Effect of Shiga toxin and Shiga-like toxins on eukaryotic cells

Microbes and Infection, 3, 2001, 493−507 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457901014058/REV

Review

Effect of Shiga toxin and Shiga-like toxins on eukaryotic cells Edward V. O’Loughlina*, Roy M. Robins-Browneb a

Department of Gastroenterology, The Royal Alexandra Hospital for Children, PO Box 3515, Parramatta 2124, Westmead NSW, Australia b Department of Microbiology and Immunology, The University of Melbourne and Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville VIC, Australia

ABSTRACT – Shigella dysenteriae and Shiga-toxin-producing Escherichia coli (STEC) elaborate the AB holotoxins, Shiga or Shiga-like toxins (Stx). Stx play a major role in the pathogenesis of haemorrhagic colitis and haemolytic uremic syndrome. This review provides an overview of the mechanisms of action of Stx and a model of the pathogenesis of Stx-induced disease. © 2001 Éditions scientifiques et médicales Elsevier SAS Shiga toxin / pathogenesis / haemolytic uremic syndrome

1. Introduction Shiga toxins (Stx), also known as verotoxins, verocytotoxins or Shiga-like toxins, are produced by several enteric pathogens, most importantly Shigella dysenteriae (serotype 1 only) and enterohaemorrhagic Escherichia coli (EHEC). These bacteria cause foodborne outbreaks of gastroenteritis and also contribute to endemic enteric infection. Stx are important factors in disease pathogenesis and are responsible for some of the severe complications, such as haemorrhagic colitis and the haemolytic uremic syndrome (HUS). This review will focus on the effect of Stx on eukaryotic cells and will provide the basis for our current understanding of pathogenesis of Stx-induced disease. 1.1. Shigellosis and EHEC infection

Shigellosis is an important bacterial cause of gastroenteritis in both the developed and developing world (reviewed in [1]). Of the Shigella spp. that infect humans, only S. dysenteriae produces Stx. This species is the usual agent for epidemic shigellosis and has been responsible for large outbreaks in Africa, southeast Asia and the Indian subcontinent, where it accounts for up to 30% of endemic dysentery. Infection with Shigella commonly occurs in children under 5 years of age and is associated with poverty and overcrowding, poor sanitation and contaminated water supply. Shigellae invade the intestinal mucosa and induce severe ileocolitis. The clinical spectrum ranges from mild watery diarrhoea to severe dysentery, complicated by neurological symptoms such as drowsiness, con-

*Correspondence and reprints. E-mail address: [email protected] (E.V. O’Loughlin). Microbes and Infection 2001, 493-507

vulsions and cerebrovascular accidents. HUS is a complication of infection with S. dysenteriae 1 and is characterised by haemolytic anaemia, thrombocytopenia and renal failure [2]. Some strains of E. coli also produce Stx and are termed Shiga-toxin producing E. coli (STEC, reviewed in [3–5]. A large variety of different STEC have been identified but only a small number of clones are significant human pathogens. However, one subset, termed EHEC, causes enteric disease ranging from mild gastroenteritis to haemorrhagic colitis and HUS, generally produces attaching and effacing lesions (usually associated with enteropathogenic E.coli) in cell culture and carry a distinctive 60-MDa pathogenicity plasmid [3]. EHEC are important agents of foodborne disease but are also an important cause of endemic gastroenteritis and HUS. These bacteria have gained wide notoriety as a result of their association with outbreaks of diarrhoea and HUS from fast food outlets and are often referred to as ‘Hamburger E. coli’ in the popular press. In contrast to Shigellae, EHEC are non-invasive, but colonise the large intestine and may cause a severe colitis. Stx contribute substantially to the haemorrhagic colitis and are the primary agents of the systemic complications such as HUS. 1.2. Discovery of Stx and related toxins

Shiga first isolated the agent of bacillary dysentery in 1898 from a patient with diarrhoea. This particular organism was S. dysenteriae. A few years after its discovery, extracts of the cultured bacillus were noted to paralyse and kill rabbits when administered parenterally [5] but it took until 1972 for the first report of partial purification of this toxin to appear [6]. Extract of S. dysenteriae caused fluid accumulation when inoculated into rabbit ileal loops 493

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Table I. Shiga-like toxin 2 variants. Stx type

2 2c 2d 2e

Also called

VT2, Slt2 Slt-2vh Slt 2v Slt 2vp

Amino acid homology to SLT-2 A

B

100 100 99 93

100 97 97 94

Receptor type

Lethal for orally infected mice

Gb3 Gb3 Gb3 Gb4

+ + + –

SLT, Shiga-like toxin; VT, verotoxin.

and was neurotoxic to mice. They suggested that both biological effects were caused by a single toxin. A separate study by these investigators demonstrated that the cytotoxic and enterotoxic effects of the ‘Shigella neurotoxin’ were due to a single toxin, Stx [7]. Evidence that the production of toxin by S. dysenteriae contributes to the dysentery was provided by studies using volunteers [8] and a monkey model of ileocolitis [9]. Stx has been purified to homogeneity [10, 11]. The purified toxin is cytotoxic for tissue culture (HeLa) cells, enterotoxic in rabbit ileal loops and lethal when injected into rabbits or mice. These biological activities of the toxin can be neutralised by specific antiserum derived by immunisation with toxoid. Neutralising antibodies later proved useful in the extraction of Stx produced by some E. coli strains. In 1977, Knowalchuk et al. reported that culture filtrates from some strains of E. coli had a cytotoxic effect on Vero cells (African green monkey kidney cells) [12]. It was later found that this effect could be neutralised by antiserum to Stx [13] suggesting that Stx or something similar was the agent responsible for cytotoxicity. O’Brien and colleagues demonstrated that a strain of E. coli O157:H7 isolated from an outbreak of haemorrhagic colitis in the USA was toxic to HeLa cells and suggested that the isolate produced a Shiga-like toxin [14]. A Shiga-like toxin was subsequently isolated from this strain [15] and its purification and characterisation were described in detail shortly thereafter [16]. A similar toxin was subsequently identified in many strains of E. coli, some of which were nonpathogenic [17]. An important milestone in the evolution of our understanding of E. coli virulence was the observation by Karmali and others that the Shiga-like toxin produced by STEC was responsible for causing HUS [18, 19]. In S. dysenteriae, Stx is chromosomally encoded. Genes for the toxin A and B subunits have been cloned and sequenced. The predicted molecular masses for the processed A and B subunits are 32 225 (293 amino acids) and 7 691 kDa (69 amino acids), respectively (reviewed in [1, 20]. On the other hand, genes encoding the production of Stx produced by E. coli are located on toxin-converting lambdoid phages [21, 22]. Two different phages encode genes for two antigenically distinct toxins, Stx-1 and 2 [21, 23]. Stx-1 is 98% homologous to Stx (from S. dysenteriae), differing by only one amino acid in the A subunit and is neutralised by anti Stx-1 antiserum. In contrast, Stx-2 has less than 60% homology with Stx-1 and is not neutralised by anti Stx antiserum. Antigenic variance has been observed in the Stx-2 subgroup (see table I) which is 494

