MARK P. STEVENS* AND TIMOTHY S. WALLIS
Division of Microbiology, Institute for Animal Health, Compton Laboratory, Berkshire RG20 7NN, United Kingdom
*Corresponding author. Phone: +44 1635 578411, Fax: +44 1635 577243, E-mail:
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Epidemiological investigation of outbreaks of a distinctive diarrheal illness in the early 1980s led to the recognition of enterohemorrhagic Escherichia coli (EHEC) as a distinct class of pathogenic E. coli. Riley et al. were the first to describe an outbreak of hemorrhagic colitis (HC) associated with ingestion of undercooked hamburgers and a previously rarely isolated E. coli serotype, O157:H7 (182). Patients presented severe abdominal pain, initially watery diarrhea, followed by grossly bloody diarrhea with little or no fever. In the same year, Karmali et al. reported an association between E. coli strains producing a toxin cytopathic for Vero cells and hemolytic-uremic syndrome (HUS), a condition characterized by acute renal failure, thrombocytopenia, and microangiopathic hemolytic anemia (111). HUS was known to be preceded by a diarrheal illness indistinguishable from HC, and Karmali et al. later concluded that cytotoxin-producing E. coli might be a common cause of HC and HUS (113). Since then EHEC and, in particular, serotype O157:H7 have emerged as a major cause of severe diarrheal illness worldwide, and EHEC infections are now the leading antecedent to pediatric acute renal failure in many countries. E. coli O157:H7 strains are estimated to cause 73,480 cases and 60 deaths per annum in the United States, with serotypes other than O157:H7 accounting for a further 36,740 illnesses (146). Non-O157:H7 EHEC are more prevalent than E. coli O157:H7 in some countries and the factors driving the emergence of new EHEC serotypes are not understood (16). EHEC O157:H7 is believed to have evolved from enteropathogenic E. coli (EPEC) serotype O55:H7 by acquisition of stx-encoding bacteriophages (180). Consequently, EPEC and EHEC share numerous conserved virulence genes (107) (Chapter Enteropathogenic Escherichia coli).
Healthy ruminants are the principal reservoir of EHEC and infections in humans are frequently associated with direct or indirect contact with ruminant feces (134, 162). A recent survey of beef cattle presented for slaughter in the United States revealed that 28% of animals shed E. coli O157:H7 in the feces (62). Outbreaks have also been associated with direct contact with the farm environment and with the consumption of water, fruit, and vegetables contaminated with ruminant manure. Indeed, the largest outbreak so far recorded involved more than 6,000 children in Sakai City, Japan, and was associated with the consumption of contaminated white radish sprouts from a single farm (147).
It has been reported that a region of lymphoid follicle-dense epithelium in the terminal rectum is the principal site of EHEC O157:H7 persistence in adult cattle (155); however, EHEC O157:H7 has been recovered from other distal sites in the intestines and the rumen (19, 33, 43, 83). In contrast EHEC serogroups O5, O26, and O111 have been observed to adhere extensively to the bovine colonic epithelium (86, 172, 199, 216) and appear not to share a tropism for terminal rectum (155). In vitro organ culture experiments using human intestinal mucosa indicate that EHEC serotypes O157:H7 and O103:H2 exhibit a tropism for follicle-associated epithelium overlying Peyer’s patches, whereas adherence of EPEC O127:H6 is not restricted to these sites (69, 70, 176, 178).
Adherence of EHEC to epithelial cells in vitro has been studied extensively and numerous putative EHEC adhesins have been identified by screening of transposon mutants for reduced adherence or by blocking attachment with specific immune sera. Several distinct in vitro adherence phenotypes have been described, but these are sensitive to the cell line, bacterial strain, and culture conditions used. Even among EHEC O157:H7 strains remarkable differences exist in the capacity to adhere to different human and bovine epithelial cell lines, with some strains adhering in microcolonies (localized adherence [LA]) and others adhering sparsely over the epithelial surface (diffuse adherence [DA]) (141, 193). Furthermore, adherence of EHEC O157:H7 to HCT-8 human ileocecal cells is characterized by a distinct phenotype termed "log jam," since the bacteria principally cluster at cell junctions (143). Interpreting the significance of such phenotypes requires caution, given that nonpolarized epithelial cells in culture, even when derived from the gastrointestinal tract, are unlikely to accurately mimic the molecular interactions that occur during colonization of mucosal surfaces by EHEC in vivo. Several groups have reported that EHEC O157:H7 is capable of significant epithelial cell invasion in vitro (141, 163, 215). The efficiency of EHEC invasion also seems to be sensitive to the bacterial strain, cell line, and culture conditions used and it remains unclear if bacterial internalization is significant in the pathogenesis of HC and HUS in humans or the persistence of EHEC in reservoir hosts.
The most intensively studied adherence phenotype of both EPEC and EHEC is the induction of attaching and effacing (A/E) lesions on intestinal epithelia, characterized by intimate bacterial adherence to the apical surface of enterocytes on raised actin-rich pedestals and localized destruction of microvilli (Fig. 1). The bacterial genes required for A/E-lesion formation are located on the chromosomal locus of enterocyte effacement (LEE) and are conserved in EPEC and Stx-producing E. coli strains capable of inducing A/E lesions, but absent in other E. coli pathotypes and nonpathogenic E. coli (142). Whereas LEE-positive Stx-producing strains are associated with the majority of cases of HC and HUS, LEE-negative Shiga toxin-producing E. coli (STEC) are also associated with acute gastroenteritis and HUS in humans. Outbreaks of HUS and HC have been caused by LEE-negative STEC O113:H21 in Australia (168) and by O104:H21 in the United States (68) and the mechanisms underlying carriage and virulence of LEE-negative STEC are poorly understood.
Our understanding of EHEC O157:H7 biology has been accelerated by determination of the complete genome sequence of strain EDL933, associated with an outbreak of HC in the United States in 1982 (175, 182; http://www.genome.wisc.edu/sequencing/o157.htm), and strain RIMD 0509952 isolated from the Sakai outbreak (90, 147; http://genome.gen-info.osaka-u.ac.jp/bacteria/o157). Independent annotation of the genomes of the two strains has revealed the strains to be largely similar; however, comparison of the EDL933 genome with that of the nonpathogenic E. coli K-12 strain MG1655 has shown that it contains ca. 1.34 Mb of additional sequence distributed in 177 "O-islands" of greater than 50 bp in length (175). The function of only 40% of O157-specific genes can be assigned at present. For a comprehensive review of the insights gained from sequencing of EHEC O157:H7 genomes and comparative genomics the reader is referred to a recent review (174). The epidemiology and pathogenesis of infections caused by EHEC and other Shiga toxin-producing E. coli is covered in detail elsewhere (171; Chapter Enterohemorrhagic Escherichia coli). This section reviews our understanding of the factors mediating adherence of EHEC and other STEC to epithelial cells, a prerequisite for intestinal colonization and the development of disease.
