INTRODUCTION

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The morbidity and mortality associated with several recent large outbreaks of gastrointestinal disease caused by Shiga toxin-producing Escherichia coli (STEC) has highlighted the threat these organisms pose to public health (10, 102, 114, 260). Such outbreaks have the potential to overwhelm acute-care resources, even in countries with advanced health care systems. Much attention has been focused upon this group of pathogens since their discovery, and there have been several excellent reviews covering either the field as a whole (156, 317) or specific aspects such as the toxin (232, 340), its structure and function (89, 139), its interaction with host cell receptors (193), and clinical aspects of disease (334). Our capacity to control STEC disease in humans and to limit the scale of outbreaks is dependent upon prompt diagnosis and identification of the source of infection. In recent years, there have been significant advances in our understanding of the pathogenesis of STEC infection, and these are contributing to the development of improved diagnostic methods, as well as to the development of therapeutic and preventative strategies. It is these aspects of STEC infection that will be the principal focus of this review.

It is now 20 years since Konowalchuk et al. (175) reported the feature which distinguishes STEC from other classes of pathogenic E. coli, namely, the production of a toxin with a profound and irreversible cytopathic effect on Vero (African green monkey kidney) cells. Of the 10 toxic strains in this initial study, 7 had been isolated from infants with diarrhea, suggesting the possibility of a role for this new Verotoxin (VT) in the pathogenesis of gastrointestinal disease. In the early 1980s, verotoxigenic E. coli strains were linked to cases of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) (160, 282). Moreover, verotoxigenic E. coli strains associated with two outbreaks of HC belonged to a previously rare serotype (O157:H7) (61, 282), as did two of eight verotoxigenic isolates from HUS patients reported by Karmali et al. (160). While it is now recognized that STEC strains belonging to a very diverse range of serotypes are capable of causing serious human disease, O157:H7 is a dominant STEC serotype in many parts of the world and historically has been the type most commonly associated with large outbreaks (127, 156, 317).

O'Brien et al. purified and characterized the cytotoxin produced by one of Konowalchuk's isolates (strain H30; serotype O26:H11) and found that it had strikingly similar structure and biological activity to Shiga toxin (Stx) produced by Shigella dysenteriae type 1 (233, 234). Moreover, it could be neutralized by anti-Stx (233, 234), resulting in the new nomenclature of Shiga-like toxin (SLT). SLT and VT nomenclature systems have been used interchangeably in the literature since this time. The situation has been complicated further by the subsequent recognition that there are two major types of SLT/VT (SLT-I and SLT-II or VT1 and VT2), with additional sequence variants within these types, as discussed below. In an attempt to avoid further confusion, Calderwood et al. (54) have proposed a rationalization of nomenclature (reproduced in Table 1), which recognizes that all of these toxins have a high degree of structural and functional homology and so belong to a Shiga toxin family. The rationalized nomenclature system will be used throughout this review; we also use STX as a generic abbreviation for the Shiga toxin family as a whole and STX to denote all stx-related genes.

Initial recognition of the presence of multiple STX types arose from the observation that anti-Stx could not neutralize the cytotoxicity of some STEC strains (308, 327). Conversely, crude antisera raised against nonneutralizable strains did not neutralize Stx. These antibody neutralization studies demonstrated that some STEC strains produced only the anti-Stx neutralizable toxin (now referred to as Stx1), others produced only the nonneutralizable toxin (now designated Stx2), while yet others produced both (308, 327). Additional studies also demonstrated that STEC isolates from piglets with edema disease produce a variant of Stx2 (designated Stx2e) (207). Although it was neutralized by polyclonal anti-Stx2, this variant could be distinguished from Stx2 on the basis of its lack of cytotoxicity for HeLa cells.

Members of the Stx family are compound toxins (the holotoxin is approximately 70 kDa), comprising a single catalytic 32-kDa A subunit and a multimeric B subunit (7.7-kDa monomers) that is involved in the binding of the toxin to specific glycolipid receptors on the surface of target cells (232). Biochemical cross-linking analysis suggested that the holotoxins of both Stx and Stx1 include five B-subunit monomers (85), and this was confirmed by X-ray crystallographic analysis of purified B subunits (322). Additional crystallographic analysis of the Stx holotoxin demonstrated that the B subunits form a pentameric ring, which encircles a helix at the C terminus of the single A subunit (97). This was consistent with the results of mutational analysis of the C-terminal region of the 293-residue Stx A subunit, which demonstrated that a sequence of nine nonpolar amino acids from residues 279 to 287 was essential for holotoxin assembly (121). These residues form an alpha-helix that penetrates the pore in the centre of the B pentamer; flanking charged residues appear to stabilize this interaction (145).

