The biochemical challenge of microbial pathogenicity

Journai of Applied Bacteriology 1984, 47, 395404 I512/2/84

The biochemical challenge of microbial pathogenicity

H. SMITH The Department of Microbiology, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U K

Received February 1984

SMITH, H. 1984. The biochemical challenge of microbial pathogenicity. Journal of Applied Bacteriology 57, 395404.

In the past decade there has been a revival of interest in microbial pathogenicity. The reasons for this revival are two-fold. First, infectious disease is still with us despite the impact of the antibiotic era; for example, the rise of bacterial and fungal infections in compromised patients and the lack of a good general antiviral drug. Second, the subject of microbial pathogenicity is ripe for application of techniques of biochemistry, molecular biology and genetics that have developed in other areas of biology over the past twenty years; and the potential of these techniques is particularly attractive to young people, who are entering the field in increasing numbers.

In this lecture I shall survey the methods and difficulties of investigating microbial pathogenicity and what we know of the main aspects of the subject at the molecular level. I shall use bacteria as examples because more is known about them than other types of microbes. Lack of space prevents quoting original papers in such a wide-ranging task; in most cases reference is made to authorative reviews.

The multifactorial nature of microbial pathogenicity

The terms pathogenicity and virulence are syn- onymous; they mean the capacity to produce disease and both will be used in this lecture.

To be pathogenic a micro-organism must be able to :

1. Infect the mucous surfaces of the respiratory, alimentary or urogenital tracts. Some microbes are introduced into the host directly through the skin by trauma or vector bite but the majority of infections start on the mucous surfaces.

2. Enter the host usually by penetration of the mucous surfaces. A few microbes can cause disease by growing on the mucous surfaces, e.g. cholera bacilli, but the majority enter the tissues to cause disease.

3. Multiply in the environment of the host’s tissues.

4. Resist or interfere with host defence mechanisms that try to remove or destroy them.

5. Cause damage to the tissues of the host. All five steps, or the last three if there is direct

introduction into the tissues, must be accom- plished for pathogenicity. Loss of ability to carry out any one of the steps causes the microbe to lose virulence. The cardinal fact about pathogenicity is its multifactorial nature.

Methods of investigating pathogenicity

The aim is to identify the determinants ofpatho- genicity i.e. the microbial components and products responsible for the five essential requirements of pathogenicity outlined above.

The first essential is a method for measuring the virulence of different strains of the chosen pathogenic species. This can be done only by infecting animals, where the pathogen is growing under conditions different from those supplied in oitro (see later). Animals, preferably the natural host or a species suffering a similar disease, are inoculated with graded doses of the pathogen and the number of organisms is obtained which produce a standard disease effect such as killing half a group of animals - the 50% lethal dose (LD,,) - or producing a skin lesion of a certain size.

396 H . Smith The next step is to identify strains of high and

low virulence by comparing naturally occurring strains or those derived by genetic manipulation. The strains are then compared in biological tests which relate to one of the five requirements of pathogenicity mentioned pre- viously, such as ability to adhere to and penetrate mucosal cells or capacity to prevent ingestion by phagocytes. When relevant biological differences have been established, e.g. a virulent strain resists ingestion by phagocytes more than an avirulent strain, the biochemical basis for this difference is sought. This can be done in two ways. First, the virulent strain can be extracted for the microbial component or product responsible for the biological activity; this requires an assay of the latter for purification of the determinant (Smith 1983). Second, genetic manipulation of strains by classical and recombinant DNA techniques can indicate the determinant concerned. Strains are constructed which should differ only in the gene which codes for a particular microbial component or product; if they also differ in virulence and the particular biological aspect under consideration, then the gene product is also the determinant concerned (Sparling 1983; Weiss & Falkow 1983). An early example was the construction of strains of Escherichia coli enteropathogenic for piglets by transferring to avirulent strains two separate plasmids, one coding for the K88 antigen which enabled the bacteria to adhere to intestinal epithelium, and the other for the enterotoxin which caused scouring (Elwell & Shipley 1980).

There are difficulties in this general plan. Two arise in fractionation and purification of a putative determinant. First, there is the com- plexity of the biochemical task; the multifactorial nature of pathogenicity means that there is more than one determinant and each must be clearly distinguished from others. The second arises from the fact that many determinants of pathogenicity are surface components of microbes which are biologically active only in situ, e.g. capsular materials that interfere with phagocytosis (Smith 1983). Thus, because re- attachment to the surface is required, it is often impossible to complete the final definitive biological test on a purified putative determinant. This is to use it to confer the relevant biological property onto either the virulent strain from which the surface component was removed, or

an avirulent strain lacking it (Smith 1983). One way round the difficulty is to see if antiserum, preferably monoclonal, raised against the determinant, neutralises the relevant biological property of the virulent strain (Smith 1983; Sparling 1983).

The genetic approach also has its difficulties. The two strains being compared biologically must differ only in the DNA segment that codes for the putative determinant. This requires criti- cal genetic analysis of both genomic and plasmid DNA (Sparling 1983). Even if this analysis is forthcoming, pleiotropy may occur; the gene product itself may not be the determinant, but influence the production of other cell constituents, one or more of which is the true determinant. Hence, the genetic approach must be applied correctly to be effective and is not completely fail-safe.