The LEE is the most intensively studied of all EHEC virulence factors and mediates intimate bacterial adherence to intestinal epithelia and A/E lesion formation. LEE-encoded proteins are required for adherence of EHEC to human intestinal mucosa in vitro and HC and HUS patients develop antibody responses to several LEE-encoded proteins. Owing to safety limitations on the testing of EHEC mutants in human volunteers the role of the LEE in EHEC pathogenesis in humans is unproven; however, studies with EHEC O157:H7 in streptomycin-treated mice, infant rabbits, gnotobiotic piglets, calves and adult ruminants have demonstrated that LEE-encoded proteins play key roles in intestinal colonization and the induction of enteritis (31, 44, 55, 56, 59, 106, 139, 183, 216). Furthermore, immunization with LEE-encoded proteins can partially protect reservoir hosts against EHEC infection (45, 106, 179).
The LEE region was first described in EPEC O127:H6 after several genes identified by transposon mutagenesis to be required for bacterial adherence to epithelial cells were mapped to a single ca. 35-kb locus. Among these were the gene for intimin (eae), which was known to be required for intimate adherence of both EPEC and EHEC to cultured epithelial cells (101, 223) and for carriage and virulence in EPEC in humans (55) and EHEC in gnotobiotic piglets (56). Probes from several regions of the EPEC O127:H6 LEE hybridized to strains of EHEC serotype O157:H7 and O26:H11 (142). Sequencing of a limited region of the LEE of the prototypic EPEC O127:H6 strain E2348/69 identified four genes predicted to encode components of a type III protein secretion system (TTSS), based on homology to Yersinia Lcr/Ysc and Shigella Mxi/Spa proteins (97). These genes were initially designated sepA-D (secretion of EPEC proteins) (97), but many have been renamed esc to conform to the nomenclature of homologous Yersinia TTSS genes (65).
Subsequent studies revealed that growth of EPEC O127:H6, EHEC O157:H7, and EHEC O26:H11 in eukaryotic cell culture medium or conditions similar to those in the gastrointestinal tract results in the secretion of several proteins, designated Esps (E. coli secreted proteins), some of which are essential for the formation of A/E lesions (60, 96, 118). Secretion of Esps required the product of the sepB (escN) gene, indicating that the export of proteins involved in A/E lesion formation occurred via the LEE-encoded TTSS (96). Type III secretion systems are key virulence factors of gram-negative enteric pathogens and the four major genera of plant pathogenic bacteria and serve to inject bacterial proteins directly into host cells (reviewed in reference 92). A subset of type III secreted proteins (translocators) is believed to interact with the eukaryotic cell membrane and mediate the delivery of secreted "effector" proteins into the target cell cytosol. Effectors typically subvert or inhibit cellular processes to the benefit of the pathogen.
The complete nucleotide sequence of the LEE has been determined in EPEC O127:H6 strain E2348/69 (65), EHEC O157:H7 strains EDL933 and RIMD 0509952 (90, 173, 175), EHEC O26:H– strain 413/89-1 (LEE accession number AJ277443), rabbit EPEC strains RDEC-1 (O15:H–), 83/39 (O15:H–), and 84/110-1 (O103:H2) (205, 227) and Citrobacter rodentium (50). LEE sequences have a significantly lower G+C content than the surrounding DNA, are inserted at tRNA loci, and contain remnants of mobile genetic elements indicating that they are likely to have been acquired by horizontal transfer. Analysis of the sequence and insertion sites of the LEE regions of A/E pathogens has aided phylogenetic analysis of EHEC evolution and is reviewed elsewhere (54). The genetic organization of the EHEC O157:H7 strain EDL933 LEE is depicted in Fig. 2. The EHEC O157:H7 LEE region is inserted at 82 minutes on the E. coli K-12 chromosome, just downstream of the selC selenocysteine tRNA locus, and it shares 41 genes in the same order as found in EPEC O127:H6 strain E2348/69 but is larger owing to the presence of a cryptic P4 family prophage at one end (173). Putative structural components of the TTSS exhibit remarkable conservation among all A/E pathogens; however, marked divergence exists among secreted proteins and other components predicted to interact with host cells. The roles played by LEE-encoded proteins in the adherence of EHEC to epithelial cells are considered below.
Intimin is a 94- to 97-kDa outer membrane adhesin produced by all EHEC and related A/E pathogens, including human EPEC, atypical EPEC from farm and domestic animals, C. rodentium, and Hafnia alvei. The gene encoding intimin (eae, for E. coli attaching and effacing) was first identified by screening EPEC O127:H6 TnphoA mutants for inability to nucleate F-actin under sites of adherence to HEp-2 cells (101). An orthologue of eae was subsequently shown to be required for intimate adherence of EHEC O157:H7 in vitro and for intestinal colonization in a porcine model and exhibits 83% amino acid sequence to EPEC O127:H6 intimin (56, 223). Intimin from both EHEC and EPEC shares significant homology with invasin proteins of Yersinia pseudotuberculosis and Y. enterocolitica with the greatest divergence at the carboxyl-terminal end. The carboxyl termini of Yersinia invasins bind to β1-chain integrins and thereby facilitate bacterial invasion of eukaryotic cells. Subsequent studies demonstrated that the host cell-binding domain of intimin is also located at to the carboxyl-terminal end. By fusing the carboxyl-terminal 280 amino acids of intimin (Int280) to maltose-binding protein (MBP) and conjugating the fusion protein to latex beads Frankel et al. demonstrated that Int280 from both EPEC O127:H6 and EHEC O157:H7 could directly bind to HEp-2 cells (72, 177). The same region also mediates binding of EHEC serogroup O26 intimin to host cells (46). Furthermore, expression of the carboxyl-terminal two-thirds of intimin is sufficient to restore adherence of an EHEC O157:H7 Δeae mutant (144), and antibodies directed against the carboxyl-terminal domain of EHEC O157:H7 intimin inhibit bacterial adherence to HEp-2 cells (76). Conflicting reports indicated that binding of purified intimin to eukaryotic cells could only be detected if the cells were preinfected with EPEC or EHEC, indicating that a bacterial "signal" may be required for intimin-mediated adherence (132, 187).
Studies to identify the host cell receptor for intimin focused on a 90-kDa membrane protein (Hp90) that became tyrosine phosphorylated during EPEC infection of epithelial cells and localized under adherent bacteria (186). EPEC intimin was later shown to bind to tyrosine-phosphorylated Hp90 but not to the nonphosphorylated Hp90, and it was hypothesized that the bacteria signal to host cells to induce phosphorylation of a membrane protein that is subsequently bound by intimin (187). Elegant studies by Kenny et al. later revealed Hp90 to be the tyrosine-phosphorylated version of a 78-kDa EPEC O127:H6 secreted protein that is translocated into the eukaryotic cell plasma membrane via the LEE-encoded type III secretion apparatus where it acts as the receptor for intimin (119). Hp90 was thus renamed Tir (for translocated intimin receptor). Tir was independently discovered in EHEC O26:H– as EspE, an 80-kDa secreted protein that is translocated into the eukaryotic cell plasma membrane under adherent bacteria (47). EHEC O157:H7 Tir is also delivered into host cells where it acts as a receptor for intimin; however, it is not tyrosine phosphorylated (47, 52). Thus, A/E pathogens insert their own receptor into the mammalian cell membrane, to which intimin then binds to promote intimate adherence (Fig. 3).