The eukaryotic cell surface receptor for members of the STX family is globotriaosylceramide (Gb₃; Gal[14]Gal[14]Glc-ceramide) (194, 360). The exception to this is the variant toxin Stx2e, which recognizes globotetraosylceramide (Gb₄; GalNAc[13]Gal[14]Gal[14]Glc-ceramide) preferentially over Gb₃ (79, 289). The interaction between the Stx1 B subunit and its receptor has been studied extensively by molecular modelling, site-directed mutagenesis, and crystallographic analysis. Phe30 was shown to play a critical role. Two possible Gb₃ binding sites have been identified on either side of this residue; one site is near the cleft between adjacent B monomers, while the other is a shallow indentation on the B subunit surface opposed to the plasma membrane (71, 193, 230, 231). A third putative Gb₃ binding site has also recently been identified in the vicinity of Trp34 (23).

Once bound to a target cell membrane, toxin molecules are thought to be internalized by a process of receptor-mediated endocytosis; this has been recently reviewed by Sandvig and van Deurs (294). Briefly, internalization involves the formation of a clathrin-coated pit within the cell membrane, which subsequently pinches off to form a sealed coated vesicle with toxin bound to the intenral surface. Subsequent intracellular trafficking has a major impact on the biological effects of STX. In some cells, the toxin-bound vesicles undergo fusion with cellular lysosomes, resulting in toxin degradation. However, in cells which are particularly sensitive to STX, the endosomal vesicles containing toxin-receptor complexes undergo retrograde transport via the Golgi apparatus to the endoplasmic reticulum before being translocated to the cytosol (291-293). During this process, the A subunit is nicked by a membrane-bound protease furin (107), generating a catalytically active 27-kDa N-terminal A1 fragment and a 4-kDa C-terminal A2 fragment, which remain linked by a disulfide bond. This disulfide bond is subsequently reduced, thereby releasing the active A1 component (294). Saleh et al. (288) have proposed that a signal sequence-like hydrophobic domain at the C terminus of the A1 fragment may function in the translocation process by directing insertion into the endoplasmic reticulum membrane. The released A1 fragment has RNA N-glycosidase activity and cleaves a specific N-glycosidic bond in the 28S rRNA, a property shared by the plant toxin ricin (93, 298, 315). This cleavage presents elongation factor 1-dependent binding of the aminoacyl-tRNA to the 60S ribosomal subunit (93, 131, 240), thereby inhibiting the peptide chain elongation step of protein synthesis and ultimately causing cell death.

The nucleotide sequences of the genes encoding Stx from S. dysenteriae, as well as Stx1 and Stx2 from E. coli, were determined in the late 1980s (55, 78, 142, 143, 177, 325). The operons had a common structure consisting of a single transcriptional unit, encoding first the A subunit followed by the B subunit. The stx B-subunit gene has a stronger ribosome binding site than that of the A-subunit gene, resulting in increased translation of B subunits, thereby satisfying the 1:5 A/B-subunit stoichiometry of the holotoxin (119). The predicted amino acid sequences were 315, 315, and 318 amino acids long for the A subunits of Stx, Stx1, and Stx2, respectively, and 89 amino acids for the B subunits of all three toxins. Both A and B subunits had hydrophobic N-terminal signal sequences characteristic of secreted proteins, and the predicted M_r values for the processed A and B subunits were in accordance with previous estimates based on analysis of purified toxins. Interestingly, a 21-bp region of dyad symmetry spanning the 10 region was found upstream of stx and stx₁, and this motif is thought to be associated with iron regulation of toxin expression (55, 78, 143, 177). Comparison of the deduced amino acid sequences indicated that Stx and Stx1 were virtually identical (there was a single amino acid difference in the A subunit) whereas Stx2 had only 56% identity to the other toxins for both the A and B subunits (142). Interestingly, a significant degree of amino acid homology was also observed between the A subunits of STX and the plant toxin ricin, which has an identical mode of action (55, 177). The most highly conserved regions were subsequently shown to be part of the active (catalytic) site (128, 379).