The final difficulty lies in the fact that pathogenicity occurs and is measured only in uiuo, where the microbe is growing under different nutritional and environmental conditions from those we provide in the laboratory cultures that we use to examine microbes. Just as any other biological property, pathogenicity is determined not only by the microbial genome but also by environmental conditions. Hence, due to pheno- typic change or selection of types, a pathogenic microbe in uitro may not produce all the determinants of pathogenicity that are found when it is growing in animals during infection. In brief, organisms grown in uitro can be deficient in virulence determinants. So, if there appear to be gaps in knowledge of the pathogenicity of a particular microbe, a close look at its behaviour when it is growing in uiuo may reveal hitherto unknown facets of pathogenicity. These facets may then be reproduced in uitro later by making appropriate changes in nutritional conditions (Smith 1964). The toxin responsible for death from anthrax was discovered in this way (Smith & Keppie 1954). Recently, in studies of gonococcal resistance to complement-mediated serum killing, gonococci grown in uiuo were shown to be resistant, and to lose this resistance phenotypically on subculture. This change from resistance to susceptibility could be reversed by incubating in uitro with a serum fraction of small molecular weight, thus facilitating the search for the determinant of gonococcal resistance (Smith 1983).

In view of these difficulties it is not surprising

Microbial pathogenicity 397 that there are many gaps in our knowledge of pathogenicity and that often we cannot be absolutely certain that a surface component is the determinant of a particular aspect of pathogenicity (Smith 1983). In the following survey of knowledge of bacterial pathogenicity, I shall take each of the five requirements in turn and try to indicate how far research is at the observational level, i.e. when relevant biological properties have been recognized; or at the determinant stage, i.e. when a microbial component or product has been identified which is either responsible for the biological property or is strongly associated with it.

Infection of mucous surfaces

When a pathogen enters the alimentary, respiratory or urogenital tract the first requirement for infection is to make contact with the epithelial surface through the overlying mucus layer (Freter 1980). This will occur by chance, but work with Vibrio cholerae, Escherichia coli and Salmonella typhimurium indicates that attraction by a chemotactic gradient can be involved and that movement along the gradient is more rapid if the bacteria are motile (Freter et al. 1981).

Having made contact, the pathogen must adhere to the surface to prevent removal by moving lumen contents or mucociliary action. Recently there has been an explosion of interest in mechanisms of adherence and in some cases the determinants are known. A few examples are summarized here and others can be found in specific reviews (Berkeley et al. 1980; Beachey 1981). Strains of E. coli which cause diarrhoea in piglets, calves and human babies produce surface antigens, K88, K99 and colonization factor antigens I and I1 respectively, which are responsible for adherence of the bacteria to the surface of the intestine (Berkeley et al. 1980; Beachey 1981). The antigens are proteins and are plasmid coded. The receptor for the K88 antigen is the product of a dominant gene inherited by piglets in a simple Mendelian manner so that some piglets lack the receptor and are resistant to infection (Beachey 1981). Escherichia coli strains that cause infection of the urinary tract adhere to its epithelial cells. The bacterial determinants appear to be pili which are of two types, mannose-susceptible and mannose-resistant according to whether or not adherence occurs in the presence of

mannose, which blocks a mannoside receptor (Beachey 1981; Hagberg et al. 1983). The mannose-resistant pili, whose receptor appears to be globotetraosylceramide, seem to be associated with those strains causing the most severe urinary infections (LeMer & Svanborg-Eden 198 1). Streptococcus pyogenes causes throat infections and adheres to buccal epithelium ; the bacterial determinant concerned is lipoteichoic acid and its receptor may be an albumin-like protein (Berkeley et al. 1980; Beachey 1981). Finally, gonococci adhere to the mucous surface of the urogenital tract; pili appear to be one of the factors involved and their receptors may contain N-acetyl galactosamine and other sugars (Berkeley et al. 1980; Beachey 1981).

To maintain infection on mucous surfaces pathogens must be able to resist the host defence mechanisms found there, such as acid or alkaline pH, bactericidal materials and extruded phagocytes. Little is known, but presumably bacteria overcome these mucosal defences by devices similar to those used later within the tissues. These devices are described below, but one is more aptly described here because it occurs only on mucous surfaces. This is production of IgA proteases which hydrolyse IgA antibodies that are excreted onto mucous surfaces t o protect them from microbial attack (Kornfeld & Plaut 1981).

In the upper respiratory tract, the lower bowel and the vagina there are many naturally occurring micro-organisms, the commensals. These commensals provide powerful protection against invading pathogens (Freter 1980; Smith 1982). They occupy mucosal space needed for adherence by pathogens, use nutrients that pathogens require and produce inhibitors of their growth such as fatty acids, lactic acid and H,S (Freter 1980; Smith 1982; Freter et al. 1983). Although we are beginning to learn some- thing about the protective mechanisms of the commensals, we are still ignorant of how pathogens overcome these mechanisms in the early stages of infection. For example, what happens when a few shigellas produce dysentery in the lower bowel despite the enormous number of commensals present in this site?.

Finally, a few bacterial species, such as cholera vibrios, remain on the mucous surface and produce their disease effects without actually entering the tissues of the host. Most bacteria however, penetrate the mucosal epithe-

398 H . Smith

lium and spread elsewhere. What determines penetration or lack of it is not known.