largely due to variation in the amino acid sequence in the B subunit of this toxin. This variation in the B subunit also accounts for differences in receptor binding specificity, and hence toxicity for toxins in animals and cell cultures [24]. This is discussed in more detail below. Most of the Stx-2 family are phage encoded though Stx-2e, associated with oedema disease of swine, is usually chromosomally encoded (likely an integrated phage) [24]. 1.3. Environmental and genetic regulation of toxin

The structural genes encoding Stx A and B subunits in S. dysenteriae are present on a single transcriptional unit, in contrast to the holotoxin, which comprises a single A subunit covalently bound to five B subunits. The biosynthetic mechanism underlying this stoichiometry of the holotoxin is unclear as the subunits are transcribed at a 1:1 ratio [1]. However, Habib and Jackson [25] mapped and characterised a promoter region upstream of the 5’ region of the Stx B gene. They identified a second promoter in the Stx B operon which was less efficient than that for the Stx A operon and, unlike the latter, is not inhibited by iron. These findings suggest that independent transcription of the B subunit may account for the 1:5 stoichiometry of the holotoxin. The Stx B subunit transcript also binds more avidly to ribosomes than the A subunit, resulting in increased translation of B subunits [26]. The operator region upstream of the gene for the A subunit for Stx-1 in S. dysenteriae and E. coli contains a binding site for Fur protein which complexes with iron, binds to DNA and blocks transcription [27, 28]. This accounts for the observation that Stx-1 in both S. dysenteriae and STEC is repressed in iron-containing media [29, 30]. Toxin production is also temperature regulated, being maximal at 37 °C. These observations suggest that Stx-1 production can be regulated in response to host-derived signals such as body temperature and a low iron environment. This environment would be expected in the distal small intestine and colon. Conceivably, production of Stx, which induces ischaemic necrosis of the gut, may facilitate bacterial survival by causing bleeding into the bowel lumen. Some strains of STEC also carry a plasmid-encoded enterohaemolysin. This has been demonstrated to lyse sheep erythrocytes. It may provide a mechanism for making iron more available to the bacteria [3, 4]. These pathogenic factors may provide essential nutrients including iron and other growth promoters, which allow for the growth of bacteria in a hostile environment. In contrast, Microbes and Infection 2001, 493-507

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Stx-2 does not appear to be regulated by environmental factors [31]. 1.4. Toxin structure

Members of the Stx family are AB holotoxins comprising one A subunit of 32 kDa, which is the active component of the toxin, non-covalently bonded to five identical B subunits each of 7.7 kDa. The latter form a pentameric structure important for toxin binding to its cellular receptor [20]. Individual B subunits are composed of antiparallel β sheets and an α helix. The cleft formed by the interaction between adjacent β sheets provides carbohydrate-binding sites for binding to the receptor. Stx have structural similarities to other toxins such as cholera toxin, the heat labile toxin of enterotoxigenic E. coli and the plant toxin ricin [20]. The A subunit of Stx is composed of two fragments, A1 and A2, which are linked by a disulphide bond. A subunit is proteolyically cleaved at this site by enzymes in the endoplasmic reticulum and cytosol [32] to form a 27-kDa A1 subunit, responsible for the enzymatic activity of Stx, and a smaller A2 fragment. Using recombinant toxin subunits, Austin et al. [33] found that intact toxin A and B subunits have the ability to form holotoxin spontaneously in vitro. However, recombinant A1 fragment is unable to combine with B subunits to form holotoxin, indicating that the A2 fragment is essential for holotoxin assembly. X-ray crystallographic studies of the B subunits indicate that they form doughnut-shaped pentamers which are penetrated by the A2 fragment [34–36]. Site directed mutagenesis to induce progressive deletions of amino acid residues at the C-terminus end of the A subunit revealed that nine amino acids which form an α helix in the A2 fragment penetrate the pore of the Stx B pentamer [37]. Flanking charged residues then allow the non-covalent association of A and the pentameric B subunits. 1.5. Toxin receptors

Fuchs et al. were the first to provide unequivocal evidence that Stx bound to a receptor on mammalian cells [38]. These investigators isolated brush border membrane vesicles from rabbit ileum, an obvious target, as it had been known for some time that Stx had enterotoxic effect in rabbit ileum. Toxin-binding experiments using 125Ilabelled Stx demonstrated specific binding of toxin to microvillus membranes. Treatment of HeLa cells with tunicamycin, a compound which inhibits N-linked glycosylation, abrogates the cytotoxic effect of Stx, indicating that the toxin bound to a carbohydrate-containing receptor on these cells. Several groups subsequently identified membrane glycolipids containing the carbohydrate sequence galactose α1-4 galactose β1-4 glucose ceramide as the putative receptor. Overlay of radiolabelled toxin on thin-layer chromatograms containing separated glycolipids and binding of toxin to glycolipid-coated microtitre plates established that the toxin bound to globotriasoyl ceramide (Gb3), globotetraosyl ceramide (Gb4) and PI (a blood group glycolipid antigen) which is present in red blood cell membranes [39–41]. Several lines of evidence established the role of these membrane glycolipids as Stx receptors and their contribuMicrobes and Infection 2001, 493-507

