Virulence determinants of Escherichia coli: present knowledge and questions HARRYSMITH
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Medical School, University of Birmingham, Birmingham BI5 2TT, United Kingdom Received June 17, 1991 Revision received December 6, 1991 Accepted January 3, 1992 SMITH,H. 1992. Virulence determinants of Escherichia coli: present knowledge and questions. Can. J. Microbiol. 38: 747-752. This paper describes the present state of research on the pathogenicity of Escherichia coli and points out the gaps in knowledge that should be filled in the future. First, the great versatility of E. coli in producing disease is noted, as well as the invaluable contributions that studies of it have made to the development of general knowledge on bacterial pathogenicity. Then, the biological requirements for pathogenicity: infection of mucous surfaces; penetration of those surfaces; multiplication in vivo; interference with host defence mechanisms; and damage to the host, are taken in turn, and an enquiry is made on how far studies have progressed toward identifying their molecular determinants and relating structure to biological action. Only for mucous surface adhesins and protein toxins are studies at the structure-function level. Some progress has been made on interference with host defence, but little is known about competition with commensals on mucous surfaces, invasion into the tissues, and growth in vivo. Key words: virulence determinants, Escherichia coli. SMITH,H. 1992. Virulence determinants of Escherichia coli: present knowledge and questions. Can. J. Microbiol. 38 : 747-752. Cet article decrit l'etat present de la recherche au sujet de la pathogenicite d'Escherichia coli et il souligne les lacunes des connaissances actuelles qui devraient &re comblees dans le futur. Premierement, l'auteur constate la grande versatilite d'E. coli a produire la maladie et il indique les contributions inestimables que les etudes sur ce sujet ont apportees au developpement des connaissances generales sur la pathogenicite bacterienne. Ensuite, les exigences biologiques de la pathogenicite sont prises en compte comme l'infection des surfaces muqueuses, la penetration de ces surfaces, la multiplication in vivo, l'interference avec les mecanismes de defense de l'h6te et le dommage a l'h6te; une investigation a ete amenee sur la progression des etudes concernant l'identification de leurs determinants moleculaires et rattachant la structure a l'action biologique. Les etudes au niveau structure-fonction ont ete realisees seulement pour les adhesines des surfaces muqueuses et pour les toxines proteiques. Un certain progres a ete accompli sur l'interference avec la defense de l'hhte, mais peu est connu au sujet de la competition avec les commensaux sur les surfaces muqueuses, l'invasion dans les tissus et la croissance in vivo. Mots clks : determinants de la virulence, Escherichia coli. [Traduit par la redaction]
Introduction When Dr. Fairbrother asked me to sum up this symposium I was taken by surprise because I have never worked on the pathogenicity of Escherichia coli. Faced with the task, I decided that, rather than summarizing what the contributors had said already, it might be more profitable to provide a view, so to speak from the outside, on the present state of research on the pathogenicity of E. coli and the gaps that should be filled in the future. I apologise in advance for any mistakes that I might make owing to lack of expertise. Before giving this view, I wish to make two points that struck me forcibly during some background reading. First, there is the versatility of E. coli. It is a normal commensal of the gut, and strains such as K12 have been used as safe tools for research and teaching over decades. Yet other strains produce severe diseases of widely differing character in man (enteritis, urinary tract infections, bacteraemia, and meningitis) and animals (pre- and post-weaning scours, oedema disease, and mastitis). Even if we focus on enteritis, there is great variation in the mechanisms whereby various strains produce their harmful effects. Thus, enterotoxigenic (ETEC; traveller's diarrhoea), enteroinvasive (EIEC; dysentery-like), enteropathogenic (EPEC; infant diarrhoea), enterohaemorrhagic (EHEC; haemorrhagic intestinal lesions), and enteroadherent aggregative (EAggEC; persistent infant diarrhoea) strains are known (Law 1988; Knutton Printed in Canada / Imprime au Canada
1990), and probably others will be discovered. For example, there is debate at present on the importance in disease of strains that show diffuse adherence (i.e., attachment over the entire cell surface) to tissue culture cells (Mathewson and Cravioto 1989). Coupled with this wonder at the versatility of E. coli is an admiration for the way in which the @rskovshave related classical 0 , H , and K serotyping of strains to disease syndromes (@rskov and @rskov 1983). In their contribution to this symposium, they distil the work of years down to a few important conclusions. There are many E. coli serotypes, but relatively few cause disease. The latter constitute two groups. One group relates to diarrhoea1 disease in man and animals, and the serotypes are largely specific to the host species in which the disease occurs. The second group of different serotypes relate to invasive extraintestinal disease and come mostly from humans. Basically, the 0 , H , and K serotyping is a marker for two groups of pathogenic E. coli, each composed of a limited number of clones with similar virulence mechanisms. My second point is the invaluable contribution that studies on E. coli have made to the general development of knowledge on bacterial virulence. Two factors have been responsible; production of similar infections in man and animals and knowledge of the genetics of E. coli. Studies of pathogenicity can be plagued by lack of valid animal
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models for human disease. Escherichia coli has the advantage that it produces similar diseases in animals and man, notably enteritis. This allows investigations on pathogenicity to proceed in the former with the probability that the results will apply to the latter. The original studies on the role of K88 fimbrial antigen in adhesion of E. coli to gut surfaces in piglet diarrhoea (Orskov and Orskov 1966; Jones and Rutter 1972) were extended to E. coli infections of man (Klemm 1985; Hacker 1992). More importantly, they sparked off the explosion of research on the determinants of bacterial adhesion to mucous surfaces that has occurred for many different pathogens over the past 20 years (Arp 1988). Also, although studies on cholera toxin led the intense interest in enterotoxins, those on the heat-labile (LT) and -stable (ST) toxins of E. coli were not far behind (Robertson 1988). The point about an animal model is underlined by Dr. Gyles (1992) in .this symposium. He notes that the role of E. coli verotoxins in disease is less firmly established than for the enterotoxins, because, unlike the latter, verotoxinrelated diseases of man are not simulated by corresponding animal diseases. Escherichia coli has, for 40 years, been the main tool for development of microbial genetics and molecular biology. When these sciences were first applied seriously to problems of pathogenicity, E. coli was the most appropriate species to use because methods of genetic manipulation were already available. The seminal experiments were those of Smith and Linggood (1971) on E. coli enteritis of piglets. They demonstrated, for the first time, plasmid-mediated transfer of virulence attributes, namely mucosal adherence and enterotoxin production. Developments were rapid (Elwell and Shipley 1980). Now, it is standard practice to use plasmids for manufacturing strains of high and low virulence, often with tailored virulence determinants. Gene cloning, perhaps the most used technique in studies of pathogenicity (Falkow 1988), first showed its strength in the field by adding weight to the evidence that the adherence antigens, the LT and ST, and the haemolysins of E. coli are virulence determinants relevant in vivo (Shipley et al. 1979; Elwell and Shipley 1980; Weiss and Falkow 1983). This was because methods for gene cloning in E. coli were well established in fields other than pathogenicity. Similarly, the difficult task of cloning all the genes responsible for production of capsular polysaccharides was first accomplished for K1 E. coli (Silver et al. 1981). Turning to the present state and possible gaps in research on the pathogenicity of E. coli, I will first explain the template on which I shall base the discussion. The biological requirements for pathogenicity are abilities to multiply in the environment of the host, to interfere with host defence mechanisms, and to damage the host. Since most diseases are contracted on or over mucous surfaces, two additional qualities are usually needed, infection and penetration of mucous surfaces. The compounds responsible for the multifactorial property are the determinants of virulence and the goal is to recognise each determinant, to identify it, and to relate its chemical structure to biological action (Smith 1988). There are seven logical steps to attaining this goal: (1) establish a method for comparing the virulence of strains in the natural host or a relevant animal model; (2) obtain strains of high and low virulence; (3) compare strains of differing virulence in biological tests related to the five requirements for pathogenicity; (4) identify the determinant that causes the chosen biological property; (5) prove relevance of the
biological property and its determinant to infection in vivo; (6) obtain the chemical structure of the determinant; and (7) relate the structure to biological action (Smith 1988). I shall now take a look at the five biological requirements for pathogenicity and enquire how far, for E. coli, we have progressed up this research ladder; I shall pay more attention to the gaps in knowledge because other symposium participants have already covered the successes.