In summary, we have made many observations on the different aspects of mucosal infection but the only facet at the determinant stage of research is adherence.

Entry to the host

Bacteria, such as gas gangrene or plague bacilli, can be introduced into the host directly by trauma or vector bite. Others, such as staphylococci and leptospiras, penetrate the skin without an external agency. Penetration is assumed to occur through minute abrasions and sweat glands, which could apply to many other bacteria that do not infect through the skin. Hence skin-penetrating bacteria may possess special attributes such as products that resist host secretions or even enzymes for penetration. If this is so, the determinants are unknown.

The main route of entry to the host is through the mucous membranes and the methods are known at the observational level. Elegant studies by light and electron microscopy show that some bacteria, such as dysentery bacilli, are ingested and retained by epithelial cells; others, such as gonococci, are ingested and pass through the cells into the sub-epithelial tissue and others, like Salmonella typhimurium pass through and between the cells (Smith 1977; McGee & Horn 1979; Formal et al. 1983).

There is much current interest in the determinants of penetration but limited knowledge. Work on Shigella flexneri is in the forefront. Cell-penetration has been studied in primates, in HeLa cells and in the conjunctivae of guinea- pigs (Smith 1977; Formal et al. 1983). The present position is summarised as follows. Entry is by engulfment and appears to depends on the bacteria being alive, possessing a complete 0- antigen with a dirhamnose repeat unit in its polysaccharide and having a large (mol. wt. 140 x lo6) plasmid which codes for proteins (Smith 1977; Sansonetti et al. 1982; Formal et al. 1983). In addition it should be noted that extracellular glycolipids (Osada & Ogawa 1977) and lipoproteins (Petrovskaya et al. 1979) from Sh. jiexneri have been reported to induce the engulfment of the bacilli by epithelial cells.

To sum up, much is known about epithelial penetration at the observational level but little at the determinant stage.

Multiplication in vivo

The fact that avirulence can arise from the ina- bility to grow in the host’s tissues was shown by injecting nutritionally deficient mutants of Sol- monella typhi into mice; lethal infection did not occur unless the purines or amino acids required by the mutants were also injected (Smith 1968). Most bacteria, however, can find sufficient nutrients in animal tissues to support some growth. The question is, at what rate? Rapid growth will increase the chance of establishing infection against the activities of the host defence mechanisms. Only in a few cases and early in infection, has the multiplication rate in uiuo been measured (Smith 1976). This rate was much slower than that seen in laboratory cultures, indicating that limiting nutritional conditions may exist in uiuo before tissue breakdown occurs. In the late stages of disease, e.g. in anthrax, the population increases, i.e. the result- ant of multiplication and destruction, can be high indicating increased availability of nutrients.

Knowledge of the factors that influence multiplication in uiuo is abysmal. The nutrients or other environmental factors that limit growth at the beginning of infection are largely unknown except for the restrictive influence of high oxygen tension on the growth of anaerobes and the influence of iron supply described below. Similarly, the actual nutrients that are made available for bacterial growth later in infection are not well known, although increased multiplication has been associated with the production of proteases, nucleases and glycosidases by Ps. aeruginosa, Streptococcus pneumoniue and Sh. Jexneri respectively (Cicmanec & Holder 1979; Firshein et a/. 1982; Prizont 1982). The only areas where we have information on the influence of nutritional factors on pathogenicity is in relation to iron supply (Griffiths 1983) and the effect of localization of nutrients on tissue specificity (Smith 1968).

Most bacteria need iron to grow and in uivo the amount of free iron is restricted by iron- complexing host proteins, transferrin and lacto- ferrin. Virulent strains of many pathogenic species grow well in uiuo, and in body fluids in uitro, but less virulent strains need extra iron. The former obtain suficient iron by secreting siderophores - iron chelating compounds - into the surrounding environment. A number of

Microbial pathogenicity 399 siderophores produced by pathogens are known (Smith 1977; Griffiths 1983) and that most investigated is enterochelin, a cyclic trimer of 2,3-dihydroxybenzoyl serine. I t is produced by E . coli and other enterobacteria in uiuo, and under iron-restricted conditions in uitrn (Griffiths 1983). Side-by-side with production of enterochelin, proteins are formed in the outer membrane of E. coli which act as receptors for the iron-bearing compound, thus facilitating its re-entry into the bacterial cell (Grifiiths 1983). The profound effect of iron restriction on the metabolism of E. coli was indicated by a change in its transfer RNAs compared with those found during growth without restriction (Griffiths 1983).

Corynebacterium renale in cattle and Proteus mirabilis in man cause severe kidney infection (Smith 1968). The growth of both bacteria is greatly stimulated by urea, which is concen- trated in the kidney. Both have powerful ureases which may aid the metabolic utilization of urea and also liberate ammonia which damages kidney tissue. Brucella spp. cause brucellosis which in humans and other animals is a rela- tively mild disease with the causative organisms showing no marked affinity for particular tissues. In pregnant cows, sheep, goats and pigs, however, there is a prolific growth of brucellas in the fetal placenta, chorion and fluids leading to gross tissue damage and the climax of the disease which is named contagious abortion. This enormous bacterial population is due to a growth stimulant for brucellas, erythritol, present in the susceptible fetal tissues but not in the maternal tissues, not even the maternal placenta (Smith 1968). Erythritol is not present in the fetal tissues of animal species such as man and mice which d o not suffer contagious abortion (Smith 1968). Thus, the presence of a small molecular weight nutrient for the pathogen determines tissue specificity in the susceptible host, and host specificity as regards the occurrence of contagious abortion.