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tion to the susceptibility of eukaryotic cells to these toxins. Inhibition of toxin-binding by glycolipid analogues or monoclonal antibodies which bound to Gb3 [40, 41], destruction of receptors by digestion of membrane glycolipids with α galactosidase [40, 42] and inhibition of glycolipid biosynthetic pathways which impair receptor expression [43] result in loss of cytotoxicity for cultured epithelial cells. In addition, artificially incorporating Gb3 into the cell membranes of toxin-resistant cells such as Daudi Burkitt lymphoma cells using liposomes containing Gb3, results in substantial incorporation of this glycolipid into the cell membrane conferring sensitivity to the toxin [41, 43, 44]. In some cells such as CHO cells, the expression of Gb3 is insufficient to confer sensitivity to Stx, implying that other key factors, such as toxin-receptor internalisation or intracellular toxin degradation is/are also important for its effect [43]. Gb3 is also developmentally regulated. For example, suckling rabbit ileum is resistant to the effect of toxin but after weaning, receptors are expressed on villus epithelial cells, which parallels the development of susceptibility to the enterotoxic and cytotoxic effect of toxin [42]. Similarly, toxin receptors can be induced in CaCo2 cells by incubating the cells with butyrate. This stimulates the biosynthetic pathways responsible for the production of Gb3 and confers toxin sensitivity on previously resistant cells [45]. The length of the Gb3 fatty acid chain has also been shown to affect toxin sensitivity and binding. Stx-1 and Stx-2c both bind to Gb3 though Stx-1 is 1 000- fold more toxic in vitro than Stx-2c. Using semisynthetic Gb3 analogues with varying fatty acid chain length, Kiarash et al. [46] demonstrated preferential binding of Stx-1 to Gb3 with fatty acid chain lengths greater than 20 carbons and Stx-2c preferentially bound to Gb3 with shorter fatty acid chain lengths. Sandvig and others [47], following a similar line of investigation, found that the butyrate-induced increase in sensitivity of A431 (human epidermoid carcinoma cell line) to Stx-1 was in part due to increases in the fatty acid chain lengths of Gb3. Moreover, these investigators found that receptor fatty acid played a key role in the intracellular trafficking and sorting of the toxin to the cytosol. Early studies utilising monoclonal antibodies directed against the B subunit provided evidence that this was the binding moiety of the toxin [41, 48]. This observation was subsequently confirmed in in vitro binding studies utilising 125 I- and fluorescein-labelled B subunit [41, 45]. Hybrid toxins [49, 50] and toxins with mutations in the putative binding regions of the B subunit [51, 52] show altered binding specificity to the receptor. Wild toxin variants such as Stx-2c and Stx-2e (the latter responsible for oedema disease in swine) [53] also demonstrate differences in receptor binding, compared to Stx-2, [54] which are due to differences in the B subunits. X-ray crystallography of the crystal structure of the B subunit pentamer of Stx complexed with a soluble trisaccharide analogue of Gb3 demonstrated three potential carbohydrate-binding sites on each B subunit within the cleft located between adjacent β sheets [55]. Site-directed mutagenesis resulting in alterations to the three putative binding sites reduces toxin binding and cytotoxicity, indi495

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cating that the binding sites identified by the structural studies are biologically significant [51]. A341 human epidermoid carcinoma and T84 human colon cancer cell lines do not express glycolipid receptors [56, 57] but are known to internalise toxin. Moreover, T84 cells are capable of apical to basolateral vectorial transport of toxin [57]. These observations suggest that other mechanisms are available for toxin uptake. Recently, studies with Gb3-deficient Vero cells (which usually express high levels of Gb3) demonstrated a saturable binding process and identified a specific membrane protein to which toxin bound. The nature of this putative protein receptor is not well understood [58]. Non-receptormediated uptake of Stx in the gastrointestinal tract may permit the toxin to gain entry into the portal and systemic circulation without destroying the epithelial cells. The tissue distribution of the Gb3 and/or Gb4 receptors in vivo determines the site of Stx-induced microangiopathy. For example, injection of rabbits with Stx-1 induces inflammation in the central nervous system and gastrointestinal tract but not the kidneys [59]. This distribution of disease correlates with expression of Gb3. Mice, on the other hand, are more susceptible to Stx-2 and develop central nervous system (CNS) and renal disease without gut involvement in keeping with the tissue distribution of Gb3 [60]. In pigs, Stx-2e induces disease in cerebellum, colon and eyelids (oedema disease) reflecting the tissue distribution of Gb4 [53, 54]. Interestingly, red blood cells contain the highest content of Gb4. Binding to the red cells does not convey protection against toxin effect, rather, red cells appear to act as a carrier of toxin. Site-directed mutagenesis of the carbohydrate-binding site of the Stx-2e B subunit, resulting in increased binding to Gb3 and reduced binding to Gb4, causes pathology more typical of Stx-induced disease, i.e. more extensive microangiopathy in the CNS and gastrointestinal tract [60] rather than typical oedema disease. 1.6. Toxin internalisation and retrograde transport to the cytosol

Shiga toxins enter epithelial cells at the apical membrane but have to traverse the cell to exert their effects on protein synthesis (figure 1). The toxins move in a retrograde fashion through the cellular machinery that transports cellular proteins from ribosomes to the apical membrane of the cell, and then inhibit protein synthesis at the level of ribosomes [61]. The toxins bind to their glycolipid receptors’ clathrincoated pits and are quickly internalised [61, 62]. Both toxin internalisation and cytotoxicity can be inhibited in epithelial cells by removal of clathrin pits by potassium depletion and treatment with the cytosolic transglutaminase inhibitor, dansyl cadaverine. A parallel pathway of toxin uptake involving caveolae has also been identified [63]. Once internalised, toxins can follow one of several routes. Using A341 cells, Sandvig et al. [64] demonstrated that toxin-insensitive cells rapidly transport internalised toxin to lysosomes probably for degradation. However, treating cells with butyrate increases their sensitivity to toxin and also results in retrograde transport of toxin to the 496

