an accidental contamination of a wound with the tetanus bacillus or the accidental ingestion of food contaminated with botulinum toxin is an individual human disaster, but it does not necessarily reveal the actual role of the toxin in the biology of Clostridium tetani or Clostridium botulinum. These organisms are not primary pathogensof humans, although their toxins presumably have evolved to play some role in their interaction with other eukaryotic life forms. Nontoxigenic variants of tetanus or the botu-linum bacterium are totally avirulent for humans.
Some toxigenic microbes are highly adapted to humans including Corynebacteriumdiphtheriae (diphtheria), and B. pertussis (whooping cough). Others such as V. cholerae are very toxic to humans but have a reservoir, presumably on or in a marine animal. For these A – B toxins, we understand the biochemical basis for toxigenicity and the indis-pensability of the toxins for the pathogenicity of the microorganism. We even understand that if we immunize individuals against these toxins, we can prevent disease. What we do not fully understand is the role of the toxin in the biology of the microorganism. The toxin cannot be so potent that it rapidly kills all of the hosts that are infected. Toxins may represent the principal determinant of bacterial virulence in some species but may not be the principal determinant of infectivity; however, it seems likely that toxins play a role in the establishment of the organism in the early phases of infection or they are elaborated only if the organism “senses” danger. Thus, V. cholerae devoid of cholera toxin does not colonize susceptible animals as well as toxigenic organisms, nor is it as efficiently trans-mitted. It is possible that the effects of cholera toxin, the induced net secretion of water and electrolytes into the lumen of the bowel, make conditions right for cholera replica-tion. On the other hand, nontoxigenic C. diphtheriae and B. pertussis can still colonize humans and be transmitted, although not as well as their toxigenic parents.
Currently, molecular cloning techniques, coupled with appropriate infection models, are leading to the elucidation of the roles of some toxins in the pathogenesis of infections. Not all toxins are essential for pathogenicity. For example, Shigella dysenteriae produces a very potent cytotoxin called Shiga toxin. Nontoxigenic variants of this organism are still pathogenic but are not as virulent. The high death rates associated with toxigenic S. dysen-teriae appear to be associated with damage done to the colonic vasculature by Shiga toxin.
The A – B toxin paradigm focused on the fact that a variety of distinct toxins harbored by a variety of distinct pathogens attached the ADP-ribose moiety from NAD to a preferred target molecule, generally a G protein that bound and hydrolyzed GTP. However, the B (binding) specificity of the toxins varies considerably. Thus, seemingly identical catalytic properties of toxin molecules have different effects in a host animal because the toxin binds to a different receptor molecule in the host. For example, the most potent neurotoxins known produced by the clostridia causing botulism and tetanus target four proteins (syntaxin, VAMP/synapto-brevin 1 and 2, and snap-25) that are involved in the docking of host cell vesicles and are involved with the release of neurotransmitters. Yet, each toxin is delivered differently and preferentially binds to different cell types when introduced into humans by accidental oral ingestion or by introduction by contaminated soil. Because these toxins were recognized to be introduced into host cells and functioned intracellularly, they became a favored reagent of cell biologists to investigate the normal biology of mammalian cells.
Some toxins, such as botulinum toxin, are used in medicine to relieve the effects of some nerve disorders. The recognition that many toxins are internalized in a membrane-bound vesicle from which the catalytically active A part has to escape into the cytoplasm led to the investigation of binding specificity within the toxin itself. In this vein, the A sub-unit of cholera toxin has a C-terminal motif that provides retention of the molecule in the endoplasmic reticulum; similar binding motifs are found in other toxic molecules. In re-cent years, the capacity of invading bacteria and other parasites to undermine the host cell biology with such exquisite sensitivity has become a hallmark of research into bacterial pathogenicity. Ten years ago, we scarcely dreamed that the study of bacterial toxins would provide such a wealth of information about human biology. The most avid medical micro-biologists did not think that such a diversity of bacterial toxins were yet to be discovered. For example, a number of bacterial toxins have been recognized that modifies proteins of the Ras superfamily, particularly the Rho subfamily. Some bacteria ADP-ribosylate Rho A, B, and C at a specific asparagine residue. Others, such as the bacterium Clostrid-ium difficile, a commensal that can cause severe diarrhea in patients whose flora has been suppressed by antibiotic therapy, glycosylate (add a glucose moiety) to their target and at-tack all members of the Rho subfamily (Rho, Rac, and CDC-42). Still others, such as the dermonecrotic toxin of the whooping cough bacillus, deamidate a glutamine residue in the Rho protein, changing it to glutamic acid, which, in the end, causes large-scale cy-toskeletal rearrangements.