In summary, little is known of the determinants of multiplication in uiuo but investigations that have been conducted show the way for future research.

Interference with host defence mechanisms

The many and varied defence mechanisms of the host cannot be described in detail here. In

summary, they consist of humoral and cellular factors. The former are in body fluids. The latter are polymorphonuclear and mononuclear phagocytes that wander all over the body and are also fixed in certain tissues such as the spleen. Some of these humoral and cellular mechanisms depend on activation of the same humoral system, complement. Both humoral and cellular defences act non-specifically at the beginning of any infection to prevent or reduce it. If infection occurs, in a few days both types of defence are strengthened by the specific immune response which usually eliminates the infection. Hence, to make progress against the host defences, a pathogenic bacterium must be able to inhibit or interfere with humoral factors, the phagocytes, complement and the immune response. These inhibitory activities will be taken in turn and a few determinants concerned with each will be described. A comprehensive coverage is not intended because the subject has received much attention recently and appropriate publications (Smith 1976, 1983 ; Schlessinger 1979; Densen & Mandell 1980; OGrady & Smith 1981; Falconi et al. 1984; Easmon et ul. 1983) will provide additional examples.

I N T E R F E R E N C E W I T H H U M O R A L B A C T E R I C I D I N S

The major humoral bactericidins are the later components of the complement cascade (see later) for Gram negative bacteria and lysozyme, 8-lysins and basic peptides for Gram positive bacteria (Smith 1976; Penn 1983). In tests with sera of various animals, where the bactericidin is often not clear, virulent strains of many bacterial species have been shown to be more resistant to killing than avirulent strains, for example Bacillus anthracis, E . coli and meningococci (Smith 1976; Braude 1981; Penn 1983). Typical determinants of this resistance are capsular poly-D-glutamic acid, a complete lipopolysac- charide (LPS) and capsular polysaccharides respectively for the three bacterial species mentioned.

I N T E R F E R E N C E W I T H THE A C T I O N

O F P H A G O C Y T E S

The four stages of the phagocytic defence system are: (1) mobilisation by inflammation; (2) chemotaxis towards bacteria; (3) attachment and

400 H . Smith ingestion by an engulfing process which is primed by humoral opsonins and places the bacteria within an intracellular vacuole - the phagosome; and (4) killing by oxygen dependent and other systems which involve discharge of cytoplasmic granules, the lysosomes, into the phagosome. Bacteria can inhibit one or more of these stages.

Mobilisation may be prevented by an active process, for example, virulent staphylococci produce an anti-inflammatory cell-wall peptidoglycan (Glynn 1972; Easmon 1984); or by a passive process, e.g. Treponema pallidum appears to be surrounded by an envelope which does not stimulate the inflammatory response (Penn 1983).

Virulent strains of many bacteria, for example meningococci, inhibit chemotaxis of phagocytes in vitro. Also, while many bacterial products stimulate chemotaxis, others are inhibitory such as the cord factor of Mycobacterium tuberculosis and the a-toxin of staphylococci (Densen & Mandell 1980; Wilton 1981). This inhibition of chemotaxis has yet to be proved relevant in vivo.

The resistance to attachment and ingestion by phagocytes of virulent, often capsulated, strains of many bacterial species, and the determinants concerned, was one of the first areas of bacterial pathogenicity to receive attention and new instances continue to be added (Smith 1976, 1977, 1983; Densen & Mandell 1980; Wilton 1981; Quie et al. 1981; Easmon 1984; Penn 1983). The capsular polysaccharides of the pneu- mococci, the M protein of Strep. pyogenes and the complete LPS of Salm. typhimurium are examples of many known determinants and exemplify the differing chemical compounds that show the same biological activity. In most cases, inhibition of ingestion has not been distinguished from inhibition of attachment but there are exceptions. For example, pilated E . coli attach to phagocytes but are not ingested if they also possess either a complete LPS or a K acid-polysaccharide capsule (Stendahl 1983). In the main, the determinants appear to work by masking specific interactions between bacterial cell-wall components and humoral opsonins (Quie et al. 1981). In some cases, however, a non-specific increase of surface hydrophilicity also appears to play a role (Stendahl 1983).

Several bacterial species, particularly those causing chronic diseases, resist intracellular killing and grow within phagocytes, thus finding

sanctuary from other host defences and some injected drugs. In some cases, the bacteria prevent phagolysosome fusion; in others they resist intraphagosome killing; in others they escape from the phagosome; and for some the mechanisms are not yet known; representative examples are Mycobacterium tuberculosis, M . lepraemurium, M . leprae and gonococci respectively (Densen & Mandell 1980; Draper 1981; Smith 1983, 1984). In a few cases, the determinants of interference with intracellular killing have been investigated but evidence for their responsibility is incomplete (Draper 198 1). However, surface sulphatides (glycol lipid sulphates), mycoside C (a peptidoglycolipid) and outer membrane proteins may play some role in the intracellular survival of M . tuberculosis, M . lepraemurium and gonococci respectively (Draper 1981; Smith 1984). The determinant of the escape of M . leprae from phagosomes is unknown.