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Figure 1. Receptor-mediated uptake and transport of Shiga toxin from the apical membrane to the cytosol. Catalytic cleavage of the A subunit occurs primarily at 1 by furin and by a secondary mechanism in the region of the TGN by calpain at 2. ER, endoplasmic reticulum; TGN, trans-Golgi network. Reproduced with permission from [61]. trans-Golgi network, Golgi stacks, endoplasmic reticulum and nuclear envelope. Other workers have used fluorescein-labelled B subunit in HeLa cells [65] to demonstrate trafficking of the toxin from apical membrane to endoplasmic reticulum via the Golgi. B subunit, rather than holotoxin, is enough to traffic the toxin to the ER [56, 65]. Brefeldin A, a compound which disrupts Golgi stacks, abrogates the cytotoxic effect of Stx in sensitive cells. Recently White et al. described a microtubule-dependent translocation pathway which traffics Stx B subunit between the Golgi and the ER [66]. This pathway is regulated by the small GTPase, Rab6. Retrograde trafficking to the Golgi and ER does occur in some proteins, such as Pseudomonas exotoxin A [67], which contain a trafficking sequence in their carboxyterminus end termed the KDEL (-Lys-Asp-Glu-Leu-) sequence [61]. However, Stx do not contain the KDEL sequence in the B subunit, indicating that there is some other mechanism driving this retrograde transport. Experimental evidence suggests that the composition of the fatty acid chain in the receptor, rather than the toxin itself, contains the trafficking signal necessary to translocate toxin from apical membranes to the cytosol. As discussed above, the addition of Na butyrate to the cell culture medium increases the expression of Gb3 receptors on the cell surface which parallels the development of Microbes and Infection 2001, 493-507

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cytotoxicity in previously resistant cells. Butyrate treatment of A341 cells also induces toxin trafficking to the ER and nuclear envelope compared with untreated cells in which toxin is directed to lysosomes [64]. These observations suggest that some intrinsic property of the receptor, which is altered by butyrate treatment, may be involved in retrograde trafficking [56]. Examination of fatty acid content of the Gb3 receptor in butyrate-treated A431 cells reveals that butyrate treatment increases the fatty acid chain lengths of Gb3 [68]. Confirmation of this mechanism was established in later studies by Sandvig et al. [47], who showed that changing Gb3 fatty acid composition or chain length induces major alterations in intracellular trafficking of the toxin. Cleavage of Stx A subunit increases its enzymatic activity [69]. Within eukaryotic cells, cleavage occurs at a trypsin-sensitive site in the carboxy-terminal end of the A subunit in the region of the disulphide bridge. Cleavage results in disassociation of A1 and A2 subunits. A derivative of Stx in which the trypsin-sensitive site is altered by site-directed mutagenesis making it resistant to proteolysis retains its cytotoxicity for cultured epithelial cells, although its effect is delayed compared to the naturally occurring toxin [70]. Stx is cleaved by epithelial cells even in the presence of brefeldin A (which disrupts the Golgi stacks), suggesting that the intracellular cleavage occurs in the early endosomes or the trans-Golgi network. However, the mutant trypsin-resistant toxin is not cleaved in the presence of brefeldin A, indicating an alternative cleavage mechanism probably in the cytosol. The alternative processing pathway requires the cytosolic enzyme calpain, because inhibition of calpain inhibited cellular toxicity of the mutant toxin. Garred et al. [52] utilised in vitro assays and in vivo cell culture models to demonstrate that furin, a calcium-sensitive serine protease localised to the transGolgi network, was the major enzyme responsible for cleaving the A subunit of Stx. This enzyme is thought to cleave precursors of various secretory and membrane proteins with the consensus motif Arg-X-Arg/Lys-Arg. The A subunits of various Stx contain a similar motif in the region of the disulphide bridge. Taken together, the experimental data indicate that holotoxin undergoes transport to endosomes and trans-Golgi network where the A subunit is cleaved by furin. Calpain and possibly other proteases, in the cytosol, provide an alternative, less efficient mechanism for proteolytic cleavage. Cytosolic cleavage may be important for some of the toxins such as Stx-2e [71]. The retrograde transport of Stx as described above is determined by initial binding to Gb3 or Gb4. However, there is evidence that in some cell lines, an alternative pathway of toxin translocation and signal transduction pathway activation can result in quite different biological effects. For example, cell lines of intestinal origin such as CaCo2 cells and T84 cells translocate toxin from apical to basolateral surfaces in the absence of glycolipid receptors and do not develop cytotoxicity [57, 72]. This pathway is likely to be important in uptake of toxin from the gastrointestinal tract. Moreover, Stx has been shown to stimulate interleukin-8 secretion by Hct-8 intestinal epithelial cells by a pathway that does not involve inhibition of protein synthesis [73]. This effect is inhibited by blockers Microbes and Infection 2001, 493-507

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of the p38/RK mitogen-activated kinase cascade, involving the ribotoxic stress response (caused by breakdown of RNA) and early activation of c-jun mRNA. Stx also stimulate the release of proinflammatory cytokines from immunocytes such as macrophages [74] and monocytes [75, 76] which express low levels of toxin receptor and are refractory to its cytotoxic effect. Stx also causes apoptosis in Daudi cells (Burkitt lymphoma cell line) by a pathway independent of its effect on protein synthesis [77]. Interestingly, the B subunit alone is enough to induce apoptosis, suggesting that the glycolipid receptor is the major mediator of this effect. 1.7. Inhibition of protein synthesis

Early reports indicated that Stx inhibits protein synthesis (reviewed in [78]). Subsequent experimentation revealed a highly specific mechanism of action. Using a cell-free system, Reisberg et al. [69] demonstrated that Stx inactivates the large 60S ribosomal subunit which is ubiquitous in eukaryotic cells. These large ribosomal subunits comprise three fragments of 5S, 5.8S and 28S ribosomal RNA (rRNA) subunits. Trypsin activated Stx in reticulocyte lysates incubated with 14C phenylalanine inhibits the synthesis of polyphenylalanine chains. This inhibition is localised to the 60S ribosomal subunits and is independent of peptidyl transferase activity which is required to elongate the peptide chains of newly synthesised polypeptides. Instead, Stx inhibits peptide chain elongation by blocking aminoacyl-tRNA binding to the acceptor site on ribosomal RNA [79, 80]. In vitro treatment of rat ribosomes with Stx-1 or 2 causes the release of an adenine base from the 28S rRNA at position 4324, indicating that Stx inactives 60S ribosomal RNA by cleaving a specific N-glycosidic bond [81]. The target of Stx is the RNA acceptor site aminoacyl-tRNA docking. In addition to its effect on eukaryotic cells, Stx has a similar effect on bacterial ribosomes, resulting in decreased proliferation of susceptible bacteria such as E. coli [82]. This raises the possibility that Stx may facilitate bacterial survival by inhibiting the growth of potential competitors in the lumen of the gastrointestinal tract. The N-glycosidase activity of Stx is very similar to that of the plant toxin ricin, implying some evolutionary conservation of these toxins.