Infection of mucous surfaces To infect mucous surfaces, a pathogen must be able to compete successfully with any commensals that may be present, to make contact with the surface through the overlying mucus, to adhere either to the surface or to the mucus, and to interfere with local humoral and cellular defences. Only in the case of adherence has there been substantial progress up the research ladder. The protective effect of the normal flora on some mucous surfaces is well proven. How it is overcome by relatively small numbers of E. coli and other pathogens, e.g., in the lower bowel, is a mystery. Research is sparse and at step 3, biological observation. The most notable worker, R. Freter, used E. coli to show some factors involved: successful competition for carbon and energy sources in anaerobic conditions and in the presence of H2S; adherence to sites not used by the indigenous flora; and a short lag phase so that multiplication occurs before removal by the moving lumen contents (Freter 1988). However, far more work is needed before the determinants of this essential stage in mucosal infection are revealed. Gaining access to epithelial surfaces through the overlying mucus is attracting more attention, but again, studies are at step 3. Collapse of the mucus layer during fixation of specimens for electron microscopy makes it difficult to distinguish bacteria in the mucus and those on the surface (Freter 1988). Chemotaxis and natural movement to the villi and crypts along the lines of stress of moving mucus appear to be two mechanisms for traversing the mucus (Freter 1988). Growth in mucus also appears important for E. coli (Cohen 1990). Mucus contains receptors for E. coli fimbriae. They can retain the bacteria in the mucus and block interaction with epithelial cell receptors (Cohen 1990; Conway et al. 1990). Some strains of E. coli produce a mucinase (Law 1988), but whether this is relevant to growth in or penetration of mucus in vivo is not clear. The amount of research on adhesion of E. coli to mucous surfaces is matched by the number of excellent reviews on the subject (Klemm 1985; Reid and Sobell 1987; Law 1988; Arp 1988; Isaacson 1988; Wadstrom et al. 1990; Hacker 1992). Hence, I will confine myself to making a few overall points. There are fimbrial adhesins, nonfimbrial adhesins, and special structures such as curlis (Olsen et al. 1989). Many of them are expressed in vivo and are relevant to pathogenicity (step 5 of the ladder). New adhesins for various types of E. coli continue to be found. Both chromosomal and plasmid genes code for adhesins, and in many instances, their regulation has been clarified. In his symposium presentation Jorg Hacker has described environmental factors that affect regulation. It is particularly intriguing that one adhesin gene cluster can influence the expression of other adhesin determinants so that cross "talk" may occur during infection. For many fimbrial adhesins, research is at the structural stage, step 6, of the research ladder.
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SMITH
Amino acid sequences have been determined for the major units and, in some cases, for the minor units that appear to determine specificity of adhesion. Research on nonfimbrial adhesins has not yet attained this structural level. Attention has also been given to the nature of host-cell receptors, such as D-mannosides for type 1 fimbriae, a-Dgalacto-(1-4)-P-D-galacto groups for P fimbriae, P-Dgalactoside groups for K88 fimbriae, and a-sialyl acid-2-3-Pgalactosamine groups for S fimbriae (Klemm 1985; Parkkiner et al. 1986; Arp 1988; Isaacson 1988; Olsen et al. 1989; Wadstrom et al. 1990). In most cases, the location and distribution of receptor sites on mucosal epithelium are not known (Klemm 1985; Isaacson 1988). Finally, the last step in the research ladder, the relationhip between molecular structure and adhesion, is just beginning; site-directed mutation has revealed the probable active sites on the sialic acid binding subunit of S fimbriae (Marschhauser et al. 1990). Compared with the effort on adhesion, the attention given to resistance to host defences on mucous surfaces is minute. During intestinal infection, neutrophils are mobilised within and on the surfaces (Hadad and Gyles 1982; Sellwood et al. 1986) and humoral bactericidins will also be present. Presumably, capsular polysaccharides and smooth (i.e., with a complete side chain) lipopolysaccharides (LPS) inhibit these defences by the mechanisms described later. Capsulated E. coli have been seen on intestinal surfaces (Hadad and Gyles 1982). The Brskovs have mentioned that 0 antigen of LPS might be a virulence factor. I am surprised, therefore, that LPS from the serogroups related to pathogenicity appear not to have been compared with the LPS of nonpathogenic serogroups for capacity to interfere with serum killing of E. coli and its ingestion by phagocytes. Another surprise, it appears that an IgA protease has not been reported for E. coli.
Penetration of mucous surfaces There are two processes to consider: penetration into the cells of the mucous surfaces by EIEC strains to cause a dysentry-like illness; and complete passage through the surface leading to bacteraemia, meningitis, and other invasive manifestations. The first aspect has received some attention but not, as far as I can judge, the second. Sparked by studies on shigellae (Sansonetti et al. 1982a; Maurelli and Sansonetti 1988; Hale and Formal 1988; Sansonetti 1990), an explosion of research on the molecular basis of epithelial cell invasion has occurred using mainly tissue culture systems but with adequate tests in vivo (Falkow et al. 1987; Finlay and Falkow 1989). Shigellae, Yersinia, and Salmonella species have received far more attention than E. coli (Hale and Formal 1988; Finlay and Falkow 1989). The seminal discovery was that large plasmids, 140 and 120 MDa, respectively, determined epithelial cell invasion by Shigella flexneri and Shigella sonnei (Sansonetti et al. 1982a, 1982b). Subsequent analysis of the plasmid for S. flexneri (Sansonetti 1990) showed that two genes code for two polypeptide invasins (ipaB, 62 kDa; ipaC, 48 kDa). Almost immediately after the discovery of the plasmid in S. flexneri, a similar large plasmid of 140 MDa was found in EIEC strains of E. coli (Sansonetti et al. 1982b; Maurelli and Sansonetti 1988). Over recent years, however, the invasion plasmid of EIEC strains has received little attention, probably on the assumption that findings on shigellae apply to E. coli.
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As far as I am aware, the mechanisms and determinants of complete penetration of mucous surfaces by invasive strains of E. coli have not been investigated. Perhaps haemolysins produced by such strains should be studied in this respect.