The three-dimensional structures of the carboxyl-terminal domain of EPEC O127:H6 intimin and the Int280-Tir complex have been resolved by nuclear magnetic resonance and X-ray crystallography (8, 115, 135). These studies indicate that EPEC intimin contains four distinct domains within the carboxyl-terminal 380 residues that protrude from the bacterial outer membrane. Domains D1, D2, and D3 belong to the immunoglobulin superfamily and are predicted to comprise an articulated rod connected to the membrane-anchored amino-terminal portion by a flexible linker comprising two glycine residues. The terminal D4 domain is predicted to be highly accessible to the target cell and shares similarity with C-type lectins, a family of proteins responsible for recognition of cell surface carbohydrates. The most distal immunoglobulin-like domain (D3) and D4 C-type lectin domain form a rigid superdomain that binds Tir (8). Tir is not glycosylated; however, the fold of the Tir-binding domain of intimin suggests that it may still be able to bind a carbohydrate moiety on the eukaryotic cell surface. The C-type lectin domain is required for Tir-independent binding of EPEC intimin to eukaryotic cells, supporting the notion that intimin may also bind to a host cell receptor(s) (87).
The intimin-binding region of Tir forms a coiled-coil structure composed of the two roughly parallel α-helices of the Tir extracellular domain connected by a β-hairpin turn at the tip of the structure. Formation of the intimin-Tir complex principally involves contacts between the Tir β-hairpin motif and an analogous region of the intimin D4 domain (135), and this portion of Tir is critical for intimin binding (41, 87). The X-ray crystal structure of the EPEC Int280-Tir complex shows the intimin-binding domain of Tir to be a dimer, with the two roughly parallel α-helices of the Tir extracellular domains coming together to form a four-helix bundle bound by two intimin molecules (135). Although it is unclear if this structure is formed in vivo, it predicts that intimin and Tir may bind in a plane roughly parallel to the surfaces of the bacteria and host cell. This is thought to be necessary for intimate bacterial contact, since the intimin-Tir complex (lacking one immunoglobulin-like domain) has a combined length of more than 140 Å, while the gap between the host cell plasma membrane and intimately attached EPEC is estimated to be only 100 Å. The molecular basis of Tir recognition by EHEC O157:H7 intimin has been studied by point mutagenesis of intimin in a yeast two-hybrid system. Four amino acid residues were identified to be critical for Tir recognition, three of which can be predicted to lie at the intimin-Tir interface based on the crystal structure of the EPEC Int280-Tir complex, suggesting that EHEC intimin may interact with its translocated receptor in a similar way (133).
Intimin Subtypes and Intimin-Mediated Intestinal Tissue Tropism.
Since the carboxyl-terminal domain of intimin mediates binding to Tir and eukaryotic cell surfaces, it was anticipated that divergence of intimin sequences may influence the avidity and specificity of adherence. Evidence that intimin may influence intestinal tissue tropism first arose from the study of EHEC O157:H7 eae mutants in a porcine model. Int-γ was known to be required for colonization of the surface and glandular epithelium of the large intestine in gnotobiotic piglets and the induction of enteritis (56). Surprisingly, trans-complementation of an EHEC O157:H7 Δeae mutant with Int-α from EPEC O127:H6 caused a shift in the intestinal tissue tropism, with colonization of the terminal ileum as well as the surface of the large intestine accompanied by severe diarrhea in a manner similar to wild-type EPEC (213). Subsequent in vitro organ culture studies using human intestinal mucosa have confirmed an important role for intimin in mediating intestinal tissue tropism. Adherence of EHEC O157:H7 to human intestinal mucosa and the formation of A/E lesions is restricted to follicle-associated epithelium (FAE) overlying ileal Peyer’s patches (178), whereas Int-α-expressing EPEC O127:H7 can adhere to small intestinal explants from a variety of sites (176). Expression of EPEC O127:H6 Int-α in an EHEC O157:H7 Δeae mutant without exchanging Tir results in an EHEC strain capable of adhering to all small intestinal explants (69). In reciprocal experiments EHEC O157:H7 Int-γ conferred upon an EPEC O127:H6 Δeae mutant a specific tropism for FAE overlying Peyer’s patches (176). It is presently unclear if intimin determines the tropism of EHEC O157:H7 for lymphoid follicle dense epithelium in the terminal rectum of cattle, or indeed if other EHEC serotypes share a tropism for this site (155).
Host Cell Receptors for Intimin.
The ability of intimin to bind to eukaryotic cell surfaces in the absence of Tir and to mediate intestinal tissue tropism suggests the existence of a cellular coreceptor(s) for intimin. Indirect evidence for the existence of a eukaryotic coreceptor also arises from analysis of Int-α mutants lacking Cys937, which forms a disulfide bridge in the carboxyl-terminal cell-binding domain. Substitution of cysteine 937 disrupts bacterial adherence to host cells, but not intimin-Tir binding in a yeast two-hybrid system or gel overlay assay (71, 73, 87). However, structural analysis of the intimin-Tir complex indicates that mutation of Cys937 may destabilize the complex sufficiently to prevent interaction in vivo but not to impair interaction in gel overlay assays (135).
Recently, Sinclair and O’Brien demonstrated that the carboxyl-terminal domain of EHEC O157:H7 intimin binds in a specific and saturable manner to HEp-2 cells with an apparent Kd of 84 nM (± 8 nM), consistent with the existence of a single host cell receptor (194). By affinity purification and sequencing of peptides derived from a 110-kDa intimin-binding HEp-2 cell protein the receptor was identified as nucleolin, a protein involved in regulation of cell growth that can be expressed at the cell surface (194). Cell surface-localized nucleolin was observed to colocalize with bound purified Int-γ and with EHEC O157:H7 adhering to HEp-2 cells. Furthermore murine anti-nucleolin antibodies partially inhibit EHEC O157:H7 adherence to cultured cells (194). Taken together, these data indicate an important role for intimin-nucleolin interactions in adherence of EHEC to epithelial cells, probably in the initial stages before Tir binding.