2. Pathophysiology of organ damage The preceding section described the cell biology of Stx. This section will describe how the toxins induce disease and will attempt to build a model of disease pathogenesis to explain the clinical manifestations of infection. Although the kidney and gastrointestinal tract are the organs most commonly affected in HUS, CNS, pancreatic, skeletal and myocardial involvement may also occur [83]. The following discussion will focus on gut, endothelium, kidney and brain. 2.1. Gastrointestinal tract

S. dysenteriae and STEC are ingested orally, often in infected food or water. S. dysenteriae first colonises and then invades the epithelium, resulting in necrosis of the 497

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mucosa (reviewed in [1, 84]. In contrast, STEC are noninvasive. Instead, they associate with the large intestinal mucosa and secrete toxins into the lumen of the gut (reviewed in [4]). Both groups of organisms cause severe intestinal damage leading to diarrhoea and dehydration. However, the pathophysiology of the gastrointestinal injury is quite complex and involves microbial and host factors in addition to the role played by the toxins. Shigellae induce severe inflammation in the ileocolonic regions of the gastrointestinal tract [2, 85]. Usually this is confined to the mucosal surface, though intestinal perforation and small vessel vasculitis is observed in humans infected with S. dysenteriae. Monkeys infected with the non-Stx-producing species, S. flexneri also develop intense inflammation in the ileocolonic region, mucosal destruction and colonic transport abnormalities primarily of reduced sodium and water absorption [85], implying the existence of pathogenic factors other than Stx. Fontaine et al. examined the role of Stx in the pathogenesis of the mucosal injury of Rhesus monkeys infected with S. dysenteriae by comparing animals infected with a wild-type strain with a group infected with an isogenic mutant that produced very low levels of toxin [86]. Both strains produced severe dysentery with similar mortality rates. Although the mutant strain produced similar severity of diarrhoea to the wild type, fewer animals experienced bloody diarrhoea. In addition, animals infected with the wild-type strain exhibited patchy subserosal haemorrhages in the colon and evidence of vasculitis in the small vessels of the mucosa and peritoneum, which was not observed in animals infected with the mutant. These findings indicate firstly, that mechanisms other than Stx account for the severe intestinal inflammation associated with Shigella infection and secondly, that toxin exacerbates mucosal damage by causing a vasculitis in the microcirculation of the mucosa and larger vessels in the mysentery. The vasculitis contributes to the development of bloody diarrhoea (haemorrhagic colitis). Similar observations to those described above have been made with STEC. Infection of suckling rabbits with STEC strains of O157:H7 leads to diarrhoea, severe intestinal inflammation and abnormalities of colonic salt and water transport which account for much of the diarrhoea [87]. Stx-1 and 2 do not play a significant role in these changes, as suckling rabbits do not express the Gb3 receptor on colonic epithelial cells [42] and animals infected with a toxin-negative mutant exhibited structural and transport changes as severe as animals infected with the wildtype strain. The hypothesis that host defence mechanisms play a role in the evolution of the intestinal injury was tested in a series of experiments designed to block the host inflammatory response to infection. Animals infected with a wild strain of O157:H7 developed diarrhoea, severe intestinal inflammation and abnormalities of mucosal electrolyte transport. Pretreatment of animals with an antibody directed against the leukocyte adhesion molecule, CD11/ CD18 inhibits polymorphonuclear leukocyte (PMN) infiltration, reduced diarrhoea and the abnormalities of mucosal structure and electrolyte transport, suggesting a major role for PMN in the production of mucosal damage [88]. 498

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Stx have been shown to directly damage the intestinal epithelium, although there are major species differences. Direct inoculation of purified Stx into ligated rabbit ileal loops causes mucosal inflammation and fluid accumulation due to reduced absorption of water and electrolytes, rather than active secretion [6]. The mucosal damage also deranges glucose and amino acid absorption [89]. Studies in isolated enterocytes from rabbit ileum show that Stx directly targets villus enterocytes, rather than crypt cells, as only the former express the Gb3 receptor [90]. In contrast, gastrointestinal epithelial cells from mice [60], and humans [91] and cells derived from some immortalised gut epithelial cell cultures [57, 72] do not express Gb3 and thus are resistant to the cytotoxic effects of Stx (see above for more detail). Pigs exhibit low levels of Gb3 expression in epithelial cells of jejunum, ileum colon which consequently bind toxin weakly or not at all [92]. Thus pigs are resistant to the enterotoxic effect of intraluminal Stx. Intestinal epithelial cells are capable of translocating intact Stx from the apical to the basolateral sides of the cell [72] without sustaining damage from the effects of toxin (figure 2; [57]). This may be of major importance in the pathogenesis of shigellosis or STEC infection, as it allows the entry of intact toxin into the systemic circulation. However, the studies which have examined Stx translocation in epithelial cells have focussed on cultured cells. It would be of interest to examine this Stx transport in a model of active colitis, as intestinal permeability to macromolecules is significantly increased by inflammation [93]. Our group has previously demonstrated increased paracellular permeability to mannitol in rabbit colon infected with STEC [94]. We postulate that the absorption of lumenal Stx is enhanced in the presence of active inflammation due to deranged mucosal barrier function. Also, bacterial products such as lipopolysaccharide (LPS) are likely to enter the circulation through a more permeable mucosa and augment the host inflammatory response. What then is the role of Stx in the gastrointestinal manifestations of shigellosis or STEC infection? Evidence suggests that toxin plays a major role in inducing vascular injury in the intestinal microcirculation rather than directly damaging the mucosa. Sjogren et al. [95] examined a rabbit model of infection with RDEC-1 (an enteropathogenic E. coli strain specific for rabbits) which had been lysogenised by an Stx-1-bearing bacteriophage derived from a strain of EHEC. The genetically modified strain produced large amounts of Stx-1 and induced more severe diarrhoea, often bloody, and more severe mucosal injury when compared to wild-type RDEC. A major difference on histological examination was the presence of microvascular thrombosis in the submucosal vessels of the cecum and colon of animals infected with the Stx-producing strains, but not in those infected with wild-type Stx-negative RDEC. Similar differences were observed in Rhesus monkeys infected with toxin-producing and toxin-negative strains of S. dysenteriae [86]. Thromboses are also found in the microcirculation of human intestine and occasionally in larger mesenteric vessels during STEC colitis [96]. Injection of purified toxin into the mesenteric artery of rats [97] or intravenously into rabbits [59] induces haemorrhagic Microbes and Infection 2001, 493-507