In 1988, the sequence of an operon encoding the variant toxin Stx2e, associated with piglet edema disease, was reported (118, 368). The deduced amino acid sequence of the A subunit of Stx2e was 1 amino acid longer than that of Stx2 and exhibited 94% homology to it. The B subunit of Stx2e was 2 amino acids shorter than that of Stx2, and there was only 87% homology. Studies involving the construction of chimeric Stx2/Stx2e operons demonstrated that the variations in the B subunit of Stx2e with respect to Stx2 were responsible for the reduced cytotoxicity of Stx2e for HeLa cells, which lack its preferred receptor, Gb₄ (367). Subsequent site-directed mutagenesis and molecular modelling studies have demonstrated that two amino acids in the mature Stx2e B subunit (Gln64 and Lys66) are critical for the distinct glycolipid binding specificity of the toxin (231, 350).

A number of other variant forms of Stx2 and also Stx1 have since been reported for human STEC isolates, illustrating the diversity of the Stx family (106, 138, 189, 214, 242, 250, 256, 258, 259, 305). One particular subgroup of Stx2 variants contains specific B-subunit amino acid differences with respect to classical Stx2 (Asp16Asn and Asp24Ala), which correlate with a lower binding affinity for the receptor Gb₃ and reduced in vitro cytotoxicity for Vero cells (191). In view of this functional distinction, these toxins are now considered a separate subgroup and have been designated Stx2c.

Studies in the early 1980s established that Stx1 and Stx2 were encoded on a variety of bacteriophages (236, 309, 316). However, toxin-converting bacteriophages have not been isolated from S. dysenteriae type 1 or from STEC strains associated with piglet edema disease (156). Several of the additional variant STX genes from other human STEC strains also do not appear to be phage encoded, although it is possible that they are carried on defective phage particles (256, 258, 259). One study has identified an IS element adjacent to an stx₁ operon in an O111:H STEC strain (253); there was no duplication of target sequence at the insertion site, which raised the possibility that the segment of DNA containing the toxin gene was part of a transposon. However, the direct involvement of such mobile elements in transmission of STX genes has yet to be demonstrated. Involvement of bacteriophages and transposons may help to explain why many STEC strains readily lose their STX genes after subcultivation in vitro (154).

It is now recognized that there is a very broad spectrum of human disease associated with STX-producing organisms. STEC-related disease may involve either sporadic cases or large outbreaks involving a common contaminated food source. Some individuals infected with STEC may be completely asymptomatic, in spite of the presence of large numbers of organisms as well as free toxin in the feces (50, 91). Very little is known of the true incidence of asymptomatic carriage. One study of Canadian dairy farm families (a group with high environmental exposure) detected carriage of STEC in about 6% of individuals (375). However, there has been little or no large-scale surveillance of healthy urban populations. Reported examples of asymptomatic carriage have usually been detected as a consequence of targeted testing of family contacts of persons with clinical STEC disease (284, 357). Many STEC-infected patients initially suffer a watery diarrhea, but in some this progresses within 1 or 2 days to bloody diarrhea and HC (235, 281, 282). Severe abdominal pain is also frequently reported. In a proportion of patients, STEC infection progresses to HUS, a life-threatening sequela characterized by a triad of acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia (158, 160). Some individuals with HUS experience neurological symptoms including lethargy, severe headache, convulsions, and encephalopathy (340). Although HUS occurs in all age groups, its incidence is higher in infants, young children, and the elderly. Indeed, it is a major cause of acute renal failure in the pediatric population (156, 317). The age distribution of HUS may be a consequence of the immunological naivety of young children and declining immune system function in the elderly (156), although age-related differences in receptor expression may contribute (193). Improved clinical management and pediatric renal dialysis techniques have reduced the mortality associated with HUS from about 50% to less than 10% over the last two to three decades (156). Nevertheless, a significant number of survivors (approximately 30%) suffer a range of permanent disabilities including chronic renal insufficiency, hypertension, and neurological deficits (156, 334). STEC infection can also result in a variant form of HUS, sometimes referred to as thrombotic thrombocytopenic purpura (TTP). This "diarrhea-associated TTP" is more common in adults than in children. The pathological features are essentially the same, but it differs from the typical form of HUS in that patients are more often febrile and have marked neurological involvement (156, 224). However, there is another form of TTP without a diarrheal prodrome, which is not associated with STEC infection.