Growth in vivo Growth rate is important in pathogenicity. If it is fast, the pathogen can overwhelm the initial nonspecific defences and cause sickness before the more powerful immune defences are fully operative. A slow-growing pathogen is more vulnerabe to both types of defence. At present, lack of knowledge on growth rate in vivo is the major gap in studies of bacterial pathogenicity (Smith 1989). Mounting the research ladder has hardly begun. There are no reliable, universally accepted, methods for measuring growth rate, and the nutrients and other environmental factors that determine it are largely unknown and unstudied. Escherichia coli has, however, been prominent in the little work that has been done. The first attempts to measure doubling times in vivo (as distinct from increase in population) relied on a marker distributing to only one of two daughter cells in each succeeding generation (Maw and Meynell 1968; Polk and Miles 1973). Genetic manipulation (abortive transduction and superinfecting phage) was needed to introduce the marker so the method was restricted to salmonellae and E. coli. It indicated slow growth in mice (doubling time of 3-5 h). More recently, the increase in ratios of wild-type organisms (which multiply in vivo) to those of temperature-sensitive mutants (which should not multiply in vivo) has been used to measure growth rate of E. coli and Pseudomonas aeruginosa in mice (Hooke et al. 1985). Doubling times of 20-33 min indicated that growth conditions in vivo may not be as limiting as first thought (Smith 1989). Such observations on mixtures of wild types and mutants that do not grow in vivo have definite potential for providing much needed information on this vital aspect of pathogenicity. The most notable exception to the lack of knowledge on nutrients and environmental factors that underpin growth rate in vivo is the influence of iron limitation (Smith 1989; Griffith 1991). The virulence of many bacterial pathogens in various animal models was enhanced by iron, which is scarce in vivo as a result of chelation by transferrin and lactoferrin. Experiments in vitro under iron-limiting conditions revealed the production of siderophores (iron chelators), new tRNAs, and hitherto unknown outer membrane proteins that act as receptors for iron-containing compounds; siderophores and receptors were then demonstrated in vivo (Griffiths 1991). Throughout, studies on E. coli have been at the forefront. Two siderophores, a phenolic enterobactin and a hydroxamate aerobactin (a product of the ColV and other plasmids, as well as chromosomal elements in some strains (Johnson et al. 1988)), were discovered and their receptors identified as proteins of 81 and 74 kDa, respectively. The genetics of siderophore production and regulation is under intensive investigation. Dr. Neiland's paper (Neiland 1992) has made the position clear for aerobactin. It is interesting that the cell level of ferrous iron governs siderophore synthesis and transport of their ferric complexes. Although knowledge on enterobactin and aerobactin is increasing in this manner, their relative role in overall virulence has yet to be clarified (Griffith 1991).
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The studies on iron limitation are superb. Unfortunately they constitute the only substantial effort on nutrition and metabolism that relates t o growth in vivo. The work should be extended to other nutrients that either limit or stimulate growth. The methods evolved for studying the effects of iron limitation could readily be transposed to other nutrients. Another avenue would be to use nutritionally deficient mutants (Smith 1989). More is known about genetics, nutrition, and metabolism for E. coli than for any other bacterial species. It is the species of choice for attempts to fill the gap in knowledge on growth rate in vivo and the underpinning metabolism.
Jann and Jann 1992) and of LPS and its 0 serotype side chains are known (Luderitz et al. 1966; Stephen and Pietrovski 1986; Volvano 1992). The molecular bases of the complement cascade (Taylor 1988) and ingestion by phagocytes (Ofek and Sharon 1988) are becoming clearer. It appears, therefore, at least for the K1, K4, and K5 capsular polysaccharides and for the LPS of E. coli, that the way is open to take step 7 of the research ladder. For example, a comparison of the specific site directed LPS mutants described by Dr. Volvano with one another and their wild-type strain in bactericidal and phagocytosis tests might be revealing.
Interference with host defence Studies have not progressed as far as those on adhesion and toxicity. Mostly, they are at steps 4 and 5, i.e., identifying determinants and proving relevance in vivo. In some cases, structures of determinants have been obtained (step 6), but they have not yet been related to biological action (step 7), other than in superficial terms. Smooth LPS and some group I1 capsular polysaccharides (Kl, K4, and K5) (Boulnois and Jann 1989; Drake et al. 1990) appear to be the main determinants of resistance in vivo to complement-mediated bactericidins. LPS interferes with the insertion of the c5b-9 lytic complement component into E. coli membranes (Taylor 1988; Valvano 1992); and capsular polysaccharide stops complement activation by either the alternate or classical pathways (Cross et al. 1984; Woolcock 1988; Jann and Jann 1992). Genes on the ColV plasmid (iss gene), the R6-5 plasmid (traT gene), and the HLY plasmid (hlyZZinsert) have also been associated with serum resistance in vitro and with virulence in mice (Chakraborty et al. 1987; Woolcock 1988). Resistance to ingestion appears to be the main mechanism of interference with the action of phagocytes. As far as I am aware, significant survival of E. coli within professional phagocytes has not been reported. Again, a complete LPS and some type I1 capsular polysaccharides seem to be the main determinants concerned (Medearis et al. 1968; Timmis et al. 1985; Czuprynski 1988; Boulnois and Jann 1989; Drake et al. 1990; Jann and Jann 1992). Their action appears to be due to inhibition of opsonisation, decreased hydrophobicity, or for the capsular polysaccharides, prevention of complement activation (Timmis et al. 1985; Czuprynski 1988; Jann and Jann 1992). The various cytotoxins produced by E. coli (see next section) may also inhibit phagocytes. Certainly, the a haemolysin is toxic to phagocytes in tests in vitro and at lower concentrations inhibits chemotaxis and ingestion (Shewen 1988). With regard to interference with the immune response, E. coli infections do not appear to be markedly immunosuppressive, but antibody is poorly stimulated by the K1, K4, and K5 capsular polysaccharides, probably because they resemble animal cell components (Widders 1988; Boulnois and Jann 1989; Drake et al. 1990; Jann and Jann 1992). The relation between the structure of known determinants and interference with defence mechanisms has not been investigated in any detailed form. This may be because the main determinants are carbohydrates, the structures of which are not so easy to elucidate or manipulate as those of peptides. Nevertheless, the general structures of some type I1 capsular polysaccharides (Boulnois and Jann 1989;
Cause of damage to the host Extracellular protein toxins are the main determinants of sickness in E. coli infections. Once only LT was known, now there are at least two LTs (LTI and LTII), two STs (STa and STb), several verotoxins (VT1, VT2, and variants of VT2), and various haemolysins. Most of them have been characterised, and they are probably relevant in vivo. This has been established more definitely for the enterotoxins than for the verotoxins (Gyles 1992). The haemolysins formed by strains that cause urinary tract disease appear to contribute to necrosis and damage of kidney cells (Chakraborty et al. 1987; Hacker and Goebel 1987). The continuing excellent work on E. coli toxins is being reviewed constantly (Stephen and Pietrovski 1986; Chakraborty et al. 1987; Law 1988; Smith and Scotland 1988; Shewen 1988; Robertson 1988; Wadstrom et al. 1990). Dr. Gyles' contribution to this symposium underlines the fact that research on LT1, STa, and the verotoxins is at the top of the research ladder. Their structures are known and their mechanisms of toxicity, including the nature of the host cell receptors, e.g., GM I gangloside for LT 1 and Gala 1-4Galfll-4glucose ceramide for VT1. The same can be said for the a haemolysin, for which the genetics of production, peptide structure, and mechanisms of action are clear, e.g., the 19 amino acid terminal peptide that determines haemolytic activity (Chakraborty et al. 1987). In the future we can expect active sites to be pinpointed by site-directed mutagenesis and possibly three-dimensional structures obtained. Also, other E. coli toxins, e.g., STb, will be taken through the same procedures. In essence, research on proteins toxins is at step 7 and ahead of other studies on the pathogenicity of E. coli. Turning to other determinants of sickness, EPEC strains of E. coli appear to cause diarrhoea by intimate attachment to epithelial cells, the so-called attaching effacing lesions. A receptor-mediated mechanism may be involved that elevates intracellular calcium levels and activates protein kinase C (Knutton 1990). Recently, a genetic locus has been found on the chromosome that may determine the attaching effacing lesions (Jerse et al. 1990). The gene product may be a 94-kDa protein. Turning to the influence of LPS, there is long-standing unresolved problem, what factors determine LPS release in vivo? If released, endotoxin will cause fever and vascular disturbances, which may lead to fatal secondary shock (Stephen and Pietrowski 1986; Rutter 1988). Normally, release from commensal E. coli does not occur in sufficient quantities to overwhelm the detoxifying action of the liver. In E. coli bacteraemia, however, fatal shock is almost certainly due to liberated endotoxin (Rutter 1988). Research
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on the factors that determine release of LPS of E. coli at different stages of infection in vivo is long overdue. Immunopathology does not appear to be a problem in most E. co[i infections, although inflammatory changes are evoked directly by some toxins such as the a haemolysin (Shewen 1988).