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Figure 2. Mechanisms of endothelial cell injury caused by Shiga toxin. NO, nitric oxide; NOS, nitric oxide synthase; PMN, polymorphonuclear leukocyte; ROM, reactive oxygen metabolite. It is controversial whether Shiga toxin increases NO production by endothelial cells or whether this is derived from inflammation in the surrounding tissues.

lesions on the serosal surface of the large intestine and thrombosis in the medium vessels of the mesentery. The effect of the toxin on the mesenteric vessels and microcirculation would compound the pathological effects by superimposing intestinal ischaemia on an already inflamed mucosa. This would account for the presence of bloody diarrhoea, and in some cases, toxic megacolon and intestinal perforation. Stx may also play a role in the induction of mucosal inflammation by stimulating the secretion of proinflammatory cytokines by epithelial cells [73]. It is unclear if this occurs in vivo as the studies which suggested this were undertaken in cell cultures and involved receptor-mediated uptake of toxins, which does not occur in the gut of most species. Nevertheless, Stx also stimulates the release of proinflammatory cytokines from immunocytes such as macrophages, including those resident in the intestinal submucosa, [74] thus potentially amplifying the intestinal inflammation induced by the bacteria. 2.2. Endothelium

Endothelial cells are the primary target of Stx (reviewed in [4, 98]. The sites of major organ damage caused by circulating toxin correspond to the distribution of toxin binding to the vascular endothelium in those organs. This is discussed in more detail above in the section on toxin receptors. The use of endothelial cell cultures has provided insights into the pathophysiology of the Stx-induced vasculitis. Cultured endothelial cells express variable levels of Gb3, bind Stx and are sensitive to the cytotoxic effects of these toxins [99–101]. As with other cells, endothelial cell susceptibility to Stx depends largely on receptor expression. Microbes and Infection 2001, 493-507

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Variability in receptor expression has been ascribed to a number of factors including the degree of confluence of the cell cultures [99, 102] and their tissue of origin, which is thought to reflect the different sensitivities observed in vivo [103]. For example, human renal and intestinal endothelial cells are very sensitive to Stx [103–105], whereas some endothelial lines derived from large vessels such as saphenous vein or human umbilical vein are relatively resistant [100, 106]. Coincubation of endothelial cell cultures with proinflammatory cytokines, interleukin-1β, (IL-1β), tumour necrosis factor-α (TNF-α) or with LPS stimulates expression of Gb3 and markedly increases cytotoxicity of the toxins [100, 101, 106]. In addition, factors other than increased Gb3 expression, such as alterations in chain lengths of the Gb3 fatty acids, may account for the increased susceptibility to the toxins [98]. There is in vivo evidence that cytokine and LPS sensitisation may enhance the effects of circulating toxin. Infection of gnotobiotic mice with O157:H7 STEC strain results in microvascular disease and inflammation in the colon, brain and kidneys. Pretreatment of the animals with systemic TNF-α markedly increases mortality and worsens microhaemorrhages and thromboses particularly in the CNS [107]. In contrast, pretreatment of mice with the TNF-α synthesis inhibitor, nafamostat mesilate, prevents mortality and attenuates organ damage particularly in the CNS. Harel and others studied transgenic mice bearing a chloramphenicol acetyl transferase reporter gene that indicates TNF-α synthesis [108]. Systemic inoculation of the mice with Stx induced TNF-α synthesis in the kidneys. These investigators also found that Stx administration enhances the nephrotoxic effect of TNF-α and LPS, suggesting that these three compounds may act synergistically. Moreover, LPS pretreatment of mice enhances mortality and nephrotoxicity caused by O157:H7 [109]. Nephrotoxicity is also increased in gnotobiotic mice infected with STEC O157:H7, and subsequently inoculated with LPS, compared to animals not given LPS [110]. The experimental data suggest that circulating or local proinflammatory cytokines amplify the vascular injury induced by Stx . LPS and possibly other bacterial products absorbed into the circulation from the inflamed colon could also enhance the injury caused by Stx by virtue of their ability to stimulate the local or systemic release of proinflammatory cytokines from mesenchymal, endothelial or circulating immune cells. Damage to endothelial cells may stimulate the expression of vascular addressins, such as intracellular cell adhesion molecule-1 (ICAM-1) or vascular cell adhesion molecule-1 (VCAM-1), which play a role in leukocyte adhesion to the endothelium (figure 3). Morigi et al. have demonstrated that low concentrations of Stx cause increased leukocyte adhesion to cultured human umbilical vein endothelial cells under flow conditions [111]. Leukocyte adhesion is inhibited by preincubation of the cells with antibodies to ICAM-1, VCAM-1 and E selectin (a β integrin expressed on the surface of leukocytes and important in their adhesion). Few studies have examined the early changes in the microcirculation after Stx administration. However we have used intravital microscopy of 499

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Figure 3. Pathogenesis of Shiga toxin-mediated disease. Stx enters the host from the lumen of the GIT through epithelial cells and possibly via the paracellular pathway (the latter hypothesised by the authors). It acts directly on submucosal immunocytes which release inflammatory mediators, thereby increasing tissue inflammation and increasing receptor expression. Stx also binds to receptors on endothelium leading to microvascular thrombosis which occurs largely in GIT, kidney and brain. Gb3, globotriaosylceramide (Stx receptor); ICAM, intracellular cell adhesion molecule; IL, interleukin; Mφ, macrophage; PMN, polymorphonuclear leukocyte; TNF, tumour necrosis factor. Reproduced with modification from [154] with permission.

mesenteric venules to show increased leukocyte adhesion and decreased red cell velocity 18 h after the intraperitoneal injection of Stx-1, 1 µg, into rats. Pretreatment with anti-ICAM-1 antibodies completely abrogated the effect of toxin (Chia, O’Loughlin Perry and others, unpublished observations). On the basis of these findings, we postulate that in the early phases of vascular injury, Stx stimulates the expression of adhesion molecules and results in increased adhesion of leukocytes to the endothelium and a reduction in flow through the inflamed vessels. Activation of adherent leukocytes would result in the release of leukocyte products, such as reactive oxygen metabolites and proteases which exacerbate the damage to the endothelium (figure 2). Later, exfoliation of damaged endothelial cells could expose the basement membrane and underlying matrix, providing a potent stimulus to the development of thrombosis. Endothelial cells also produce vasoactive substances and factors that regulate coagulation and could also be affected by Stx. Damage to the endothelium will activate platelet adhesion, resulting in platelet consumption and thrombocytopenia, one of the major clinical manifestations of HUS. Fibrin deposition follows and causes the mechanical lysis of erythrocytes, another key feature of 500