Whether STEC-associated diarrheal disease progresses to life-threatening complications depends upon an interplay between bacterial and host factors. In an outbreak setting, the age of infected persons will have a significant influence on the proportion of infected persons who develop HUS, as well as the mortality rate. Characteristics of individual STEC strains will also have a major impact, and these are discussed below. Notwithstanding these considerations, studies of large outbreaks caused by O157:H7 STEC indicate that roughly 5 to 10% of individuals with diarrhea progress to HUS (114, 115, 156).

Species and serotype distribution of STX producers. It has been recognized for a number of years that STEC strains causing human disease belong to a very broad range of O:H serotypes. Karmali (156) listed 32 O serogroups (approximately 60 distinct O:H types), and the list has grown considerably since then (109, 179, 267). Although not represented in the initial group of STEC isolates described by Konowalchuk (175), serotype O157:H7 was the first STEC type to be linked to outbreaks of HC and HUS (156, 282). In many parts of the world, STEC strains belonging to this serotype (as well as O157:H) appear to be the most common causes of human disease. However, the relative ease of isolation of this serotype on the basis of its inability to ferment sorbitol may be contributing to an overestimation of its prevalence with respect to other STEC serotypes. Other common STEC serogroups include O26, O91, O103, and O111, and in several studies, non-O157 STEC serotypes such as these have been the predominant cause of human disease (39, 109, 267). There have been several reports of multiple STEC serotypes being isolated from a single patient, and in such circumstances, the contribution of each type to the pathogenesis of disease is difficult to ascertain (39, 260, 344). When one of the isolated types is O157, there is a (perhaps mistaken) tendency to ignore the potential etiological significance of the other(s). Other members of the family Enterobacteriaceae are known to produce STX and to cause serious gastrointestinal disease and HUS in humans. The most notable of these is S. dysenteriae type 1, the causative agent of bacillary dysentery, which is frequently complicated by HUS (232). It is the principal cause of HUS in parts of Africa and Asia (16, 37). Disease due to S. dysenteriae type 1 may be particularly severe, because the organism is capable of invading the colonic mucosa, and this might result in more efficient delivery of Stx to the bloodstream, as well as significant endotoxemia. Stx2-producing Citrobacter freundii also causes diarrhea and HUS in humans, including one outbreak in a German child care centre (304, 348). Haque et al. (124) have described the production of an Stx1-related cytotoxin by strains of Aeromonas hydrophila and A. caviae, as judged by stx₁-specific PCR and neutralization of Vero cytotoxicity with Stx1 antiserum. Enterobacter cloacae has also been associated with transient expression of an stx₂-related gene, although its role in disease is unproven (254, 263).

Sources of STEC. Cattle have long been regarded as the principal reservoir of STEC strains, including those belonging to serotype O157:H7. However, epidemiological surveys have revealed that STEC strains are also prevalent in the gastrointestinal tracts of other domestic animals, including sheep, pigs, goats, dogs, and cats (31, 58, 156, 180, 377). Estimation of the incidence of carriage of STEC is complicated by the fact that fecal shedding may be transient and is almost certainly influenced by a range of factors including diet, stress, population density, geographical region, and season (72, 180). Serological studies have suggested that the vast majority of cattle have been exposed to STEC at some point during their lives (72, 269). STEC isolates from animal sources include the important human disease-causing serotypes, as well as a number of O:H types that have yet to be associated with human infections (31, 72, 156).