Conclusions Research on the pathogenicity of E. coli is concentrated, perhaps too concentrated, on adhesion and toxicity. In these two areas it is at the highest level of endeavour, the relation between determinant structure and biological activity. Effort on inhibition of host defences is some distance behind, probably because bacterial polysaccharides are involved and defence mechanisms are very complex. Neglected areas in mucosal infection are competition with commensals, gaining access to the surfaces through mucus, and interference with host defence. Sooner or later, the determinants of epithelial-cell invasion by EIEC E. coli will have to be checked to see whether they are the same as those for shigellae. Above all, E. coli infections could be used to close the major gap in studies of bacterial pathogenesis-lack of knowledge on growth rate in vivo and the underpinning metabolism. Acknowledgement The author thanks Dr. S. Knutton for a critical reading of this manuscript. Arp, L.H. 1988. Bacterial infection of mucosal surfaces: an overview of cellular and molecular mechanisms. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 3-27. Boulnois, G. J ., and Jann, K. 1989. Bacterial polysaccharide capsule synthesis, export and evolution of structural diversity. Mol. Microbiol. 3: 1819- 1823. Chakraborty, T., Kathariou, S., Hacker, J., et al. 1987. Molecular analysis of bacterial cytolysins. Rev. Infect. Dis. 9: S456-S466. Cohen, P.S. 1990. The role of large intestinal mucus in colonization of the mouse large intestine by Escherichia coli F-18 and Salmonella typhimurium. Abstracts of FEMS symposium on molecular pathogenesis of gastro-intestinal infections. Holger Danske, Denmark. Conway, P.L., Blomberg, L., and Cohen, P.S. 1990. The role of piglet intestinal mucus in the pathogenesis of E. coli K88. Abstracts of FEMS symposium on molecular pathogenesis of gastro-intestinal infections. Holger Danske, Denmark. Cross, A.S., Gemski, P., Sadoff, J.C., et al. 1984. The importance of the KI capsule in invasive infection caused by Escherichia coli. J . Infect. Dis. 149: 184-193. Czuprynski, C.J. 1988. Bacterial evasion of cellular defense mechanisms; an overview. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 141-160. Drake, C.R., Roberts, I.S, Jann, B., et al. 1990. Molecular cloning and expression of the genes encoding the Escherichia coli K4 capsular polysaccharide a fructose-substituted chondroitin. FEMS Microbiol. Lett. 66: 227-230. Elwell, L.P., and Shipley, P.L. 1980. Plasmid-mediated factors associated with virulence of bacteria to animals. Annu. Rev. Microbiol. 14: 465-490. Falkow, S. 1988. Molecular Koch's postulates applied to microbial pathogenicity. Rev. Infect. Dis. 10: S274-S276. Falkow, S., Small, P., Isberg, R., et al. 1987. A molecular strategy for study of bacterial invasion. Rev. Infect. Dis. 9: S450-S455.
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Finlay, B.B., and Falkow, S. 1989. Common themes on microbial pathogenicity. Microbiol. Rev. 53: 210-230. Freter, R. '1988. Mechanisms of bacterial colonization of the ~ U C O Surfaces S ~ ~ of the gut. In Vir~lencemechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology~Washington, D.C. pp. 45-60. Griffiths, E. 1991. Iron and bacterial virulence-a brief overview. Biol. Metals, 4: 7-13. Gyles, C.L. 1992. Escherichia coli cytotoxins and enterotoxins. Can. J . Microbiol. This issue. Hacker, P.D. J. 1992. Role of fimbrial adhesins in the pathogenesis of Escherichia coli infections. This issue. Hacker, P.D. J., and Goebel, W. 1987. Mechanisms and methods for analysing pathogenicity. Swiss Biotechnol. 2a: 21-3 1. Hadad, J. J., and Gyles, C.L. 1982. Scanning and transmission electron microscopic study of the small intestine of colostrum-fed calves infected with selected strains of E. coli. Infect. Immun. 40: 340-350. Hale, L.T., and Formal, S.B. 1988. Virulence mechanisms of enteroinvasive pathogens. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 61-69. Hooke, A.M., Sordelli, D.O., Cerquetti, H.C., and Vagt, A. J . 1985. Quantitative determination of bacterial replication in vivo. Infect. Immun. 49: 424-427. Isaacson, R.E. 1988. Molecular and genetic basis of adherence for enteric Escherichia coli in animals. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 28-44. Jann, K., and Jann, B. 1992. Capsules of Escherichia coli, expression and biological significance. Can. J. Microbiol. This issue. Jerse, H.E., Yu, J., Tall, B.D., and Kaper, J.B. 1990. A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc. Acad. Natl. Sci. U.S.A. 87: 7839-7843. Johnson, J.R., Moseley, S.L., Roberts, P.C., and Stamm, W .E. 1988. Aerobactin and other virulence factor genes among strains of Echerichia coli causing urosepsis: association with patient characteristics. Infect. Immun. 56: 405-412. Jones, G.W., and Rutter, J.M. 1972. Role of the K88 antigen in the pathogenesis of neonatal diarrhea caused by E. coli in piglets. Infect. Immun. 6: 918-927. Klemm, P. 1985. Fimbrial adhesins of Escherichia coli. Rev. Infect. Dis. 7: 321-340. Knutton, S . 1990. Intestinal colonization of diarrheic Escherichia coli. Abstracts of FEMS symposium on molecular pathogenesis of gastro-intestinal infections. Holger Danske, Denmark. Law, D. 1988. Virulence factors of enteropathogenic Escherichia coli. J . Med. Microbiol. 26: 1-10. Luderitz, O., Staub, A.M., and Westphal, 0. 1966. Immunochemistry of 0 and R antigens of Salmonellae and related Enterobacteriaceae. Bacteriol. Rev. 30: 192-255. Mathewson, J . J., and Cravioto, A. 1989. HEp-2 cell adherence as an assay for virulence amongst diarrheagenic Escherichia coli. J. Infect. Dis. 159: 1057-1060. Maurelli, A.T., and Sansonetti, P. J. 1988. Genetic determinants of Shigella pathogenicity. Annu. Rev. Microbiol. 42: 127- 150. Maw, J., and Meynell, G.C. 1968. The true division and death rates of Salmonella typhimurium in the mouse spleen determined with superinfecting phage P,,. Br. J. Exp. Pathol. 49: 597-613. Medearis, D.N., Jr., Camitta, B.M., and Heath, E.C. 1968. Cell wall composition and virulence in Escherichia coli. J. Exp. Med. 128: 399-414. Morschhauser, J., Horchutzky, H., Jann, K., and Hacker, J. 1990. Functional analysis of the sialic acid-binding adhesin SfaS of pathogenic Escherichia coli by site-specific mutagenesis. Infect. Immun. 58: 2133-2138. Ofek, I., and Sharon, N. 1988. Lectinophagocytosis: a molecular mechanism of recognition between cell surface sugars and lectins
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in the phagocytosis of bacteria. Infect. Immun. 56: 539-547. Olsen, A., Jensson, A., and Normark, S. 1989. Fibronectin binding mediated by a novel class of surface organelles on Escherichia coli. Nature (London), 338: 652-656. Orskov, I., and Orskov, F. 1966. Episome carried surface antigen K88 of Escherichia coli. I. Transmission of the determinant of the K88 antigen and influence on the transfer of chromosomal markers. J. Bacteriol. 91: 69-75. Orskov, I., and Orskov, F. 1983. Serology of Escherichia coli fimbriae. Prog. Allegy, 33: 80-105. Parkkinen, J., Rogers, G.N., Korhonen, T., et al. 1986. Identification of the 0-linked sialyloligosaccharides of glycophorin as the erythrocyte receptors for S-fimbriated Escherichia coli. Infect. Immun. 54: 37-42. Polk, H.C., and Miles, A.A. 1973. The decisive period in the primary infection of muscle by Escherichia coli. Br. J. Exp. Pathol. 54: 99-109. Reid, G., and Sobel, J.D. 1987. Bacterial adherence in the pathogenesis of urinary tract infection: a review. Rev. Infect. Dis. 9: 470-487. Robertson, D.C. 1988. Pathogenesis and enterotoxins of diarrheagenic Escherichia coli. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D .C. pp. 241-263. Rutter, J.M. 1988. Bacterial toxins as virulence determinants of veterinary pathogens: an overview. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 213-227. Sansonetti, P.J. 1990. Strategies of invasion of intestinal cells by Shigella flexneri. Abstracts of FEMS symposium on molecular pathogenesis of gastro-intestinal infections. Holger Danske, Denmark. Sansonetti, P. J., Kopecko, D. J., and Formal, S.B. 19821. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect. Immun. 35: 852-860. Sansonetti, P.J., d'Hauteville, H., Formal, S.B., and Toucas, M. 1982b. Plasmid mediated invasiveness of shigella-likeEscherichia coli. Ann. Microbiol. (Inst. Pasteur), 132: 351-355. Sellwood, R., Hall, G., and Anger, H. 1986. Emigration of polymorphonuclear leucocytes into the intestinal lumen of the neonatal piglet in response to challenge with K88-positive Escherichia coli. Res. Vet. Sci. 40: 128-135. Shewen, P.E. 1988. Cytocidal toxins of Gram-negative rods. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 228-240. Shipley, P.L., Dallas, W.S., Dougan, G., and Falkow, S. 1979.