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this condition. Karch and others demonstrated that Stx-1 and Stx-2 inhibited prostacyclin synthesis by rat aortic endothelial cells [112]. Prostacyclin plays a role in the inhibition of platelet aggregation and its production is reduced in children with HUS [113]. The application of Stx-1 to rat glomerular endothelial cells stimulates synthesis of the proconstrictor, procoagulant arachidonic acid metabolites, thromboxane A2 and 12-(S)-HETE [114]. Stx treatment of human renal glomerular endothelial cells decreases expression of fibrinolysis factor [115] which may increase the risk of thrombosis in the renal micocirculation. In addition, studies in children with HUS have revealed the presence of a circulating inhibitor of fibrinolysis (plasminogen activator type 1) [116]. In another study, Bitzan et al. examined the effect of Stx on endothelin and nitric oxide (NO) production by bovine aortic endothelial cells [117]. Both mediators have important roles in the regulation of vascular tone and in the case of NO, platelet aggregation and leukocyte adhesion. Stx inhibited endothelin production but did not effect NO production. Thus, Stx and SLTs impair a variety of important endothelial cell functions, resulting in abnormal leukocyte adhesion, reduced blood flow in small vessels of affected organs and an increased tendency towards coagulation and thrombosis formation. 2.3. Immunocytes

Stx modulate important physiological functions of immunocytes. For example they induce apoptosis in Burkitt lymphoma cells which are a subset of B-lymphocytes [77]. This effect is independent of the effect of Stx on protein synthesis. Stx also inhibits bovine lymphocyte function by blocking activation and proliferation in vitro [118], although this property has not been examined in vivo. Stx-1 and Stx-2 stimulate the secretion of inflammatory cytokines from monocytes [75, 119] and macrophages [74] increasing cytotoxicity of circulating toxins and tissue inflammation and aggravating organ damage. Depletion of splenic and liver macrophages by liposome encapsulated clodronate reduces the lethal effect of Stx-2 [120], suggesting that macrophage products such as TNF-α, play an important role in the pathophysiology of Stx-mediated tissue damage. Toxins also inhibit PMN apoptosis and phagocytosis [121, 122] and stimulate bone marrow granulopoeisis [123]. The latter probably accounts for the granulocytosis observed in patients with HUS [124]. Taken together, the data indicate that Stx causes a complex modulation of the immune system which, while dampening specific immunity, increases local inflammation and enhances Stx-induced cytotoxicity. 2.4. Kidney

Human kidney expresses high levels of Gb3 in both cortex and medulla with correspondingly high levels of toxin binding [125]. Microscopic examination of renal tissue from patients with HUS demonstrates a variety of histopathological changes. Capillary wall thickening, endothelial cell swelling and thrombosis of capillaries are present in most glomeruli [126]. Cortical necrosis is also prominent. Larger preglomerular arterioles and even medium-sized vessels may be involved with intimal thickening, fibrin thrombi and extensive fibrin deposition. Microbes and Infection 2001, 493-507

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While endothelial cells in the kidney appear to be a major target of the toxins, there is substantial evidence that epithelial and mesangial cells also express receptors and are affected by Stx. Patients with HUS excrete much higher levels of β2 microglobulin in their urine than healthy individuals, indicating significant tubular dysfunction [127]. Lingwood [128] examined the binding of fluorescein-conjugated Stx-1 to frozen human renal sections taken from patients with a variety of renal disorders other than HUS. He observed specific binding of the toxin to distal convoluted tubules, collecting ducts and the occasional proximal tubule with little binding in the glomeruli of adult patients. Sections from children and infants, however, demonstrated more pronounced toxin binding in the glomerulus, presumably to endothelial cells. Others have reported the presence of apoptotic glomerular and tubular epithelial cells in renal biopsies and autopsy material from infants with HUS [129]. Uchida et al. used imunohistochemistry to examine renal biopsy material from an infant with HUS and found both Gb3 receptor and bound Stx in endothelial cells and epithelial cells of distal tubules [130]. Some animals develop renal failure as a result of STEC infection. Streptomycin-treated mice infected with STEC develop renal failure due to acute cortical tubular necrosis [60, 131]. Animals can be protected from this effect by passive immunisation with anti-Stx-2 but not anti-Stx-1 antibodies suggesting that Stx-2 is the toxin involved. A laboratory strain of E. coli lysogenised with a bacteriophage encoding Stx-2 also caused renal damage and death when inoculated into mice [131] adding further support to the proposal that Stx-2 is the major toxin causing renal disease in this animal model. When administered to mice, Stx-2 is approximately 400-fold more potent than Stx-1 [60]. The basis of the increased susceptibility to Stx-2 is unknown, given that Stx-1 binds more avidly to Gb3. Proteolytic processing of the A2 fragment of Stx-2d by intestinal mucus increases toxin cytotoxicity [132], although this is not the explanation, as other Stx-2 variants are not processed in this fashion, but nevertheless are more potent than Stx-1. These animal studies are of interest as epidemiological studies of outbreaks of HUS in humans indicate that STEC strains producing Stx-2 only are more likely to be associated with disease than strains producing Stx-1 alone [133]. Circulating proinflammatory cytokines and bacterial products such as LPS enhance renal damage in animal models (described earlier). While systemic proinflammatory cytokine levels are increased in children with HUS [134–136], it is not yet known whether they play a role in disease pathogenesis in humans. Cell culture studies indicate that Stx-1 and 2 are cytotoxic to renal endothelial [102, 104], epithelial [127, 137–140] and mesangial cells [140, 141]. Interestingly, primary cultures of human renal endothelial cells are 1 000-fold more susceptible to the cytotoxic effect of Stx-2 than they are to the effect of Stx-1 despite binding less avidly than the [104]. The increased potency of Stx-2 involve cellular internalisation and intracellular processing of the toxin. Stx treatment of primary cultures of human proximal tubule cells [142] and human mesangial cells [141] also stimulates the release of proinflammatory cytoMicrobes and Infection 2001, 493-507