Before leaving interference with the action of phagocytes, it should be mentioned that several bacterial toxins kill phagocytes in vitro; examples are the staphylococcal leucocidin, the anthrax toxin, the streptolysins and the leucocidin of Pseudomonas aeruginosa (Densen & Mandell 1980; Wilton 1981; Arbuthnott 1981). In smaller quantities, some of these toxins inhibit chemotaxis of leucocytes in vitro and prevent bacterial ingestion by phagocytes (Arbuthnott 1981). This action of these toxins has yet to be demonstrated during infection in vivo.

I N T E R F E R E N C E W I T H THE A C T I O N

O F C O M P L E M E N T

Resistance to one humoral bactericidin and to some aspects of the phagocyte defences can have the following common humoral origin. The complement cascade of nine or more components can be activated either by direct reaction with some bacterial cell-wall and surface components (the alternative pathway) or after their reaction with ‘natural’ antibody (the classical pathway), the latter being evoked by normal exposure to microbes and part of the ‘non-specific’ defence (Penn 1983). Acting alone or in consort with lysozyme, the terminal components of the complement cascade form the most important humoral bactericidin against Gram negative organisms (Braude 1981 ; Penn

Microbial pathogenicity 40 1

1983; Easmon 1984). Mobilisation of phagocytes and their chemotaxis towards bacteria are promoted by earlier components of the cascade which also opsonise the bacteria for attachment and ingestion by phagocytes (Wilton 1981; Quie et al. 1981; Penn 1983). Hence, preventing activation of complement or destroying its components can interfere with both humoral and cellular defences.

Some surface or capsular materials of bacteria have been strongly implicated in masking cell- wall components which would otherwise activate complement directly or after reaction with natural antibody. These surface materials include some of the determinants of interference with humoral and phagocytic defences mentioned above and additional examples are the capsular polysaccharides of type KI E . coli, Group B streptococci and Group B Haemo- philus injiuenzae (Smith 1983).

Some bacteria destroy the components of complement thereby preventing its action. For example, the elastase of Ps. aeruginosa destroys C,, C,, C, , C, and C, of the complement cascade (Densen & Mandell 1980; Quie et ul. 1981).

INTERFERENCE WITH THE IMMUNE

RESPONSE

Bacteria can interfere with the immune response either by directly suppressing it or by subverting its action.

Many pathogenic bacteria particularly those causing chronic infections, suppress the action of B and T cells by various mechanisms (Falconi & Campa 1981; Schwab 1983). In some cases the determinants are known, for example, the LPS of Ps. aeruginosa and peptidoglycan- polysaccharide complexes of Strep. pyogenes (Falconi & Campa 1981; Schwab 1983). Some- times the mechanism is clear, e.g., the LPS of Ps. aeruginosa appears to stimulate the production of suppressor B cells (Falconi & Campa 1981).

A full discussion of the methods whereby bacteria can subvert rather than suppress the immune response can be found elsewhere (Falconi & Campa 1981; Smith 1984). Three of these methods should be mentioned. First, the determinants of virulence can be bad antigens, i.e. they stimulate the immune response poorly or not at all. Thus, the determinants will not be completely neutralized by the immune response

and infection will persist. This may happen in chronic staphylococcal mastitis. Second, bacteria can undergo antigenic shift; when the host has responded immunologically to their surface antigens, new bacterial types emerge with different surface antigens and the immune response is rendered ineffective. This may happen in relaps- ing fever caused by Borrelia recurrent i s . Finally, the bacteria may hide away in cells such as epithelial cells or impaired mononuclear phagocytes (Smith 1984). Within these cells the bacteria are protected against host defence mechanisms and some injected drugs. This may happen in the carrier-state of typhoid fever and in chronic tuberculosis and brucellosis.

In summary, much knowledge on interference with host defence mechanisms is a t the determinant stage of research but many observed phe- nomena still require biochemical explanations.

Damage to the host

Pathogenic bacteria damage the host tissues in two ways, production of toxins and evocation of harmful immunological reactions.

P R O D U C T i O N OF TOXINS

Most pathogenic bacteria form poisons or toxins, many of which are produced in vivo and are responsible for the harmful, sometimes lethal, effects of disease (Stephen & Pietrowski 1981; Arbuthnott 1983). In a few cases, e.g. diphtheria and tetanus toxins, they are so powerful as to dominate the multifactorial nature of virulence. In most cases, however, toxins are one of a number of equally important determinants of virulence (Stephen & Pietrowski 198 1).

Most toxins are extracellular. Such toxins have been intensely studied and knowledge is often at the most sophisticated level of molecular biology. A few representatives of many toxins described elsewhere (Stephen & Pietrowski 1981 ; Arbuthnott 1983) are included here. Toxins can inhibit protein synthesis, produce fluid loss from the gut (enterotoxins), interfere with nerve action (neurotoxins), lyse cells (cytotoxins) and cause vascular effects. The diphtheria, cholera, tetanus, Clostridium perfringens and anthrax toxins, respectively, are examples of such toxins. Many toxins consist of