Expression of plasmid genes in pathogenic bacteria. In Microbiology 1979. Edited by D. Schlessinger. American Society for Microbiology, Washington, D.C. pp. 176-1 80. Silver, R.P., Finn, C.W., Vann, W .F., et al. 1981. Molecular cloning of the K1 capsular polysaccharide genes of E. coli. Nature (London), 289: 696-698. Smith, H . 1988. The state and future of studies on bacterial pathogenicity. In Virulence mechanisms of bacterial pathogens. Edited by J . A. Roth. American Society for Microbiology, Washington, D.C. pp. 365-382. Smith, H. 1989. Pathogenicity and the microbe in vivo. J . Gen. Microbiol. 136: 377-393. Smith, H.R., and Scotland, S.M. 1988. Vero cytotoxin-producing strains of Escherichia coli. J. Med. Microbiol. 26: 77-85. Smith, H.W., and Linggood, M.A. 1971. Obervations on the pathogenic properties of the K88, HLY and ENT plasmids of Escherichia coli with particular reference to porcine diarrhoea. J . Med. Microbiol. 4: 467-485. Stephen, J., and Pietrowski, R.A. 1986. Bacterial toxins. 2nd ed. Thomas Nelson and Sons, Ltd., Waltham-on-Thames, England. Taylor, P. W. 1988. Bacterial resistance to complement. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 107-120. Timmis, K.N., Boulnois, G.J., Bitter-Suerman, D., and Carbello, F.C. 1985. Surface components of Escherichia coli that mediate resistance to bactericidal activities of serum and phagocytes. Curr . Top. Microbiol. Immunol. 118: 197-2 18. Valvano, M.A. 1992. Pathogenicity and molecular genetics of 0-specific side-chain lipopolysaccharides of Escherichia coli. Can. J . Microbiol. This issue. Wadstrom, T., Makela, P.H., Svennerholm, A.M., et al. 1990. Abstracts of papers in the FEMS symposium on molecular pathogenesis of gastro-intestinal infections. Holger Danske, Denmark. Weiss, A.A., and Falkow, S. 1983. The use of molecular techniques to study microbial determinants of pathogenicity. Philos. Trans. R. Soc. London, Ser. B, 303: 219-225. Widders, P . R. 1988. Bacterial resistance to antibody-dependent host defences. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. Am. Soc. Microbiol. Washington D.C. pp. 94-106. Woolcock, J.B. 1988. Bacterial resistance to humoral defense mechanisms: an overview. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 73-93.
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Medical School, University of Birmingham, Birmingham BI5 2TT, United Kingdom Received June 17, 1991 Revision received December 6, 1991 Accepted January 3, 1992 SMITH,H. 1992. Virulence determinants of Escherichia coli: present knowledge and questions. Can. J. Microbiol. 38: 747-752. This paper describes the present state of research on the pathogenicity of Escherichia coli and points out the gaps in knowledge that should be filled in the future. First, the great versatility of E. coli in producing disease is noted, as well as the invaluable contributions that studies of it have made to the development of general knowledge on bacterial pathogenicity. Then, the biological requirements for pathogenicity: infection of mucous surfaces; penetration of those surfaces; multiplication in vivo; interference with host defence mechanisms; and damage to the host, are taken in turn, and an enquiry is made on how far studies have progressed toward identifying their molecular determinants and relating structure to biological action. Only for mucous surface adhesins and protein toxins are studies at the structure-function level. Some progress has been made on interference with host defence, but little is known about competition with commensals on mucous surfaces, invasion into the tissues, and growth in vivo. Key words: virulence determinants, Escherichia coli. SMITH,H. 1992. Virulence determinants of Escherichia coli: present knowledge and questions. Can. J. Microbiol. 38 : 747-752. Cet article decrit l'etat present de la recherche au sujet de la pathogenicite d'Escherichia coli et il souligne les lacunes des connaissances actuelles qui devraient &re comblees dans le futur. Premierement, l'auteur constate la grande versatilite d'E. coli a produire la maladie et il indique les contributions inestimables que les etudes sur ce sujet ont apportees au developpement des connaissances generales sur la pathogenicite bacterienne. Ensuite, les exigences biologiques de la pathogenicite sont prises en compte comme l'infection des surfaces muqueuses, la penetration de ces surfaces, la multiplication in vivo, l'interference avec les mecanismes de defense de l'h6te et le dommage a l'h6te; une investigation a ete amenee sur la progression des etudes concernant l'identification de leurs determinants moleculaires et rattachant la structure a l'action biologique. Les etudes au niveau structure-fonction ont ete realisees seulement pour les adhesines des surfaces muqueuses et pour les toxines proteiques. Un certain progres a ete accompli sur l'interference avec la defense de l'hhte, mais peu est connu au sujet de la competition avec les commensaux sur les surfaces muqueuses, l'invasion dans les tissus et la croissance in vivo. Mots clks : determinants de la virulence, Escherichia coli. [Traduit par la redaction]
Introduction When Dr. Fairbrother asked me to sum up this symposium I was taken by surprise because I have never worked on the pathogenicity of Escherichia coli. Faced with the task, I decided that, rather than summarizing what the contributors had said already, it might be more profitable to provide a view, so to speak from the outside, on the present state of research on the pathogenicity of E. coli and the gaps that should be filled in the future. I apologise in advance for any mistakes that I might make owing to lack of expertise. Before giving this view, I wish to make two points that struck me forcibly during some background reading. First, there is the versatility of E. coli. It is a normal commensal of the gut, and strains such as K12 have been used as safe tools for research and teaching over decades. Yet other strains produce severe diseases of widely differing character in man (enteritis, urinary tract infections, bacteraemia, and meningitis) and animals (pre- and post-weaning scours, oedema disease, and mastitis). Even if we focus on enteritis, there is great variation in the mechanisms whereby various strains produce their harmful effects. Thus, enterotoxigenic (ETEC; traveller's diarrhoea), enteroinvasive (EIEC; dysentery-like), enteropathogenic (EPEC; infant diarrhoea), enterohaemorrhagic (EHEC; haemorrhagic intestinal lesions), and enteroadherent aggregative (EAggEC; persistent infant diarrhoea) strains are known (Law 1988; Knutton Printed in Canada / Imprime au Canada
1990), and probably others will be discovered. For example, there is debate at present on the importance in disease of strains that show diffuse adherence (i.e., attachment over the entire cell surface) to tissue culture cells (Mathewson and Cravioto 1989). Coupled with this wonder at the versatility of E. coli is an admiration for the way in which the @rskovshave related classical 0 , H , and K serotyping of strains to disease syndromes (@rskov and @rskov 1983). In their contribution to this symposium, they distil the work of years down to a few important conclusions. There are many E. coli serotypes, but relatively few cause disease. The latter constitute two groups. One group relates to diarrhoea1 disease in man and animals, and the serotypes are largely specific to the host species in which the disease occurs. The second group of different serotypes relate to invasive extraintestinal disease and come mostly from humans. Basically, the 0 , H , and K serotyping is a marker for two groups of pathogenic E. coli, each composed of a limited number of clones with similar virulence mechanisms. My second point is the invaluable contribution that studies on E. coli have made to the general development of knowledge on bacterial virulence. Two factors have been responsible; production of similar infections in man and animals and knowledge of the genetics of E. coli. Studies of pathogenicity can be plagued by lack of valid animal