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kines such as IL-1 and monocyte chemoattractant peptide, which clearly have the potential to amplify the inflammatory response to endothelial and epithelial cell injury. Thus, circulating toxin has the capacity to bind to and injure endothelial, glomerular and tubular cells of the kidney. Cytotoxicity is probably enhanced by the action of systemic and locally released inflammatory mediators, bacterial factors and chemoattractant peptides which increase cellular susceptibility of the toxin and attract and activate immunocytes into the region of injury. The diverse biological effects of Stx in the kidney appear to account for the broad clinical spectrum of renal disease which can vary from anuria to azotemia without reduced urine output. 2.5. CNS

Approximately 20% of children with HUS have CNS manifestations which can vary from encephalopathy with or without seizures to major cerebrovascular accidents which occur in up to 5% of children with HUS involvement (reviewed in [143]). CNS disease is present in the majority of children who die from HUS and is the commonest cause of death. Post mortem examination reveals a range of pathology from cerebral oedema and diffuse hypoxic ischaemic changes to medium vessel thrombosis and cerebral infarction [143]. Lesions are most common in the cerebral hemispheres and basal ganglia. While Stx is thought to play a major role in the pathogenesis of CNS manifestations, studies in humans with shigellosis reveal a high incidence of CNS involvement even when infection is due to non-Stx-producing strains [144, 145]. This implies that microbial factors other than Stx are important in pathogenesis of the CNS disease. The effect of parenteral administration of culture extracts of S. dysenteriae on the CNS in animals has been known since the early 1900s. Studies in the 1950s established that ’Shigella neurotoxin’ directly damaged endothelial cells of small cerebral blood vessels in rabbits [146]. Most species studied to date develop CNS complications from infection with STEC or the parenteral administration of Stx. Piglets infected with E. coli O157:H7 develop convulsions and encephalopathy accompanied by cerebral microangiopathy, thrombosis and infarction [147]. Mice infected with STEC develop severe neurotoxicity and die [23, 148]. When Stx-1 is injected into rabbits, it localises to spinal cord, brain, cecum, colon and small bowel [59] mainly targeting endothelial cells of small blood vessels. The resultant histological damage correlates with tissuebinding of the toxin. Most animals develop CNS signs in the form of paresis, opisthotonic posturing and abnormal respirations. Cerebral swelling, focal grey matter haemorrhage and ischaemic change with necrosis are seen on pathological examination. The most severe lesions exhibit extensive oedema, haemorrhage and infarction present in association with thrombotic microangiopathy. Similar lesions occur within the brain particularly in midbrain, medulla and cerebellum. Inoculation of rabbits with Stx-2 causes similar changes to those described above [149]. Immunohistochemical study of affected brain tissue reveals toxin localised primarily to endothelial cells of small blood vessels, where it causes ultrastructural dam501

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age resulting in increased permeability of the blood–brain barrier to the tracer molecule, horseradish peroxidase [149]. Altered macromolecular permeability of the blood brain barrier would increase access of toxin to the CNS where it could cause direct damage to neurons. Indeed, toxin can be identified within the destroyed myelin sheaths of nerves particularly in the medulla. Similar observations have been made in mice infected with an E. coli which produced Stx-2e [150]. Further evidence implicating Stx in direct damage of neurons comes from recent identification of Stx receptors in small sensory neurons of dorsal root ganglia in humans, rabbits and rodents [151] and astrocytoma cells [152]. Treatment of the astrocytoma cells with Stx inhibits cellular proliferation and induces apoptosis. Inflammatory mediators such as cytokines and bacterial products may amplify the damage caused by Stx, although this mechanism has not been investigated in brain tissue to the same extent as it has in other organs. TNF-α [148] and LPS [107] increase CNS pathology in mice infected with STEC. Moreover, the cytotoxic effect of Stx in cultured human cerebral endothelial cells is also increased by coincubating the cells with TNF-α or IL-1β [62, 153] by increasing Gb3 expression and increasing toxin binding. TNF-α production in the CNS is increased following infection and a TNF-α protease inhibitor attenuates the brain injury [107] implicating an important role for this inflammatory mediator in the pathophysiology of the CNS insult. Taken together, the data suggest that Stx cause CNS disease by inducing endothelial cell damage. It is also likely that toxin traversing the damaged blood–brain barrier may also directly damage neurons by inducing cytotoxicity or apoptosis. The effect of toxins is likely to be modified by inflammatory mediators. 2.6. Model of disease pathogenesis

A model of Stx-induced disease is slowly evolving (figure 3). Although S. dysenteriae invades the gastrointestinal epithelium and E. coli adheres to the mucosal surface, both species excite host defence mechanisms and induce intestinal inflammation. S. dysenteriae and STEC secrete toxins into the lumen of the gut where they may play a role in inhibiting the growth of commensal bacteria. Stx is then absorbed through the mucosa into the circulation, probably through epithelial cells, but possibly also through the paracellular pathways. Intestinal inflammation may increase toxin uptake by making the mucosal barrier more permeable. Within the host, the toxins circulate, where they bind to receptors on endothelial cells and immunocytes. This results in endothelial cell damage, increased adherence of leukocytes to the endothelium and the initiation of a cycle of damage which activates prothrombotic elements leading to thrombosis in small and medium-sized vessels. Toxicity is increased by the local and systemic release of inflammatory mediators such as TNF-α which increase the expression of Gb3 receptors and thus enhance binding of toxin to susceptible cells. Infarction of the intestinal mucosa leads to bleeding into the bowel and bloody diarrhoea. This may provide essential nutrients such as iron to the pathogens within the gut lumen. Toxin also binds to circulating immunocytes lead502

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ing to greater release of inflammatory mediators. Toxins damage organs, such as kidney and brain, by binding to endothelial cells and inducing thrombotic microangiopathy. Some cells such as kidney epithelial cells and neurons may also express receptors, which bind Stx and may be affected by it. Fibrin deposition in the microcirculation leads to mechanical damage to erythrocytes with resultant haemolytic anaemia, while the widespread thrombosis in small blood vessels causes thrombocytopenia. Possible therapies such as passive immunisation and the use of agents to bind toxins are based on the concept that the major pathogenic factor is circulating toxin. However, by the time most patients present with symptoms of disease, a considerable amount of toxin may have been absorbed into the circulation. Further research is required to understand the pathogenic events after toxin has bound to the endothelium. Inhibiting these events, for example by inhibiting leukocyte-binding with antibodies to cellular adhesion molecules, may provide an opportunity to ameliorate Stx-mediated disease.

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