While the A – B exotoxins and the toxins described thus far are, strictly speaking, intracel-lular toxins, a plethora of other bacterial toxins are described in the medical literature. Most of these are not well characterized, although many of them act directly on the sur-face of host cells to lyse or to kill them. They may facilitate penetration of host epithelial or endothelial barriers, and some toxins can kill white cells or paralyze the local immune system. Many bacteria elaborate substances that cause hemolysis of erythrocytes, and this property has been postulated to be an important virulence trait. In fact, some bacterial he-molysins are representative of general classes of bacterial exotoxins (the cytotoxins) that kill host cells by disrupting the host cell membrane. Moreover, hemolysins may liberate necessary growth factors such as iron for the invading microorganisms.
Among Gram-negative bacteria, a surprising number of these cytotoxins are members of a single family called the RTX (repeats in toxin) group based on a recurrent theme of a nine-amino-acid tandem duplication. RTX toxins are calcium-dependent proteins that act by creating pores in eukaryotic membranes, which may cause cellular death or at least a perturbation in host cell function. Such toxins are thought to be particularly effective against phagocytic cells. Other exotoxins contribute to the capacity of an organism to in-vade and spread. The lecithinase α-Toxin of Clostridium perfringens, for example, dis-rupts the membranes of a wide variety of host cells, including the leukocytes that might otherwise destroy the organism, and produces the necrotic anaerobic environment in which it can multiply.
Many bacteria produce one or more enzymes that are nontoxic per se but facilitate tissue invasion or help protect the organism against the body’s defense mechanisms. For exam-ple, various bacteria produce collagenase or hyaluronidase or convert serum plasminogen to plasmin, which has fibrinolytic activity. Although the evidence is not conclusive, it is reasonable to assume that these substances facilitate spread of infection. Some bacteria also produce deoxyribonuclease, elastase, and many other biologically active enzymes, but their function in the disease process or in providing nutrients for the invaders is uncertain. All are proteins and have most of the characteristics of exotoxin, except specific toxicity. Although many such factors have been thought to be involved in bacterial virulence, for-mal proof that they may contribute to pathogenicity has not been obtained in many cases.
In the past 5 years there has been a growing recognition that many Gram-negative bacteria have blocks of genes called pathogenicity islands (PAIs) , which are composed of a secretory pathway that delivers virulence factors into the cytoplasm of host cells. Several of these were described earlier when considering Salmonella invasion. The difference between these molecules and the classical bacterial toxins is that these vir-ulence molecules are not in and of themselves toxic but they induce host cellular damage like apoptosis. These factors are described in considerable detail later that de-scribes Salmonella, Shigella, and Yersinia .
It has become clear in recent years some microbial exotoxins have a direct effect on cells of the immune system and this interaction leads to many of the symptoms of disease. Thus, the enterotoxins causing staphylococcal food poisoning, the group A streptococcal exotoxin A responsible for scarlet fever, and the TSS exotoxin responsible for the staphy-lococcal toxic shock syndrome interact directly with the T-cell receptor. The effect of this interaction is dramatic. Cytokines such as IL-1 and TNF are produced, which leads to their familiar effects systemically and to local skin and gastrointestinal effects (depending on the toxin and its site of action). In addition, after binding to class II major histocom-patibility complex (MHC) molecules on antigen-presenting cells, these exotoxins act as polyclonal stimulators of T cells so that a significant proportion of all T cells respond by dividing and releasing cytokines. This eventually leads to immunosuppression for reasons that are not totally clear. When trying to assess these findings from the standpoint of bacte-rial pathogenicity, it is important to divorce the disease entity seen in ill patients from the potential role of these toxins in the normal life of the microorganism.
For example, staphylococcal food poisoning is an intoxication and does not involve infection by living microbes but rather the ingestion of the products produced by staphy-lococci in improperly handled food. The toxins that cause such food poisoning are resis-tant to digestive enzymes. Staphylococcal enterotoxins are also resistant to boiling, so that disease may follow ingestion of contaminated foods in which the organism has al-ready been killed. What then is the role of the toxin in the normal biology of the microbe? Although the complete answer to this question is unknown, it seems likely that the toxins would play a role in the interaction of the microorganism with local host defenses in its preferred human niche, on the skin and the mucosal surface. Here, at the microscopic level, the capacity to neutralize the antigen-presenting cells in the microcosm of the pores of the skin is clearly more important than the induction of vast systemic symptoms. Not all staphylococci carry the enterotoxin genes. Indeed, enterotoxin genes may be carried on plasmids or bacterial viruses. Perhaps, the staphylococci that carry such “superanti-gens” have an advantage over their competing brethren. Such questions need to be answered at the experimental level. We must examine the determinants of bacterial patho-genicity with an eye to their role in the biology of the microbe, as well as from the view that they play an essential role in relatively rare cases of overt disease.
Superantigens are not restricted simply to bacterial toxins of Gram-positive bacteria. Increasingly, they are reported as potential factors in the pathogenesis of viral infection and in a number of other bacteria. Moreover, polyclonal activation of other immune cells is seen, as in the activation of B cells by the Epstein – Barr virus. Hence, the interaction of microbial products directly with cells of the immune system that leads to immunosup-pression may be a common theme of microbial pathogenicity.
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