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models for human disease. Escherichia coli has the advantage that it produces similar diseases in animals and man, notably enteritis. This allows investigations on pathogenicity to proceed in the former with the probability that the results will apply to the latter. The original studies on the role of K88 fimbrial antigen in adhesion of E. coli to gut surfaces in piglet diarrhoea (Orskov and Orskov 1966; Jones and Rutter 1972) were extended to E. coli infections of man (Klemm 1985; Hacker 1992). More importantly, they sparked off the explosion of research on the determinants of bacterial adhesion to mucous surfaces that has occurred for many different pathogens over the past 20 years (Arp 1988). Also, although studies on cholera toxin led the intense interest in enterotoxins, those on the heat-labile (LT) and -stable (ST) toxins of E. coli were not far behind (Robertson 1988). The point about an animal model is underlined by Dr. Gyles (1992) in .this symposium. He notes that the role of E. coli verotoxins in disease is less firmly established than for the enterotoxins, because, unlike the latter, verotoxinrelated diseases of man are not simulated by corresponding animal diseases. Escherichia coli has, for 40 years, been the main tool for development of microbial genetics and molecular biology. When these sciences were first applied seriously to problems of pathogenicity, E. coli was the most appropriate species to use because methods of genetic manipulation were already available. The seminal experiments were those of Smith and Linggood (1971) on E. coli enteritis of piglets. They demonstrated, for the first time, plasmid-mediated transfer of virulence attributes, namely mucosal adherence and enterotoxin production. Developments were rapid (Elwell and Shipley 1980). Now, it is standard practice to use plasmids for manufacturing strains of high and low virulence, often with tailored virulence determinants. Gene cloning, perhaps the most used technique in studies of pathogenicity (Falkow 1988), first showed its strength in the field by adding weight to the evidence that the adherence antigens, the LT and ST, and the haemolysins of E. coli are virulence determinants relevant in vivo (Shipley et al. 1979; Elwell and Shipley 1980; Weiss and Falkow 1983). This was because methods for gene cloning in E. coli were well established in fields other than pathogenicity. Similarly, the difficult task of cloning all the genes responsible for production of capsular polysaccharides was first accomplished for K1 E. coli (Silver et al. 1981). Turning to the present state and possible gaps in research on the pathogenicity of E. coli, I will first explain the template on which I shall base the discussion. The biological requirements for pathogenicity are abilities to multiply in the environment of the host, to interfere with host defence mechanisms, and to damage the host. Since most diseases are contracted on or over mucous surfaces, two additional qualities are usually needed, infection and penetration of mucous surfaces. The compounds responsible for the multifactorial property are the determinants of virulence and the goal is to recognise each determinant, to identify it, and to relate its chemical structure to biological action (Smith 1988). There are seven logical steps to attaining this goal: (1) establish a method for comparing the virulence of strains in the natural host or a relevant animal model; (2) obtain strains of high and low virulence; (3) compare strains of differing virulence in biological tests related to the five requirements for pathogenicity; (4) identify the determinant that causes the chosen biological property; (5) prove relevance of the
biological property and its determinant to infection in vivo; (6) obtain the chemical structure of the determinant; and (7) relate the structure to biological action (Smith 1988). I shall now take a look at the five biological requirements for pathogenicity and enquire how far, for E. coli, we have progressed up this research ladder; I shall pay more attention to the gaps in knowledge because other symposium participants have already covered the successes.
Infection of mucous surfaces To infect mucous surfaces, a pathogen must be able to compete successfully with any commensals that may be present, to make contact with the surface through the overlying mucus, to adhere either to the surface or to the mucus, and to interfere with local humoral and cellular defences. Only in the case of adherence has there been substantial progress up the research ladder. The protective effect of the normal flora on some mucous surfaces is well proven. How it is overcome by relatively small numbers of E. coli and other pathogens, e.g., in the lower bowel, is a mystery. Research is sparse and at step 3, biological observation. The most notable worker, R. Freter, used E. coli to show some factors involved: successful competition for carbon and energy sources in anaerobic conditions and in the presence of H2S; adherence to sites not used by the indigenous flora; and a short lag phase so that multiplication occurs before removal by the moving lumen contents (Freter 1988). However, far more work is needed before the determinants of this essential stage in mucosal infection are revealed. Gaining access to epithelial surfaces through the overlying mucus is attracting more attention, but again, studies are at step 3. Collapse of the mucus layer during fixation of specimens for electron microscopy makes it difficult to distinguish bacteria in the mucus and those on the surface (Freter 1988). Chemotaxis and natural movement to the villi and crypts along the lines of stress of moving mucus appear to be two mechanisms for traversing the mucus (Freter 1988). Growth in mucus also appears important for E. coli (Cohen 1990). Mucus contains receptors for E. coli fimbriae. They can retain the bacteria in the mucus and block interaction with epithelial cell receptors (Cohen 1990; Conway et al. 1990). Some strains of E. coli produce a mucinase (Law 1988), but whether this is relevant to growth in or penetration of mucus in vivo is not clear. The amount of research on adhesion of E. coli to mucous surfaces is matched by the number of excellent reviews on the subject (Klemm 1985; Reid and Sobell 1987; Law 1988; Arp 1988; Isaacson 1988; Wadstrom et al. 1990; Hacker 1992). Hence, I will confine myself to making a few overall points. There are fimbrial adhesins, nonfimbrial adhesins, and special structures such as curlis (Olsen et al. 1989). Many of them are expressed in vivo and are relevant to pathogenicity (step 5 of the ladder). New adhesins for various types of E. coli continue to be found. Both chromosomal and plasmid genes code for adhesins, and in many instances, their regulation has been clarified. In his symposium presentation Jorg Hacker has described environmental factors that affect regulation. It is particularly intriguing that one adhesin gene cluster can influence the expression of other adhesin determinants so that cross "talk" may occur during infection. For many fimbrial adhesins, research is at the structural stage, step 6, of the research ladder.