H . Smith more than one component and in some cases the individual role of each component is known. One component of the diphtheria toxin promotes entry of the other component into host cells. The second component then shuts off host cell protein synthesis by catalysing an ADP ribosylation reaction between nicotinamide adenosine dinucleotide (NAD ') and elongation factor 2 (EF2), thereby inactivating the latter which is required for protein synthesis (Stephen & Pietrowski 1981). Similarly, cholera toxin has one component which promotes entry of a second component into intestinal epithelial cells where it causes fluid excretion by increasing the production of cyclic adenosine monophosphate (AMP). Again, an ADP ribosylation reaction with NAD' is involved, but here an inhibitor which normally keeps adenyl cyclase, the enzyme that produces cyclic AMP, in check, is inactivated by the reaction (Stephen & Pietrowski 1981 ; Arbuthnott 1983). Tetanus toxin may have two components; it acts in the central nervous system to prevent the release or action of the inhibiting neurotransmitter, glycine. Spastic paralysis of muscle occurs because stimulation of motor neurones which lead to muscle contraction is not abolished by the glycine (Stephen & Pietrowski 1981). The toxin of C1. perfringens is a single component enzyme ~ phospholipase C - which lyses cells by hydrolysing lecithin in host-cell membranes. The anthrax toxin consists of three components (Stephen & Pietrowski 1981) one of which is an adenylcyclase (Leppla 1982), although its role in toxicity is not yet clear.

The most important cell-bound toxins are the endotoxins in the cell-walls of Gram negative bacteria such as E . cofi, V . cholerae, Salm. typhi and meningococci. They are lipopolysaccharides whose toxicity appears to lie in the lipid portion, lipid A (Stephen & Pietrowski 1981). When extracted, purified endotoxins from a variety of different bacteria cause similar toxic effects, notably fever and vascular disturbances which can lead to fatal secondary shock (Stephen & Pietrowski 1981). The relevance of endotoxins to infectious disease depends on their release in uiuo. In some cases, e.g. cholera, endotoxin is not released. In others, e.g. typhoid fever, meningococcal meningitis and 'Gram negative shock' caused by a variety of species, endotoxin is released to produce profound and often lethal effects (Stephen & Pietrowski 1981).

D A M A G E B Y I M M U N O P A T H O L O G I C A L

M E C H A N I S M S

Bacteria can produce severe, even fatal, effects on the host by stimulating an immune response to otherwise non-toxic bacterial components, either during a primary infection or early in a chronic infection; and then interacting with this response in a subsequent infection or late in a chronic infection in a manner which damages tissue - immunopathology (Parish 1972; Zab- riskie 1983). There are four types of immunopathological reaction (Gel1 & Coombs 1968).

Type I reaction is anaphylaxis or immediate hypersensitivity where IgE antibody is present on mast cells and reaction with antigen releases histamines. The latter produce the vascular and respiratory effects seen in hay fever and asthma. Anaphylaxis is rare in bacterial infection but may occur in lobar pneumonia (Parish 1972).

Type I1 reaction is a cytotoxic reaction where antigen is present on host cells and antibody reacts with these cells to prime them for lysis by complement or destruction by phagocytes. This reaction is often seen in virus disease where viral components are inserted into the membrane of infected host cells (Mims 1983). In bacterial disease it is seen when the antigens on host cells are similar to those of the bacteria; the bacterial antigens evoke antibody, which then reacts with normal host cells - an autoimmune effect. This appears to happen in heart damage and other rheumatoid sequelae to streptococcal infection (Parish 1972; Zabriskie 1983).

Type 111 or Arthus type reactions occur when antigen-antibody complexes are deposited on tissue, for example on the glomerulo-membranes of kidney tissue. The complexes fix complement and attract phagocytes which release their enzymes to damage tissue. This seems to happen in kidney damage following infections with Pr. mirabilis and streptococci (Parish 1972; Zab- riskie 1983).

Type IV are delayed hypersensitivity reactions. Cell mediated immunity (CMI) stimulated by previous interaction with the bacteria, mobi- lises mononuclear phagocytes to the site of additional or persistent infection; enzymes released from the phagocytes damage tissue. This occurs in tuberculosis.

Another damaging phenomenon should be mentioned here because the effects are similar to immunopathological effects. Sometimes after

Microbial Pathogenicity 403 live organisms have been killed by host defences, the remains of these organisms, for example cell-wall components from streptococci, may persist in the tissues for long periods. The persisting antigens activate complement by the alternative pathway and the resulting chronic inflammation can produce arthritis (Chetty et al. 1982; Schwab 1983).

In summary, the study of toxins is clearly a t the determinant level but the determinants of immunopathological damage are far less clear.

Conclusions: studies on viruses, fungi and protozoa

This survey of knowledge of bacterial pathogenicity shows that although much remains to be done, some determinants of all five of the important aspects of pathogenicity have been identified. The position is much less satisfactory with regard to studies on the pathogenicity of viruses, fungi and protozoa. For example, in a recent symposium (Smith et al. 1983) where the determinants of bacterial and viral pathogenicity were compared and contrasted, only one aspect of viral pathogenicity ~ multiplication in vioo - was clearly a t the determinant level. Little is known about viral interaction with and penetration of mucous surfaces. Also, although there is much information on the host defence mechanisms against virus infection and viral interference with these defences has been observed, the determinants are largely unknown. Similarly, damage of host cells by direct viral cytotoxicity and by immunopathological mechanisms have been observed but studies on the biochemistry involved have only just begun.

Clearly, the progress on pathogenic bacteria should be extended to other types of micro- organisms.