Although it is known that intimin plays a crucial role in the carriage and virulence of EHEC O157:H7 in mice, infant rabbits, gnotobiotic piglets, neonatal calves, and adult cattle and sheep (31, 44, 56, 106, 139, 183), the relative importance of the intimin binding to Tir as apposed to cellular coreceptors has received little attention. It is known that Tir is required for carriage and enteropathogenicity of rabbit EPEC in infant rabbits (139), C. rodentium in mice (48), and EHEC O157:H7 in infant rabbits and calves (183, 198), indicating that the role of intimin in intestinal colonization can be explained, at least in part, by it binding to Tir as apposed to cellular coreceptors. However, it is possible that interactions between intimin and host cell receptors determine which intestinal sites are colonized. Recent studies have shown that intimin subtypes α,β, and γ bind to nucleolin with equal affinity, indicating that the distribution of nucleolin along the gastrointestinal tract is unlikely to explain the differential tissue tropism of EHEC and EPEC (195). All three intimin subtypes bind to nucleolin with a lower avidity than to Tir. Furthermore, binding of intimin α, β, or γ to Tir in vitro blocks the interaction between intimin and nucleolin (195). Taken together, these findings suggest that nucleolin may be involved in adherence mediated by all three intimin subtypes and that Tir and nucleolin compete for intimin during adherence.
It is conceivable that the interaction between intimin and cellular coreceptors activates signal transduction pathways to elicit beneficial responses from host cells. Binding of intimin-coated latex spheres to HEp-2 cells has been reported to facilitate the elongation of microvillus-like processes at the site of initial bacterial adherence to HEp-2 cells and formation of cage-like structures engulfing the bacteria (177). It has been postulated that this may be a consequence of displacement of laminin from nucleolin-laminin complexes upon intimin binding (194). Like intimin, laminin can induce the extension of filopodia; however, the significance of these cytoskeletal rearrangements in EHEC adherence is unknown.
Several reports indicate that, in addition to its role as an adhesin, intimin may modulate mucosal immune responses. During C. rodentium infection of mice intimin is required to induce a pronounced T-helper cell-type 1 (TH1) mucosal immune response characterized by mucosal thickening and infiltration of CD4+ T cells (91). A C. rodentium null eae mutant does not induce colonic hyperplasia, whereas killed wild-type C. rodentium and E. coli K-12 strains engineered to express intimin induce inflammatory responses similar to those seen in wild-type C. rodentium infection (91), suggesting that intimin alone drives the mucosal inflammatory response. Indeed, purified Int280 can augment mitogen-stimulated proliferation of spleen CD4+ T lymphocytes and cells from organized lymphoid tissues (81, 91). It is not known if EHEC intimins modulate mucosal immune responses or what role such modulation may play in intestinal colonization.
Intimin-Based Vaccines.
The key role played by intimin in EHEC-intestinal interactions has prompted the development and testing of intimin-based subunit vaccines. Intimin-specific antibodies can be detected in sera from convalescent HC and HUS patients (98, 114, 131), and partially inhibit adherence of EHEC O157:H7 to HEp-2 cells (76, 144). Recent studies by Dean-Nystrom et al. demonstrate that passively acquired intimin-specific antibodies are protective. Pregnant pigs vaccinated with EHEC O157:H7 Int-γ produce high intimin-specific antibody titers in the serum and colostrum (45). Neonatal piglets allowed to suckle vaccinated dams exhibit increased resistance to colonization and intestinal damage following experimental inoculation with EHEC O157:H7 compared with piglets allowed to suckle mock-vaccinated dams (45).
Immunization with purified intimin subunits also shows promise. Mice primed parenterally with the carboxyl-terminal portion of Int-γ expressed from the plant cells, then fed transgenic Int-γ-expressing plant cells, generate intimin-specific mucosal immune responses and shed EHEC O157:H7 in the feces for a shorter duration than mock-vaccinated animals after challenge (106). However, the protection conferred by such responses appears to be subtype specific. Immunization of mice with purified Int280 from EPEC O127:H6 (Int-α) induced protection against a C. rodentium strain engineered to express Int-α but not against wild-type C. rodentium expressing Int-β (79). Immunization of mice with a conserved domain of intimin (Int388-667) did not induce protective immunity against C. rodentium infection, indicating that polyvalent vaccines comprising the carboxyl-terminal domains of several intimin subtypes may be required to provide broad protection against the predominant EHEC serotypes (79). The ability of intimin-based subunit vaccines to control EHEC infection in humans or reservoir hosts remains to be tested. Recent evidence indicates that intimin is not required for persistence of EHEC O157:H7 in adult pigs (105) or specific-pathogen-free chicks (15), indicating that intimin-based vaccines may not be effective in all livestock at all ages.
Mutations affecting the type III secretion apparatus greatly reduce the ability of EHEC O157:H7 and O26:H– to colonize the intestines of calves (59, 216). Furthermore, mutations affecting structural components of the C. rodentium LEE-encoded TTSS are highly attenuating in mice (49, 150). This indicates that type III secreted proteins and the functions they perform in eukaryotic cells are crucial to the outcome of infection. Type III secreted proteins relevant to EHEC adherence are considered below. A more complete description of the repertoire and function of known or putative EHEC type III secreted effectors can be found elsewhere (Chapter Enterohemorrhagic Escherichia coli). The regulation of LEE expression is highly complex and is described by Mellies (chapter Virulence Gene Regulation in Escherichia coli).
EspA.
EspA is a major structural component of a filamentous organelle that is transiently expressed on the surface of EPEC and EHEC during the early stages of A/E lesion formation (61, 124). EspA filaments are a sheath-like cylindrical extension of the type III secretion system needle complex and in the case of EPEC are ca. 12 nm in diameter, varying in length up to 600 nm (40, 188). EPEC EspA binds directly to the needle complex protein EscF, which is required for type III secretion and the assembly of EspA filaments (188, 221). Secretion of EPEC EspA requires the chaperone CesAB, encoded by orf3 of the LEE, which is essential for stability of intracellular EspA (34). EspA filaments form a physical bridge between the bacteria and host cells and are required for the translocation into host cells of EspB and Tir (61, 119, 124, 222). This led to the hypothesis that EspA filaments may form a hollow channel through which effector proteins are injected into host cells. This notion is supported by resolution of the three-dimensional structure of EPEC O127:H6 EspA filaments, which showed EspA subunits to be polymerized in a helical tube of 120-Å diameter with 5.6 subunits per turn enclosing a central channel of 25-Å diameter (39). This arrangement is strikingly similar to that of R-type straight flagella of Salmonella and likely reflects the common evolutionary origin of flagellar and type III secretion systems. It was recently demonstrated by immunogold electron microscopy that Tir is secreted from the tips of EspA filaments with only low levels of background staining in association with EspA filaments of a Δtir strain (37). This provides the first direct evidence that EspA filaments are hollow conduits through which effector proteins are secreted. Furthermore it was reported by the same authors that EspA filaments are elongated by addition of EspA subunits to the tip of the growing filament and that filament length is modulated by the availability of intracellular EspA subunits (37).