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Amino acid sequences have been determined for the major units and, in some cases, for the minor units that appear to determine specificity of adhesion. Research on nonfimbrial adhesins has not yet attained this structural level. Attention has also been given to the nature of host-cell receptors, such as D-mannosides for type 1 fimbriae, a-Dgalacto-(1-4)-P-D-galacto groups for P fimbriae, P-Dgalactoside groups for K88 fimbriae, and a-sialyl acid-2-3-Pgalactosamine groups for S fimbriae (Klemm 1985; Parkkiner et al. 1986; Arp 1988; Isaacson 1988; Olsen et al. 1989; Wadstrom et al. 1990). In most cases, the location and distribution of receptor sites on mucosal epithelium are not known (Klemm 1985; Isaacson 1988). Finally, the last step in the research ladder, the relationhip between molecular structure and adhesion, is just beginning; site-directed mutation has revealed the probable active sites on the sialic acid binding subunit of S fimbriae (Marschhauser et al. 1990). Compared with the effort on adhesion, the attention given to resistance to host defences on mucous surfaces is minute. During intestinal infection, neutrophils are mobilised within and on the surfaces (Hadad and Gyles 1982; Sellwood et al. 1986) and humoral bactericidins will also be present. Presumably, capsular polysaccharides and smooth (i.e., with a complete side chain) lipopolysaccharides (LPS) inhibit these defences by the mechanisms described later. Capsulated E. coli have been seen on intestinal surfaces (Hadad and Gyles 1982). The Brskovs have mentioned that 0 antigen of LPS might be a virulence factor. I am surprised, therefore, that LPS from the serogroups related to pathogenicity appear not to have been compared with the LPS of nonpathogenic serogroups for capacity to interfere with serum killing of E. coli and its ingestion by phagocytes. Another surprise, it appears that an IgA protease has not been reported for E. coli.
Penetration of mucous surfaces There are two processes to consider: penetration into the cells of the mucous surfaces by EIEC strains to cause a dysentry-like illness; and complete passage through the surface leading to bacteraemia, meningitis, and other invasive manifestations. The first aspect has received some attention but not, as far as I can judge, the second. Sparked by studies on shigellae (Sansonetti et al. 1982a; Maurelli and Sansonetti 1988; Hale and Formal 1988; Sansonetti 1990), an explosion of research on the molecular basis of epithelial cell invasion has occurred using mainly tissue culture systems but with adequate tests in vivo (Falkow et al. 1987; Finlay and Falkow 1989). Shigellae, Yersinia, and Salmonella species have received far more attention than E. coli (Hale and Formal 1988; Finlay and Falkow 1989). The seminal discovery was that large plasmids, 140 and 120 MDa, respectively, determined epithelial cell invasion by Shigella flexneri and Shigella sonnei (Sansonetti et al. 1982a, 1982b). Subsequent analysis of the plasmid for S. flexneri (Sansonetti 1990) showed that two genes code for two polypeptide invasins (ipaB, 62 kDa; ipaC, 48 kDa). Almost immediately after the discovery of the plasmid in S. flexneri, a similar large plasmid of 140 MDa was found in EIEC strains of E. coli (Sansonetti et al. 1982b; Maurelli and Sansonetti 1988). Over recent years, however, the invasion plasmid of EIEC strains has received little attention, probably on the assumption that findings on shigellae apply to E. coli.
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As far as I am aware, the mechanisms and determinants of complete penetration of mucous surfaces by invasive strains of E. coli have not been investigated. Perhaps haemolysins produced by such strains should be studied in this respect.
Growth in vivo Growth rate is important in pathogenicity. If it is fast, the pathogen can overwhelm the initial nonspecific defences and cause sickness before the more powerful immune defences are fully operative. A slow-growing pathogen is more vulnerabe to both types of defence. At present, lack of knowledge on growth rate in vivo is the major gap in studies of bacterial pathogenicity (Smith 1989). Mounting the research ladder has hardly begun. There are no reliable, universally accepted, methods for measuring growth rate, and the nutrients and other environmental factors that determine it are largely unknown and unstudied. Escherichia coli has, however, been prominent in the little work that has been done. The first attempts to measure doubling times in vivo (as distinct from increase in population) relied on a marker distributing to only one of two daughter cells in each succeeding generation (Maw and Meynell 1968; Polk and Miles 1973). Genetic manipulation (abortive transduction and superinfecting phage) was needed to introduce the marker so the method was restricted to salmonellae and E. coli. It indicated slow growth in mice (doubling time of 3-5 h). More recently, the increase in ratios of wild-type organisms (which multiply in vivo) to those of temperature-sensitive mutants (which should not multiply in vivo) has been used to measure growth rate of E. coli and Pseudomonas aeruginosa in mice (Hooke et al. 1985). Doubling times of 20-33 min indicated that growth conditions in vivo may not be as limiting as first thought (Smith 1989). Such observations on mixtures of wild types and mutants that do not grow in vivo have definite potential for providing much needed information on this vital aspect of pathogenicity. The most notable exception to the lack of knowledge on nutrients and environmental factors that underpin growth rate in vivo is the influence of iron limitation (Smith 1989; Griffith 1991). The virulence of many bacterial pathogens in various animal models was enhanced by iron, which is scarce in vivo as a result of chelation by transferrin and lactoferrin. Experiments in vitro under iron-limiting conditions revealed the production of siderophores (iron chelators), new tRNAs, and hitherto unknown outer membrane proteins that act as receptors for iron-containing compounds; siderophores and receptors were then demonstrated in vivo (Griffiths 1991). Throughout, studies on E. coli have been at the forefront. Two siderophores, a phenolic enterobactin and a hydroxamate aerobactin (a product of the ColV and other plasmids, as well as chromosomal elements in some strains (Johnson et al. 1988)), were discovered and their receptors identified as proteins of 81 and 74 kDa, respectively. The genetics of siderophore production and regulation is under intensive investigation. Dr. Neiland's paper (Neiland 1992) has made the position clear for aerobactin. It is interesting that the cell level of ferrous iron governs siderophore synthesis and transport of their ferric complexes. Although knowledge on enterobactin and aerobactin is increasing in this manner, their relative role in overall virulence has yet to be clarified (Griffith 1991).
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The studies on iron limitation are superb. Unfortunately they constitute the only substantial effort on nutrition and metabolism that relates t o growth in vivo. The work should be extended to other nutrients that either limit or stimulate growth. The methods evolved for studying the effects of iron limitation could readily be transposed to other nutrients. Another avenue would be to use nutritionally deficient mutants (Smith 1989). More is known about genetics, nutrition, and metabolism for E. coli than for any other bacterial species. It is the species of choice for attempts to fill the gap in knowledge on growth rate in vivo and the underpinning metabolism.
Jann and Jann 1992) and of LPS and its 0 serotype side chains are known (Luderitz et al. 1966; Stephen and Pietrovski 1986; Volvano 1992). The molecular bases of the complement cascade (Taylor 1988) and ingestion by phagocytes (Ofek and Sharon 1988) are becoming clearer. It appears, therefore, at least for the K1, K4, and K5 capsular polysaccharides and for the LPS of E. coli, that the way is open to take step 7 of the research ladder. For example, a comparison of the specific site directed LPS mutants described by Dr. Volvano with one another and their wild-type strain in bactericidal and phagocytosis tests might be revealing.