STEC strains are a diverse group in terms of their capacity to cause serious disease in humans, and their ability to adhere to intestinal epithelial cells and to colonize the human gut is undoubtedly one of the key determinants of virulence. Estimates of the infectious dose for some STEC strains (O111:H and O157:H7) are of the order of 1 to 100 CFU (114, 260); these estimates are many orders of magnitude lower than that for enterotoxigenic E. coli (ETEC) or enteropathogenic E. coli (EPEC) strains. At present, the processes involved in establishment and maintenance of gut colonization by STEC are poorly understood. However, an increased knowledge of the mechanisms at the cellular and the molecular level and identification of the bacterial products involved may provide targets for vaccination strategies, or opportunities for therapeutic intervention.

Acid resistance of STEC. An important feature of STEC strains that may impact upon their capacity to colonize the human gut, particularly at low infectious doses, is resistance to the acidity of the stomach. It is now known that exposure of certain enteric bacteria, including E. coli, to low pH induces an acid tolerance response (110), and this has been shown to increase the survival of O157:H7 STEC in mildly acidic foods (187). A distinct phenotype referred to as acid resistance has also been described and is mediated by rpoS, which encodes a stationary-phase sigma factor. This factor regulates genes enabling stationary-phase E. coli organisms to survive for extended periods below pH 2.5 (111). Interestingly, Waterman and Small (365) have recently reported heterogeneity in acid resistance phenotype among STEC strains which correlated with mutations in rpoS. Such differences may contribute to apparent differences in infectivity of STEC strains.

Epithelial cell adherence phenotypes. Having survived the harsh conditions of the stomach, STEC must establish colonization of the gut by adhering to intestinal epithelial cells. It is generally assumed that the colon and perhaps also the distal small intestine are the principal sites of STEC colonization in humans, although this has not been demonstrated directly. In vitro adherence of STEC has been examined by using several different epithelial cell lines under a range of experimental conditions, and several adherence phenotypes have been described. However, interpretation of the significance of these studies is complicated by the fact that adherence to nonpolarized epithelial cells in tissue culture (even those of human colonic origin) may not be an accurate reflection of molecular interactions that occur between STEC and human colonic epithelium in vivo. Even within STEC strains belonging to serotype O157:H7, there is heterogeneity in adherence, and this may reflect differences in mechanisms. Indeed, Sherman et al. (313) reported marked quantitative differences (up to 250-fold) in the adherence of five O157:H7 strains to both HEp-2 (human laryngeal epithelioma) and Henle 407 (human colonic carcinoma) cell lines. Some strains adhered in a diffuse fashion, with bacteria distributed evenly over the surface of the epithelial cells (diffuse adherence [DA]). Other strains formed tight clusters or microcolonies at a limited number of sites on the epithelial surface (localized adherence [LA]). Moreover, a given strain did not necessarily exhibit the same pattern of adherence on both cell lines (313). A LA phenotype is also exhibited by EPEC and is mediated by type IV fimbriae (bundle-forming pili), which are encoded by a cluster of 14 bfp genes carried by the EAF plasmid (319, 324). However, STEC strains do not carry this plasmid and lack bfp genes (32, 371, 373). McKee and O'Brien (210) also described a distinct pattern of adherence of O157:H7 STEC to HCT-8 (human ileocecal) cells, which they termed "log jam." Since adherence occurred principally at junctions between the cells, it is possible that this is a consequence of interaction with the basolateral surface. Moreover, the log jam phenotype was also observed in some commensal E. coli strains. The best-characterized STEC adherence phenotype, however, is intimate or attaching and effacing (A/E) adherence. This property is exhibited by a subgroup of STEC strains and is discussed in more detail below.

Role of the 60-MDa plasmid in STEC adherence. The involvement of the 60-MDa STEC plasmid, referred to as pO157, in the adherence of O157:H7 STEC was initially suggested by Karch et al. (150). These investigators found that the presence of the plasmid correlated with expression of fimbriae and adherence to Henle 407 but not HEp-2 cells. However, subsequent studies have produced conflicting results (98, 148, 347), and there is no consistent in vitro evidence for a role for pO157 in STEC adherence. The discordant findings may be attributable to differences in growth or assay conditions, as well as to differences between O157:H7 strains, and possibly also to the fact that the large plasmid itself appears to be heterogeneous, even within serotype O157:H7 (21, 86). The potential contribution of pO157 to pathogenesis has also been examined in animal models. Tzipori et al. (353) found that the presence or absence of the plasmid had no effect on the capacity of STEC strains to colonize the colon or to cause A/E lesions in gnotobiotic piglets. On the other hand, Wadolkowski et al. (361) demonstrated that both O157:H7 strain 933 and its plasmid-cured derivative 933cu could individually colonize the gut of streptomycin-treated mice but that 933cu could not establish colonization when used together with 933. Although the same strains were used in the above piglet experiments, competitive colonization studies were not performed. Therefore, it is not possible to determine whether the apparent contribution of pO157 is influenced by host species. Moreover, the degree to which either of these animal models reflects colonization mechanisms in humans is uncertain. Clearly, more research is needed to determine whether pO157 or related plasmids play a role in adherence of STEC to colonic epithelium.