It remains unclear how EspA filaments are connected to the host cell surface and the translocation pore created. It is believed that the type III secreted EspB and EspD proteins mediate formation of the translocation pore on the basis that they are homologous to the Yersinia YopB and YopD proteins, respectively, translocated into the host cell plasma membrane, and can form pores in erythrocyte membranes (93, 124, 126, 219, 220, 222). EspD is required for the assembly of EspA filaments (124, 126), and is itself dependent on the chaperones CesD and CesD2 for secretion (157). EspB interacts with EspA and is required for the translocation of Tir (88). However, EspA filaments can bind to eukaryotic cell membranes in the absence of EspB, indicating that they may interact directly with the cellular components (88). Indeed, the evidence suggests that EspA filaments play an important role in initial adherence of A/E pathogens to host cells. A nonpolar deletion of espA abolished adherence of EHEC O26:H– to HeLa cells (61) and EspA filaments connect EHEC O157:H7 and O26:H– with host cells (61, 156, 190). However, it is difficult to separate these effects from EspA-dependent translocation of Tir and thus adherence via intimin-Tir interactions. Recent studies using single, double, or triple mutants of EPEC O127:H6 lacking EspA filaments, intimin, and bundle-forming pili indicate that EspA filaments play a role in intimin-independent adherence to HEp-2 and Caco-2 cells, in that a bfpA espA eae triple mutant was less adherent than a bfpA espA+eae strain (28). Consistent with a role in adherence and the translocation of LEE-encoded effectors, EspA is required for carriage and virulence of rabbit EPEC in an infant rabbit model (2) and EHEC O157:H7 in mice (153). Antibodies specific to EspA can be detected in HC and HUS convalescent patient sera (98, 114, 131); however, EspA filaments of EPEC and EHEC are antigenically distinct and this may have important implications for the design of vaccines based on EspA and other type III secreted proteins (156).
Recent studies indicate that not all bacteria in a given population of EHEC express EspA filaments at any one time and that marked differences exist in EspA filament expression exist between EHEC O157:H7 strains isolated from cattle and humans (185). There is no evidence that heterogenous expression of EspA filaments occurs in EPEC O127:H6, and the basis of regulation of EspA filament expression in EHEC is incompletely understood.
Tir (EspE).
Tir is delivered into the apical leaflet of the host cell plasma membrane by an unknown mechanism. Current data favor the possibility that soluble Tir may initially be translocated into the cytoplasm and then inserted into the membrane, since phosphorylated intermediates of EPEC O127:H6 Tir can be detected in the cytoplasm and there is an apparent delay between Tir injection and intimin-Tir interaction (116, 120). The stability and secretion of EPEC O127:H6 Tir requires the chaperone CesT (1, 66), which also serves as a chaperone for the effector protein Map (35). Recent data indicate that Tir and its chaperone CesT can bind to EscN, a predicted inner membrane ATPase required for type III secretion, indicating that it may direct Tir to the secretion apparatus (78).
In addition to its role as the translocated receptor for intimin, Tir activates actin-signaling pathways and recruits cytoskeletal components to the site of bacterial adherence, resulting in pedestal formation (reviewed in reference 24). In EPEC O127:H6 Tir, these events require the phosphorylation of tyrosine residue 474 in the carboxyl-terminal cytoplasmic domain (116). Insertion of Tir into enterocyte membranes and subsequent tyrosine phosphorylation has been detected in vivo during C. rodentium infection of mice and is crucial for pedestal formation in vitro (48). Y474 is part of a 12-amino-acid motif that is both necessary and sufficient to recruit the host cell adaptor protein Nck and in turn the neural-Wiskott Aldrich syndrome protein (N-WASP) that stimulates the actin-nucleating activity of the cellular Arp2/3 complex (23, 27, 51, 84). Recent evidence suggests that actin assembly by EPEC Tir may also involve an Nck-independent pathway that depends on the phosphorylation of Y474 and Y454 (25). EPEC O127:H6 Tir also recruits the host adaptor proteins Grb2 and CrkII and other actin-regulatory proteins (cofilin, cortactin, gelsolin, and VASP), focal adhesion proteins (α-actinin, talin, and vinculin), and lipid raft-associated proteins (annexin 2, CD44, and GPI-anchors) (82). Recent studies also show that EPEC Tir interacts directly with the intermediate filament associated protein cytokeratin 18 (7). Cytokeratin 18 is recruited to the EPEC-induced pedestal and has a direct role in actin accretion and microfilament reorganization in pedestals.
In marked contrast to EPEC Tir, EHEC O157:H7 Tir lacks the critical Y474 residue and is not tyrosine phosphorylated upon entry into host cells (51, 52). Furthermore, the sequence of the 12-amino-acid motif required for Nck recruitment is not conserved in the Tir molecules from EHEC O157:H7 and O157:H–, although Y474 and the immediate context are conserved in EHEC O26:H– and O111:H– Tir (169). Consistent with this, EHEC O157:H7 pedestals do not contain the host adaptor proteins Nck, Grb2, and CrkII (23, 82, 84). Recent studies have shown that a type III secreted protein encoded within cryptic prophage 933U (EspFU/TccP) is required to recruit N-WASP, α-actinin, and Arp3 to EHEC O157:H7 pedestals and for subsequent actin assembly (26, 77). EspFU/TccP is not required for EspA filament assembly or the translocation and focusing of Tir at the site of bacterial adherence. EspFU/TccP associates with Tir in infected cells and activates N-WASP, stimulating actin assembly in the presence of the purified Arp2/3 complex (26, 77). EHEC O157:H7 Tir cannot restore the ability of EPEC O127:H6 or C. rodentium tir mutants to form pedestals on cultured epithelial cells, although both EPEC and C. rodentium Tir can replace EHEC Tir (23, 48, 117). This correlates with the absence of EspFU/TccP in EPEC O127:H6 and C. rodentium and the key role of this effector in coupling EHEC O157:H7 Tir to N-WASP/Arp2/3.
Initial interaction of EPEC O127:H6 with cultured epithelial cells is characterized by the transient appearance of filopodia at the site of bacterial attachment. Both intimin and the effector proteins Map appear to be required for the induction of filopodia, which can lead to uptake of the bacteria by nonphagocytic cells (100, 120, 177). Tir appears to repress the Cdc42-dependent formation of filopodia and may therefore play a role in preventing bacterial invasion (120). This activity requires a putative GTPase-activating protein (GAP) motif in the carboxyl-terminal cytoplasmic domain, suggesting that Tir may possess such activity (120). It is not known whether EHEC Tir possesses such activity, or what role it may play in intimate bacterial adherence. The predicted Tir sequences from EHEC O157:H7, O157:H–, O111:H–, and O26:H– share only 59.5%, 56.7%, 80.2%, and 65.4% amino acid identity with EPEC O127:H6 Tir, respectively (169). Given the observations that EPEC and EHEC Tir subvert cellular actin dynamics in distinct ways and that events crucial for Tir activity in vitro may be irrelevant in vivo, caution is required when predicting the activities of EHEC Tir molecules.
EspF.