References

ARBUTHNOTT, J.P. 1981 Membrane-damaging toxins in relation to interference with host defence mechanisms. In Microbial Perturbation of Host Defences ed. O’Grady, F. & Smith, H., pp. 97-120, London: Academic Press.

ARBUTHNOTT, J.P. 1983 Host damage by bacterial toxins. Philosophical Transactions of the Royal Society, Series B 303, 149-165.

BEACHEY, E.H. 1981 Bacterial adherence: adhesin- receptor interactions mediating the attachment of

bacteria to mucosal surfaces. Journal of Infectious Diseuses 143, 325-345.

BERKELEY, R.C.W., LYNCH, J.M., MELLING, J., RUTTER, P.R. & VINCENT, B. 1980 Microbial Adhe- sion to Surfaces. Chichester: Ellis Horwood Ltd.

BRAUDE, A.I. 1981 Bacterial interference with non- specific non-phagocytic defences. In Microbial Per- turbation ofIIost Defences ed. O’Grddy, F. & Smith, H., pp. 31-48, London: Academic Press.

Soluble peptidoglycan-polysaccharide fragments of the bacterial cellwall induce acute inflammation. Infection and Immunity 38, 1010-1019.

CICMANEC, J.F. & HOLDER, LA. 1979 Growth of Pseudomonas aeruginosa in normal and burnt skin extracts: role of extracellular proteases. Injection and Immunity 25,477483.

DENSEN, P. & MANDELL, G.I. 1980 Phagocyte strategy vs. microbial tactics. Reviews qf Infectious Diseases

DRAPER, P. 1981 Microbial inhibition of intracellular killing. In Microbial Perturbation of Host D<fenc,es ed. OGrady, F. & Smith, H., pp. 143-164, London: Academic Press.

EASMON, C.S.F. 1984 Inhibition of non-specific humoral and phagocytic defence mechanisms by bacteria. In Bacterial and Viral Inhibition and Immuno-modulation of Host Defences ed. Falconi, G., Campa, A,, Smith, H. & Scott, G.M., pp. 61-74, London: Academic Press.

EASMON, C.S.F., JELJASZCWICZ, J., BROWN, M.R.W. & LAMBERT, P.A. 1983 Medical Microbiology 3 - The Role of the Entielope in Survival of Bacteria in Jnfec- tion. London: Academic Press.

ELWELL, L.P. & SHIPLEY, P.L. 1980 Plasmid mediated factors associated with virulence of bacteria in animals. Annual Reviews of Microbiology 34, 465- 495.

FALCONI, G. & CAMPA, A. 1981 Bacterial interference with the immune response. In Microbial Pertur- bation of Host Defences ed. O’Grady, F. & Smith, H., pp. 185-210, London: Academic Press.

FALCONI, G., CAMPA, A,, SMITH, H. & SCOTT, G.M. 1984 Bacterial and Viral Inhibition and Immuno- modulation of Host Defences. London: Academic Press.

FIRSHEIN, W., GROSS, D. & Russo, J. 1982 Changes in the serum of mice infected with Streptococcus pneu- moniae that stimulate in vitro multiplication of virulent but not avirulent strains. Journal of Medical Microbiology 15, 163-172.

FORMAL, S., HALE, T.L. & BOEDEKER, E.C. 1983 Inter- actions of enteric pathogens and the intestinal mucosa. Philosophical Transactions of the Royal Society, Series B 303, 65-73.

FRETER, R . 1980 Prospects for preventing association of harmful bacteria with host mucosal surfaces. In Bacterial Adherence ed. Beachey, E.H., pp. 441458, London: Chapman Hall.

M.M. & CAREY, K.E. 1983 Survival and implanta- tion of Escherichia coli in the intestinal tract. Infec- tion and Immunity 39,686-703.

FRETER, R., O’BRIEN, P.C.M. & MACSAI, M.S. 1981

CHETTY, c . , KLAPPER, D.G. & STHWAB, J.H. 1982

2,817-838.

FRETER, R., BRICKNER, H., FEKETE, J., VICKERMAN,

H . Smith Role of chemotaxis in the association of motile bacteria with intestinal mucosa: in vivo studies. lnfec- tion and Immunity 34,234-240.

CELL, P.G.H. & COOMBS, R.R.A. 1968 Clinical Aspects of Immunology. Oxford: Blackwell Scientific Pub- lications.

GLYNN, A.A. 1972 Bacterial factors inhibiting host defence mechanisms. Symposium of the Society for General Microbiology 22, 75-1 12.

GRIFFITHS, E. I983 Adaptation and multiplication of bacteria in host tissues. Philosophical Transactions ofthe Royal Society, Series B 303, 85-96.

HAGBERG, L., HULL, R., HULL, S., FALKOW, S., FRETER, R. & SVANBORG-ED~N, C. 1983 Contribu- tion of adhesion to bacterial persistence in the mouse urinary tract. Infection and Immunity 40, 265-272.

KORNFELD, S.J. & PLAUT, A.G. 1981 Secretory immunity and bacterial IgA proteases. Reviews of lnfec- tious Diseases 3, 521-534.

LEFFLER, H. & SVANBORG-ED~N, C. 1981 Glycolipid receptors for uropathogenic Escherichia coli on human erythrocytes and uroepithelial cells. lnfec- tion and Immunity 34,920-929.