Interference with host defence Studies have not progressed as far as those on adhesion and toxicity. Mostly, they are at steps 4 and 5, i.e., identifying determinants and proving relevance in vivo. In some cases, structures of determinants have been obtained (step 6), but they have not yet been related to biological action (step 7), other than in superficial terms. Smooth LPS and some group I1 capsular polysaccharides (Kl, K4, and K5) (Boulnois and Jann 1989; Drake et al. 1990) appear to be the main determinants of resistance in vivo to complement-mediated bactericidins. LPS interferes with the insertion of the c5b-9 lytic complement component into E. coli membranes (Taylor 1988; Valvano 1992); and capsular polysaccharide stops complement activation by either the alternate or classical pathways (Cross et al. 1984; Woolcock 1988; Jann and Jann 1992). Genes on the ColV plasmid (iss gene), the R6-5 plasmid (traT gene), and the HLY plasmid (hlyZZinsert) have also been associated with serum resistance in vitro and with virulence in mice (Chakraborty et al. 1987; Woolcock 1988). Resistance to ingestion appears to be the main mechanism of interference with the action of phagocytes. As far as I am aware, significant survival of E. coli within professional phagocytes has not been reported. Again, a complete LPS and some type I1 capsular polysaccharides seem to be the main determinants concerned (Medearis et al. 1968; Timmis et al. 1985; Czuprynski 1988; Boulnois and Jann 1989; Drake et al. 1990; Jann and Jann 1992). Their action appears to be due to inhibition of opsonisation, decreased hydrophobicity, or for the capsular polysaccharides, prevention of complement activation (Timmis et al. 1985; Czuprynski 1988; Jann and Jann 1992). The various cytotoxins produced by E. coli (see next section) may also inhibit phagocytes. Certainly, the a haemolysin is toxic to phagocytes in tests in vitro and at lower concentrations inhibits chemotaxis and ingestion (Shewen 1988). With regard to interference with the immune response, E. coli infections do not appear to be markedly immunosuppressive, but antibody is poorly stimulated by the K1, K4, and K5 capsular polysaccharides, probably because they resemble animal cell components (Widders 1988; Boulnois and Jann 1989; Drake et al. 1990; Jann and Jann 1992). The relation between the structure of known determinants and interference with defence mechanisms has not been investigated in any detailed form. This may be because the main determinants are carbohydrates, the structures of which are not so easy to elucidate or manipulate as those of peptides. Nevertheless, the general structures of some type I1 capsular polysaccharides (Boulnois and Jann 1989;
Cause of damage to the host Extracellular protein toxins are the main determinants of sickness in E. coli infections. Once only LT was known, now there are at least two LTs (LTI and LTII), two STs (STa and STb), several verotoxins (VT1, VT2, and variants of VT2), and various haemolysins. Most of them have been characterised, and they are probably relevant in vivo. This has been established more definitely for the enterotoxins than for the verotoxins (Gyles 1992). The haemolysins formed by strains that cause urinary tract disease appear to contribute to necrosis and damage of kidney cells (Chakraborty et al. 1987; Hacker and Goebel 1987). The continuing excellent work on E. coli toxins is being reviewed constantly (Stephen and Pietrovski 1986; Chakraborty et al. 1987; Law 1988; Smith and Scotland 1988; Shewen 1988; Robertson 1988; Wadstrom et al. 1990). Dr. Gyles' contribution to this symposium underlines the fact that research on LT1, STa, and the verotoxins is at the top of the research ladder. Their structures are known and their mechanisms of toxicity, including the nature of the host cell receptors, e.g., GM I gangloside for LT 1 and Gala 1-4Galfll-4glucose ceramide for VT1. The same can be said for the a haemolysin, for which the genetics of production, peptide structure, and mechanisms of action are clear, e.g., the 19 amino acid terminal peptide that determines haemolytic activity (Chakraborty et al. 1987). In the future we can expect active sites to be pinpointed by site-directed mutagenesis and possibly three-dimensional structures obtained. Also, other E. coli toxins, e.g., STb, will be taken through the same procedures. In essence, research on proteins toxins is at step 7 and ahead of other studies on the pathogenicity of E. coli. Turning to other determinants of sickness, EPEC strains of E. coli appear to cause diarrhoea by intimate attachment to epithelial cells, the so-called attaching effacing lesions. A receptor-mediated mechanism may be involved that elevates intracellular calcium levels and activates protein kinase C (Knutton 1990). Recently, a genetic locus has been found on the chromosome that may determine the attaching effacing lesions (Jerse et al. 1990). The gene product may be a 94-kDa protein. Turning to the influence of LPS, there is long-standing unresolved problem, what factors determine LPS release in vivo? If released, endotoxin will cause fever and vascular disturbances, which may lead to fatal secondary shock (Stephen and Pietrowski 1986; Rutter 1988). Normally, release from commensal E. coli does not occur in sufficient quantities to overwhelm the detoxifying action of the liver. In E. coli bacteraemia, however, fatal shock is almost certainly due to liberated endotoxin (Rutter 1988). Research
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on the factors that determine release of LPS of E. coli at different stages of infection in vivo is long overdue. Immunopathology does not appear to be a problem in most E. co[i infections, although inflammatory changes are evoked directly by some toxins such as the a haemolysin (Shewen 1988).
Conclusions Research on the pathogenicity of E. coli is concentrated, perhaps too concentrated, on adhesion and toxicity. In these two areas it is at the highest level of endeavour, the relation between determinant structure and biological activity. Effort on inhibition of host defences is some distance behind, probably because bacterial polysaccharides are involved and defence mechanisms are very complex. Neglected areas in mucosal infection are competition with commensals, gaining access to the surfaces through mucus, and interference with host defence. Sooner or later, the determinants of epithelial-cell invasion by EIEC E. coli will have to be checked to see whether they are the same as those for shigellae. Above all, E. coli infections could be used to close the major gap in studies of bacterial pathogenesis-lack of knowledge on growth rate in vivo and the underpinning metabolism. Acknowledgement The author thanks Dr. S. Knutton for a critical reading of this manuscript. Arp, L.H. 1988. Bacterial infection of mucosal surfaces: an overview of cellular and molecular mechanisms. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 3-27. Boulnois, G. J ., and Jann, K. 1989. Bacterial polysaccharide capsule synthesis, export and evolution of structural diversity. Mol. Microbiol. 3: 1819- 1823. Chakraborty, T., Kathariou, S., Hacker, J., et al. 1987. Molecular analysis of bacterial cytolysins. Rev. Infect. Dis. 9: S456-S466. Cohen, P.S. 1990. The role of large intestinal mucus in colonization of the mouse large intestine by Escherichia coli F-18 and Salmonella typhimurium. Abstracts of FEMS symposium on molecular pathogenesis of gastro-intestinal infections. Holger Danske, Denmark. Conway, P.L., Blomberg, L., and Cohen, P.S. 1990. The role of piglet intestinal mucus in the pathogenesis of E. coli K88. Abstracts of FEMS symposium on molecular pathogenesis of gastro-intestinal infections. Holger Danske, Denmark. Cross, A.S., Gemski, P., Sadoff, J.C., et al. 1984. The importance of the KI capsule in invasive infection caused by Escherichia coli. J . Infect. Dis. 149: 184-193. Czuprynski, C.J. 1988. Bacterial evasion of cellular defense mechanisms; an overview. In Virulence mechanisms of bacterial pathogens. Edited by J.A. Roth. American Society for Microbiology, Washington, D.C. pp. 141-160. Drake, C.R., Roberts, I.S, Jann, B., et al. 1990. Molecular cloning and expression of the genes encoding the Escherichia coli K4 capsular polysaccharide a fructose-substituted chondroitin. FEMS Microbiol. Lett. 66: 227-230. Elwell, L.P., and Shipley, P.L. 1980. Plasmid-mediated factors associated with virulence of bacteria to animals. Annu. Rev. Microbiol. 14: 465-490. Falkow, S. 1988. Molecular Koch's postulates applied to microbial pathogenicity. Rev. Infect. Dis. 10: S274-S276. Falkow, S., Small, P., Isberg, R., et al. 1987. A molecular strategy for study of bacterial invasion. Rev. Infect. Dis. 9: S450-S455.
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