Studies using EPEC O127:H6 have shown that EspF is not required for adhesion to eukaryotic cells or actin nucleation under sites of attachment (32, 145). However translocation of EPEC O127:H6 EspF into polarized T84 monolayers causes a loss of transepithelial resistance (TER) and an increase in paracellular permeability likely to be associated with redistribution of occludin, a transmembrane tight-junction protein that contributes to barrier function (145), and reorganization of the host intermediate filament network owing to EspF-cytokeratin 18 interactions (218). The loss of cell polarity that results from disruption of tight junctions has been reported to cause migration of β1-integrin from the basolateral to the apical surface of cells and it has been suggested that this may facilitate Tir-independent bacterial adherence via intimin (152). Intestinal barrier dysfunction also depends on a chaperone encoded by rorf10 (CesF) (64), intimin, and the effector protein Map (42). Expression of EspF in COS or HeLa cells causes cell death via a mechanism with features of apoptosis, including chromatin condensation and fragmentation, membrane blebbing, enhanced surface expression of phosphatidylserine, and elevated caspase activity (32). Such events may be due to the ability of EspF to localize to mitochondria, disrupt mitochondrial membrane potential, and induce cytochrome c release and the cleavage of caspases-3 and -9 (161). Caution is required in interpreting the significance of such findings in vivo because the intracellular levels of EspF during EHEC infection may be very different. It has been suggested that disruption of barrier function and/or the induction of apoptosis by EspF may contribute to carriage and the induction of enteritis; however, EspF and CesF play only a subtle role in colonization of the murine intestine by C. rodentium and the production of colonic hyperplasia (49, 151).
Recent studies have shown that EHEC O157:H7 also induces occludin redistribution and reduces the TER of infected monolayers, but to a lesser extent and at a slower rate than EPEC O127:H6 (217). Surprisingly, mutation of EHEC O157:H7 espF and cesF did not dramatically affect the reduced TER phenotype (217). This may be explained by the presence of two EspF-like homologues identified by sequencing of the EHEC O157:H7 genome: Z3072 (EspFU/TccP, encoded in cryptic prophage CP-933U and 31% identical over 312 amino acids to EHEC O157:H7 EspF) and Z1385 (M-EspF, encoded in CP-933M, 36% identical over 203 amino acids to EHEC O157:H7 EspF). The role played by EHEC EspF and EspF-like proteins individually and in concert during infection merits further investigation.
Sequencing of the genomes of EHEC O157:H7 strains EDL933 and RIMD 0509952 identified a second cluster of type III secretion-associated genes (ETT2, O-island 115) homologous to the Salmonella inv-spa-prg (SPI-1) and Shigella ipa-mxi-spa loci required for epithelial cell invasion (90, 175). ETT2 genes are widely distributed among Shiga-toxin producing E. coli strains, other pathogenic E. coli, and commensal strains, but they have been subject to varying degrees of mutational attrition (89, 137, 181). The EHEC O157:H7 ETT2 locus lacks homologs of the Salmonella avrA, sptP, and sipABCD genes, the latter of which are required for translocation of effectors, and also contains frameshift mutations in the homologs of spaR, prgH, and orgB, suggesting that it is unlikely to encode a functional type III secretion apparatus. Homologs of the Sip translocator proteins have been identified in a genomic island (eip) in enteroaggregative E. coli strain 042 and in some other ETT2-positive strains (181). It is presently unclear if these strains produce a functional type III secretion system. Recent data indicate that two ETT2-encoded genes of EHEC O157:H7 (etrA and eivF) negatively regulate expression of the LEE (224), indicating that cross-talk between the loci occurs.
Efa1 and ToxB.
The existence of a predicted 365-kDa LCT homolog had already been demonstrated in EPEC serotype O127:H6 by Klapproth et al., but in this case the protein was shown to be required for inhibition of the mitogen-activated proliferation of human peripheral blood lymphocytes and the synthesis of certain proinflammatory cytokines (121). The product, termed lymphocyte inhibitory factor (LifA, or lymphostatin) is 97.4% identical at the amino acid level to the EHEC O111:H– Efa1 protein (159). LifA also inhibits the proliferation of human and murine intraepithelial lymphocytes and it has been suggested that it may facilitate intestinal colonization by modulating mucosal immunity in the gut (122, 138). Efa1/LifA are also highly homologous to complete and partial genes required for Chlamydia trachomatis cytotoxicity (11).
Almost all LEE-positive strains tested to date contain either Efa1 or ToxB and indeed in EHEC O26 strain 413/89-1 and rabbit EPEC strains RDEC-1 and 83/89 the LEE and efa1 are adjacent on the chromosome (205, 227) (EHEC O26 LEE accession number AJ277443). This close genetic linkage occurs in many other EPEC and EHEC strains (149) and may imply that Efa1 and/or ToxB are required for complete activity of the LEE-encoded type III secretion system.
Iha.
Iha (IrgA homolog adhesin) was identified by screening a cosmid library of EHEC O157:H7 DNA fragments for their ability to confer an adherent phenotype to E. coli K-12 (202). Iha is a 67-kDa outer membrane protein in EHEC O157:H7 and is 53% similar at the amino acid level to the product of Vibrio cholerae iron-regulated gene A (irgA), an outer membrane protein implicated in pathogenesis that shares homology with TonB-dependent proteins involved in siderophore transport. An in-frame deletion in iha reduced adherence of EHEC O157:H7 to HeLa cells, but not significantly so (202). The iha gene is located adjacent to an O-island encoding urease and tellurite resistance in EHEC O157:H7 and is conserved in many EHEC serotypes isolated from cattle or humans (201, 207) and among extraintestinal E. coli isolates from patients with urosepsis (103). Iha is required for full virulence of UPEC in ascending urinary tract infections of mice (102); however, the importance of Iha in intestinal colonization has yet to be determined.
OmpA.
Evidence for the involvement of outer membrane porins in EHEC adherence was first provided by the demonstration that antisera raised against 36- and 40-kDa major outer membrane proteins of EHEC O157:H7 block adherence to HEp-2 cells (192). Amino-terminal peptide sequencing subsequently identified the proteins to be OmpA and OmpF, respectively (58). More recently, the role of OmpA in EHEC adherence was confirmed by screening EHEC O157:H7 transposon mutants for increased adherence to epithelial cells (208). Mutation of the tcdA gene, a transcriptional activator of the tdc operon which mediates L-threonine transport and degradation, results in elevated expression of OmpA and hyperadherence of EHEC O157:H7 to HeLa and Caco-2 cells in vitro (208). Inactivation of ompA in the tdcA mutant abolished the hyperadherent phenotype, indicating that OmpA and not other targets of TdcA is required for the adherent phenotype. In support of this conclusion, mutation of ompA alone in the wild-type EHEC O157:H7 strain reduced adherence by 13.5%, and OmpA-specific antiserum inhibited the adherence of three different EHEC O157:H7 strains to HeLa cells by ca. 25% (208). Adherence of an EPEC O127:H6 strain was not affected by OmpA-specific antiserum and it remains to be determined if non-O157 EHEC rely to a similar extent on OmpA or other outer membrane porins for adherence. Furthermore, OmpA is present in high-copy number in the E. coli cell envelope (estimated to be >105 molecules per cell) and it is unclear whether OmpA acts as an adhesin per se or facilitates the correct insertion, folding, or stability of other membrane proteins by playing a structural role.