Attaching and effacing adherence. It has been known for more than a decade that certain strains of STEC are capable of causing A/E lesions on enterocytes (96, 312). A/E lesions involve ultrastructural changes, including loss of enterocyte microvilli and intimate attachment of the bacterium to the cell surface. Beneath the adherent bacteria, there is accumulation of cytoskeletal components, resulting in the formation of pedestals; this is recognizable by electron microscopy and by fluorescence microscopy after staining with phalloidin-fluorescein isothiocyanate (172). The capacity to produce A/E lesions was initially recognized in EPEC strains, and recent studies have elucidated the molecular events involved in their generation, as reviewed by Donnenberg et al. (81). All of the genes necessary for generation of A/E lesions in EPEC are located on a 35.5-kb "pathogenicity island" termed the locus for enterocyte effacement (LEE), which is inserted at 82 min in the E. coli chromosome. Binding of EPEC to epithelial cells (initially via the bundle-forming pili) triggers intracellular signals including release of inositol triphosphate, phosphorylation of myosin light chains, and tyrosine phosphorylation of certain proteins in the epithelial cell membrane (81). In contrast to earlier reports, generation of A/E lesions does not require changes in intracellular Ca²⁺ levels (17). LEE includes a cluster of genes (sepA to sepI) which encode a type III secretion system. This machinery is responsible for secretion of other LEE-encoded proteins, including EspA, EspB, and EspD, which are necessary for initiation of the signal transduction events referred to above. LEE also includes the eaeA gene, which encodes intimin, a 939-amino-acid outer membrane protein (OMP) which mediates intimate attachment to the enterocyte (81, 181). Interestingly, Kenny et al. (168) have recently reported that the receptor for intimin is also encoded by LEE. This protein was previously referred to as Hp90 but has now been renamed Tir (translocated intimin receptor). Tir is secreted from EPEC as a 78-kDa species, and efficient delivery into the host cell is dependent upon the type III secretion system and other LEE-encoded secreted proteins. Tyrosine phosphorylation of Tir after insertion into the epithelial cell membrane increases its apparent size to 90 kDa, a phenomenon which can be reversed by alkaline phosphatase treatment. However, tyrosine phosphorylation is not essential for intimin binding, at least in vitro (168).

Other adherence mechanisms. Factors implicated in the adherence of other enteric pathogens include fimbriae, OMPs, and lipopolysaccharide (LPS). Studies by Sherman and Soni (311) showed that antibodies to whole cells or outer membranes, but not to H7 flagella, significantly inhibited the adherence of O157:H7 STEC to HEp-2 cells. Moreover, exogenous addition of OMP extracts inhibited adhesion in a concentration-dependent manner but addition of isolated flagella and LPS did not. Subsequent studies demonstrated that polyclonal antiserum raised against a purified 94-kDa OMP also blocked adhesion (310). This protein was subsequently shown to be distinct from intimin (88). Recently, an 8-kDa O157:H7 OMP has also been implicated in adherence. A TnphoA insertion mutant deficient in production of this protein had a significantly reduced adherence to Henle 407 cells in vitro and was less able to colonize chicken ceca than was the wild-type O157:H7 STEC. Furthermore, preincubation with a monoclonal antibody specific for the 8-kDa OMP blocked subsequent in vitro adherence of the bacteria (382). In another recent study, Tarr et al. (335) described the isolation of a chromosomal E. coli O157:H7 gene, designated iha, which appears to encode the capacity to adhere to HeLa cells. The gene was found in all 20 O157:H7 STEC strains tested and in 4 of 5 eaeA⁺ non-O157:H7 human STEC isolates but was not found in 10 eaeA-negative meat isolates. Interestingly, however, iha was not part of the LEE, and the 696-amino-acid product of this gene is a surface protein with approximately 40% homology to IrgA, an iron-regulated protein of Vibrio cholerae. It is not yet known whether iha encodes the capacity of STEC to adhere to epithelial cells of intestinal origin. Maneval et al. (204) have also recently reported the isolation of 21-kDa fimbrial subunits from O157:H7 and O26:H11 STEC strains. N-terminal sequence analysis indicated a degree of homology to Bordetella pertussis and E. coli F17 fimbriae. However, there was evidence of antigenic variation between fimbriae from the two STEC strains, and their role in adherence remains to be determined.