LEPPLA, S.H. 1982 Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclic AMP concentrations in eukaryotic cells. Proceedings of the National Academy of Sciences, U S A 79, 3162- 3 166.

MCGEE, Z.A. & HORN, R.G. 1979 Phagocytosis of gonococci by non-professional phagocytes. In Microbiology - 1979 ed. Schlessinger, D., pp. 158- 161, Washington DC: American Society for Micro- biology.

MIMS, C.A. 1983 Immunopathology of virus diseases. Philosophical Transactions of the Royal Society, Series B 303, 189-198.

O’GRADY, F. & SMITH, H. 1981 Microbial Pertur- bation of Host Defences. London: Academic Press.

OSADA, Y . & OGAWA, H. 1977 A possible role of glycolipids in epithelial cell penetration by virulent Shigella fiexneri 2a. Microbiology and Immunology

PARISH, W.E. 1972 Host damage resulting from hypersensitivity to bacteria. Symposium for the Societyfor General Microbiology 22, 157-192.

PENN, C.W. 1983 Envelope and humordl defences. In Medical Microbiology 3 - Role of the Envelope in the Survival of Bacteria in Infection ed. Easmon, C.S.F., Jeljaszewicz, J., Brown, M.R.W. & Lambert, PA., pp. 109-135, London: Academic Press.

PETROVSKAYA, V.G., BONDARENKO, V.M. & MIROLYU- BOVA, L.V. 1979 The problem of interaction of Shi- gella with epithelial cells. Zentralblatt f u r Bakteriologie und Hygiene I Abt. Orig. 243, 57-73.

PRIZONT, R. 1982 Degradation of intestinal glycopro- tein by pathogenic Shigella ,$exneri. Infection and Immunity 36,615-620.

QUIE, P.G., GIELBKINK, G.S. & PETERSON, P.K. 1981 Bacterial mechanisms for inhibition of ingestion by phagocytic cells. In Microbial Perturbation of Host Defences ed. O’Grady, F. & Smith, H., pp. 121-142,

21,405-41 1 .

London: Academic Press. SANSONETTI, P.J., KOPECKO, D.J. & FORMAL, S.B. 1982

Involvement of a plasmid in invasive ability of Shi- qellajlexneri. Infection and Immunity 35, 852-860.

S~HLESSINGER, D. 1979 Microbiology - 1979. Wash- ington DC: American Society for Microbiology.

SCHWAB, J. 1983 Bacterial interference with immuno- specific defences. Philosophical Transactions of the Royal Society, Series B 303, 123-135.

SMITH, H. 1964 Microbial behaviour in natural and artificial environments. Symposium of the Society for General Microbiology 14, 1-29.

SMITH, H. 1968 Biochemical challenge of microbial pathogenicity. Bacteriological Reuiews 32, 1641 84.

SMITH, H. 1976 Survival of vegetative bacteria in animals. Symposium of the Society for General Microbiology 26,299-326.

SMITH, H. 1977 Microbial surfaces in relation to pathogenicity. Bacteriological Reviews 41,475-500.

SMITH, H. 1982 The role of microbial interactions in infectious disease. Philosophical Transactions vf the Royal Society, Series B 297, 551-561.

SMITH, H. 1983 The elusive determinants of bacterial interference with non-specific host defences. Philo- sophical Transactions of the Royal Society, Series B

SMITH, H. 1984 Bacterial subversion rather than sup- pression of immune defences. In Bacterial ba id Viral Inhibition and Immunomodulation of Hosr Drfhnces ed. Falconi, G., Campa, A., Smith, H. & Scott, G.M., pp. 171-190, London: Academic Press.

SMITH, H., ARBUTHNOTT, J.P. & MIMS, C.A. 1983 The Determinants of Bacterial and Viral Pathogenicity. London: The Royal Society.

SMITH, H. & KEPPIE, J. 1954 Observations on experi- mental anthrax, demonstration of a specific lethal factor produced by Bacillus anthracis in oivo. Nature, London 173,869-871.

SPARLING, P.F. 1983 Applications of genetics to studies of bacterial virulence. Philosophiral Truns- actions ofthe Royal Society, London B 303, 199-207.

STENDAHL, 0. 1983 The physicochemical basis of surface interaction between bacteria and phagocytic cells, In Medical Microbiology 3 - Rolc of the Envelope in the Survival uf Bacteria in Infection ed. Easmon, C.S.F., Jeljaszewicz, J., Brown, M.R.W. & Lambert, PA., pp, 137-152, London: Academic Press.

STEPHEN, J. & PIETROWSKI, R.A. 1981 Bacterial Toxins. Walton-on-Thames: Thomas Nelson.

WEISS, A.A. & FALKOW, S. 1983 The use of molecular techniques to study microbial determinants of pathogenicity. Philosophical Transactions of the Royal Society, Series B 303,219-225.

WILTON, J.M.A. 1981 Microbial interference with inflammation and phagocyte function. In Microbial Perturbation of Host Defences ed. O’Grady, F. & Smith, H., pp. 67-96, London: Academic Press.

ZABRISKIE, J.B. 1983 Immunopathological mechanisms in bacteria-host interactions. Philosophical Transactions of the Royal Society, Series B 303, 177-187.

303,99-113.

Documents

The biochemical challenge of microbial pathogenicity