Saa.
Paa.
Paa (porcine attaching and effacing associated) was identified as an adhesin of porcine nontoxigenic E. coli O45 associated with postweaning diarrhea by transposon mutagenesis, but is highly conserved among EPEC and EHEC strains (5). Sequence analysis of the porcine EPEC O45 paa gene predicts a 27.6-kDa protein which is 100% conserved in EHEC O157:H7 EDL933 and RIMD 0509952 and which exhibits 51.3% similarity to PEB3, a major antigen of Campylobacter jejuni, and 49% similarity to the AcfC, an accessory colonization factor of V. cholerae required for production of the toxin-coregulated pilus (9). Inactivation of the paa gene of porcine EPEC O45 strain 86-1390 or rabbit EPEC strain E22 resulted in a ca. 50% reduction in the number of ileal villi exhibiting adherent bacteria in an in vitro porcine ileal explant model, with no evidence of A/E lesion formation (9). Introduction of the cloned paa gene on a plasmid restored adherence and the A/E phenotype and chicken anti-Paa antibodies specifically inhibited adherence to porcine ileal explants (9). Such antibodies also specifically labeled a surface-exposed porcine EPEC protein by immunogold electron microscopy. The presence of eae and paa is highly correlated and this has led to speculation that Paa may be a primary adhesin or be directly required at an early stage of A/E lesion formation. In support of this conclusion, three porcine EPEC O45 strains positive for eae but lacking paa were found to be A/E negative, although it is not known if the entire LEE region or its regulators were intact in these strains (9).
AIDA.
AIDA (adhesin involved in diffuse adherence) was first described in diffusely adhering E. coli O126:H27 and is frequently found in Stx2e-positive STEC strains isolated from pigs with postweaning diarrhea and edema disease (12, 85, 160) (chapter Adhesins of Diffusely Adherent and Enteroaggregative Escherichia coli). AIDA belongs to the growing family of type V secreted (autotransporter) proteins, and two genes (aah and aidA) are required for full adherence. The aidA gene encodes a pre-pro-protein of 132-kDa, which after amino- and carboxyl-terminal processing, matures to the adhesin AIDA-I which remains noncovalently associated with the cell surface (ca. 100 kDa, α-domain) and the autocatalytically cleaved membrane-integrated β-domain of 47.5 kDa (13, 200). Full activity of AIDA-I requires the posttranslational addition of heptose residues at multiple sites, mediated by the aah encoded autotransporter adhesin heptosyltransferase (14). The cellular receptor of AIDA-I has been identified as an integral N-glycosylated membrane protein of about 119 kDa (gp119) with a pI of 5.2 (130). EHEC O157:H7 strain EDL933 lacks the aidA and aah genes but encodes two identical copies of an AIDA-I homolog (Cah), which mediates interbacterial interactions leading to microcolony formation but not adherence per se (211).
Fimbriae and fibrillae are proteinaceous surface-associated appendages produced by many gram-negative bacteria and mediate important interactions with host cells. Fimbriae are rod-like and 5 to 10 nm in diameter, whereas fibrillae are shorter, 2 to 4 nm in diameter and are sometimes mistaken for afimbrial adhesins. Fibrillae tend to be either long and wiry or curly and flexible. Analysis of the complete genome sequence of the EHEC O157:H7 Sakai outbreak strain RIMD 0509952 identified 14 putative fimbrial loci. Five fimbrial loci of RIMD 0509952 and three of EDL933 are conserved in E. coli K-12, including the genes for type I fimbriae and curli fibers. Four fimbrial loci in RIMD 0509952 are absent or poorly conserved in EDL933 (90, 174, 175). The remaining five loci of RIMD 0509952 are partially conserved in E. coli K-12 and have orthologues in EDL933 but the sequences and/or gene order of the genes in the two EHEC O157:H7 may differ (see supplementary material in references 90 and 174). Two similar loci resemble the Salmonella lpfABCDE locus encoding long polar fimbriae (see below). Genes encoding Afa8, F17, Cs31A fimbriae that are conserved in other pathogenic E. coli have not been detected in human or bovine EHEC strains (201). F4 and F18 (F107) fimbriae are often associated with Stx2e-producing STEC strains from pigs with postweaning diarrhea and edema disease, and it likely they play a role in pathogenesis (38, 85, 94, 154, 160, 164). The FedF adhesin of F18 fimbriae is required for receptor binding during in vitro adherence of enterotoxigenic E. coli to porcine epithelial cells (196).
Sequencing of a region of the EHEC O157:H7 EDL933 genome corresponding to the 76- to 81.5-minute region of the E. coli K-12 chromosome identified a 6-kb locus (lpf1) predicted to contain six genes (lpfABCC'DE) similar in sequence and gene order to the Salmonella lpfABCDE genes required for the synthesis of long polar fimbriae (z4965-z4971; O-island 141) (209). Long polar fimbriae of Salmonella enterica serovar Typhimurium mediate adhesion to murine Peyer’s patches and are required for full virulence (10). Moreover, expression of the Salmonella lpf operon in the nonfimbriated E. coli K-12 strain ORN172 results in increased bacterial adherence to lymphoid follicles in thin sections of murine Peyer’s patches (10). Expression of the EHEC O157:H7 lpf1 operon in ORN172 has also been reported to increase adherence to HeLa cells (by ca. 60%) and is associated with the appearance of peritrichous short fimbriae, similar in appearance to Salmonella long polar fimbriae and E. coli type I fimbriae (209). stx-Positive and stx-negative EHEC O157:H7 mutant strains harboring insertions in the lpfA1 gene, predicted to encode the major fimbrial subunit, exhibit a modest reduction in adherence to HeLa cells (by ca. 20%), and adhere in a diffuse rather than localized manner (209, 210). LpfB is a putative chaperone, LpfC is a putative outer membrane usher protein, and LpfD and LpfE are putative minor fimbrial proteins. The lpfC gene is disrupted in O-island 141, with two open reading frames, lpf and lpfC', predicted to encode 40.2- and 17.8-kDa proteins. In serovar Typhimurium lpfC encodes a 94.4-kDa protein and it is presently unclear if EHEC O157:H7 produces both lpfC variants or a single protein by readthrough translation.