Adherence mechanisms of non-O157 STEC. The mechanism of adherence of the class of STEC responsible for piglet edema disease to intestinal cells has been studied extensively (133). These strains do not generate A/E lesions, and adhesion to isolated porcine intestinal villi is mediated by a specific fimbrial adhesin referred to as F107. The gene cluster encoding F107 biosynthesis has been cloned, and the structural gene encoding the 15-kDa fimbrial subunit (designated fedA) has been sequenced (134). fedA has been found in the majority of edema disease STEC isolates but is also present in a small number of ETEC strains associated with postweaning diarrhea in piglets (132).

Uptake and translocation of STX by intestinal epithelial cells. Studies with rabbits have shown that STX has direct enterotoxic properties which result from selective targeting of Gb₃-containing absorptive villus epithelial cells in the ileum. Interestingly, this susceptibility of rabbit intestinal cells is age related and correlates with upregulation of net Gb₃ biosynthesis in the third week of life (218, 219). It is possible that the diarrhea seen in human STEC infections is due at least in part to direct exposure of enterocytes to STX in the gut lumen. However, the presence of Gb₃ in human enterocytes has yet to be demonstrated. Other studies suggest that many of the gastrointestinal pathological findings may be caused by systemic toxin. Intravenous injection of Stx1 into rabbits caused diarrhea with edematous and hemorrhagic lesions in the mucosa and submucosa of the cecum (280). Tashiro et al. (336) also demonstrated that local intra-arterial injection of Stx1 or Stx2 caused hemorrhagic lesions in the rat small intestine. In both studies, microvascular endothelium appeared to be the principal cytotoxic target. Since STEC strains appear to be unable to invade gut epithelial cells to any significant extent, the generation of systemic sequelae must presumably involve translocation of STX produced by colonizing bacteria from the gut lumen to underlying tissues and the bloodstream. One possible route might be through lesions in the mucosal barrier caused either by the direct effects of STX or other factors such as intimin or perhaps through gaps between adjacent epithelial cells. An alternative route from gut lumen to tissues might be through intact epithelial cells. This possibility has been examined by using polarized human colonic carcinoma cells (CaCo-2A and T84) grown on collagen-coated polycarbonate membranes (4). When grown for extended periods, these cells form tight junctions and the monolayers exhibit high transepithelial electrical resistance. For both cell lines, a significant proportion of active Stx1 added to the culture medium on the apical side was translocated to the medium on the basolateral side over 24 h; during this time, there was no toxin-induced damage to the epithelial barrier as judged by electrical resistance. This process appeared to be energy dependent, since it was blocked by low temperature or an uncoupler of oxidative phosphorylation. The total amount of Stx1 that could be translocated appeared to be saturable, suggesting the involvement of a cellular receptor, but this is unlikely to be the specific STX receptor Gb₃, since T84 cells lack this glycolipid. Moreover, induction of Gb₃ synthesis in CaCo-2A cells by treatment with sodium butyrate actually reduced toxin translocation. Thus, at least Stx is capable of translocation across intestinal epithelial cells without apparent cellular disruption via a transcellular pathway (4).

Interaction of STX with its glycolipid receptor. Once having crossed the epithelial barrier and presumably entered the bloodstream, STX targets tissues expressing the appropriate glycolipid receptor. The specificity of this interaction and the distribution of receptors among various cell types has a major impact on the pathogenesis of disease, both in humans and in various animal models, as recently reviewed by Lingwood (193). High levels of Gb₃ are found in the human kidney, particularly in the cortical region, the principal site of renal lesions in patients with HUS (47). However, overall levels were higher in adult kidneys than in infant kidneys, which contrasts with the age susceptibility to the disease. A subsequent study involving overlaying human renal sections with fluorescence-tagged Stx1 indicated that in tissue from adults, the toxin bound principally to distal convoluted tubules, particularly those closely apposed to a glomerulus, whereas in infants, overall Stx1 staining was less intense, in parallel with Gb₃ content. However, toxin bound to glomeruli as well as to distal tubules, which is consistent with the increased susceptibility to HUS in this age group (192). Tesh et al. (342) found similar levels of Gb₃ in the cortex and medulla of human, baboon, and mouse kidneys, as well as comparable binding of Stx1; both receptor and bound toxin were associated primarily with tubular epithelial cells. Although the pathological findings of HUS in humans are strongly indicative of significant endothelial cell (EC) disturbance (discussed below), there is also evidence for the involvement of other renal cell types in pathogenesis of disease. For example, Takeda et al. (332) reported that HUS patients have elevated levels of markers of tubular injury during the early stages of disease. Tubular necrosis is also seen in a proportion of patients with HUS (120, 279), and a significant number of patients with HUS present with acute anuric renal failure consistent with tubular necrosis but without obvious signs of coagulopathy. In rabbits, intravenous administration of Stx1 results in vascular damage (thrombotic microangiopathy), particularly in the cecum, colon, and central nervous system (280, 383). EC in these tissues were also the principal sites of uptake of ¹²⁵I-labelled Stx1. However, rabbit renal tissue was unaffected and did not bind toxin (280), which is consistent with its lack of Gb₃ (383).

Effects of STX on endothelial cells. It is now generally agreed that the major portion of the histopathological lesions associated with both HC and HUS is a consequence of the interaction of STX with endothelial cells. The typical features of HUS include swollen and detached glomerular EC, and deposition of fibrin and platelets in the renal microvasculature (particularly in the glomerulus) (279). Capillary occlusion results in reduced blood flow to the kidneys and hence to renal insufficiency and may also cause physical damage to RBCs. Thrombotic lesions are also observed systemically, particularly in the microvasculature of the bowel, brain, and pancreas. Analogous histopathological lesions in the brain and gastrointestinal tract are seen in animal models of STX-induced disease (280, 336), although there is evidence that in both mice and rabbits, neurological damage by Stx2-related toxins involves direct neuronal injury in addition to microangiopathy (100, 101, 217). Although the precise molecular mechanisms whereby microangiopathic lesions are generated are not fully elucidated, STX may prevent the production of molecules critical for maintenance of the procoagulant-anticoagulant balance of the endothelium and/or an imbalance between vasodilators and vasoconstrictors produced by these cells (149).

Influence of STX type on pathogenesis. Epidemiological studies have indicated that STEC strains producing Stx2 only are more commonly associated with serious human disease, such as HUS, than those producing Stx1 alone or Stx1 and Stx2 (171, 244). One possible explanation for this is that the level of transcription of stx₂ in vivo is higher than that of stx₁. Transcription of stx₁ is known to be iron repressible in vitro, and its promoter region includes a recognition site for the fur gene product (56, 366). However, iron levels in the gut are very low, and so it is likely that transcription of stx₁ will be fully derepressed in vivo. Sung et al. (329) have shown that in vitro transcription of stx₂-related genes is constitutive and at a level commensurate with that of derepressed stx₁. Mühldorfer et al. (225) also found that stx₂ promoter activity was unaffected by osmolarity, pH, oxygen tension, acetates, iron level, or carbon source but that there was a slight effect of growth temperature. However, treatment with mitomycin, which induces the lytic cycle of STX-converting bacteriophages, has been shown to significantly increase both Stx1 and Stx2 production in lysogenized E. coli strains (6, 126). For Stx2, this increase appears to be due to a combination of amplification of stx₂ as the phage DNA is replicated and an increase in stx₂ promoter activity mediated by a phage-encoded positive regulatory factor (225). This increase in Stx2 production may be clinically significant, since mitomycin is used to treat certain neoplastic disorders and these patients are at increased risk of HUS (185).