Type IV pili bundle-forming pili are a key colonization factor of EPEC O127:H6 in humans and mediate interbacterial interactions as well as direct attachment to epithelial cells and the localized adherence (LA) phenotype (17, 28). A homologous type IV pilus encoded by the cfc locus is required for carriage and virulence of C. rodentium in mice (150); however, sequencing of the genomes of EHEC O157:H7 strains EDL933 and RIMD 0509952 has failed to identify homologs of type IV pili (90, 175). However, sequencing of the pO113 large plasmid of LEE-negative STEC O113:H2 associated with an outbreak of HUS identified a cluster of 11 contiguous genes (designated pilL through pilV), with an additional gene (pilI) predicted to be encoded upstream (197). The pO113 pil locus is homologous to the type IV pilus biosynthesis loci of IncI plasmids R721, R64, and ColIb9 and mediates the formation of long thin pili in STEC O113:H21 and agglutination of guinea pig erythrocytes (197). Probes based on the pO113-encoded pilS gene hybridize to all STEC serogroup O113 strains tested to date and to 11 of 14 LEE-negative STEC belonging to other serogroups; however, LEE-positive strains appear to lack the pil locus. The cloned pO113 pil locus could not restore adherence of a plasmid-cured derivative of STEC O113:H21 to HEp-2 or Hct-8 cells in vitro, indicating that it may not be involved in adherence (197).
Flagella influence intestinal colonization, invasion, and persistence by several enteric pathogens, including E. coli O78:K80 in a day-old chick model of avian colibacillosis (127). Consistent with a role in adherence, inactivation of the EPEC O127:H6 fliC flagellin structural gene or addition of flagella-specific antibodies significantly impaired adherence to cultured HeLa cells (80). However, the fliC mutant also exhibited reduced expression of the type IV bundle-forming pilus when adhering to HeLa cells and adherence of the fliC mutant was not fully restored by introduction of the cloned fliC gene on a plasmid (80). An EPEC O127:H6 motB mutant capable of synthesizing intact flagella but not flagella rotation adhered to HeLa cells as well as the parent strain, indicating that motility is not required for adherence and that the flagellum per se may act as an adhesin (80). In support of this notion, purified EPEC H6 and H2 flagella can bind to epithelial cells and flagella are highly expressed on adherent bacteria (80). Purified H7 flagella produced by many EHEC serogroup O157 strains associated with human disease do not adhere to HeLa cells and H7-specific antibodies do not significantly inhibit adherence of EHEC O157:H7 in vitro (80, 191). One caveat to this finding is that the flagella of avian collibacillosis-causing E. coli O78:K80 were found to be important for adherence to cultured cells only when a mucous-secreting cell line was used, indicating that some epithelial cell adherence assays may be incapable of identifying adhesins (127). Flagella were recently shown to be required for persistence of an stx-minus EHEC O157:H7 strain in the intestines of specific-pathogen-free chicks (15). However, flagella may not be essential for EHEC pathogenesis in humans, as nonmotile sorbitol-fermenting O157:H– strains are associated with up to 40% of HUS cases in Germany and are an emerging problem in Europe (108). These strains contain a 12-bp in-frame deletion in flhC, which is required for transcription of flagella biosynthesis genes (148).
Antibodies directed against the O111 and O157 lipopolysaccharide (LPS) O-antigens have been reported to block adherence of the homologous EHEC serogroup to Henle 407 cells in vitro (167). However, preincubation of cells with purified LPS does not competitively inhibit EHEC adherence (167, 191), suggesting that LPS is unlikely to be acting as an adhesin per se. Indeed, EHEC O157:H7 mutants deficient in synthesis of the O157 polysaccharide side chain actually exhibit increased adherence to HEp-2 or HeLa cells in vitro and still form A/E lesions (18, 29, 208). It is possible that this is the result of unmasking of underlying adhesins and/or alterations in surface hydrophobicity. Inhibition of adherence by anti-LPS antibodies was independent of bactericidal or agglutinating activity and it has been suggested that LPS-specific antibodies may have therapeutic value if administered early in EHEC infection (167). LPS-specific antibodies can be readily detected in sera from convalescent HC and HUS patients and it unclear what role such humoral responses play in the resolution of EHEC infection.
A 60-MDa (ca. 92-kb) plasmid (pO157) is highly conserved among EHEC serotype O157:H7 strains associated with human disease. Contradictory data exist on the role of pO157 in EHEC adherence. Initially it was reported that pO157 is required for adherence to intestinal Henle 407 cells and for the elaboration of fimbriae with a major subunit of 16 kDa (109). Toth et al. confirmed that pO157 was required for full adherence of EHEC and further demonstrated that pO157 was able to confer a weak adherence phenotype upon E. coli K-12 (212). However by transmission electron microscopy the authors could find no evidence for loss of fimbriae in plasmid-cured EHEC O157:H7, or gain of fimbriae by E. coli K-12 strain HB101 upon transformation with a Tn801-marked derivative of pO157 (212). Fratamico et al. also reported no difference in the number or type of fimbriae in pO157-bearing and plasmid-cured strains, and moreover reported that pO157 did not influence adherence to Henle 407 or HEp-2 cells (74). Sequencing of the large plasmids of EHEC O157:H7 strains EDL933 and the Sakai outbreak strain RIMD 0509952 later confirmed that pO157 does not encode any fimbrial operons and the origin of the fimbriae observed in early experiments is unknown (22, 136). Subsequent studies have shown that the pO157-encoded toxB gene is required for full adherence to cultured epithelial cells, probably as a result of posttranscriptional control of the expression and secretion of LEE-encoded effector proteins (198, 204). Experimental infection studies indicate that pO157 is not required for EHEC O157:H7 virulence in gnotobiotic piglets (214), and that the pO157-encoded toxB gene is not required for intestinal colonization in calves or sheep (198).
By screening transposon mutants of EHEC for reduced adherence to cultured epithelial cells or using specific immune sera to block adherence, numerous other candidate adhesins have been identified. Tatsuno et al. screened 4,677 EHEC O157:H7 mini-Tn5Km2 transposon mutants for their ability to adhere to Caco-2 cells (203). Twenty-nine mutants that reproducibly exhibited reduced adherence in the absence of growth defects were examined further and their results confirm the key role played by LEE-encoded proteins in EHEC adherence (203). Sixteen known or hypothetical non-LEE-encoded genes were found to influence EHEC O157:H7 adherence without impairing the function of the LEE-encoded type III secretion system (203). Proteins implicated in the transport of potassium, arsenite, and lysine were required for full adherence, together with two putative transcriptional regulators. The remainder of insertions mapped to hypothetical membrane proteins. The roles played by these genes in EHEC pathogenesis are unknown.
Studies using immune sera raised against EHEC O157:H7 outer membrane proteins have identified several candidate adhesins including OmpA, OmpF, and 30- and 94-kDa outer membrane proteins (58, 192). Amino-terminal sequencing of the 94-kDa EHEC O157:H7 protein revealed that it was distinct from intimin; however, no matches could be identified in databases at the time (58). A search of the predicted proteomes of the sequenced O157:H7 strains with the peptide sequence from the 94-kDa protein (AEGFVV) reveals an identical match to residues 21 to 26 of a hypothetical protein YaeT (Z0188/ECs0179) predicted to be a 90.5-kDa Sec-dependent outer membrane protein. The peptide sequence from the 30-kDa protein identified by Dytoc et al. (AENDKPQYLGD) is an almost identical match with residues 23 to 33 of the predicted 33.6-kDa outer membrane protein Tsx (Z0512/ECs0464), which is known to function as a nucleoside-specific channel-forming protein and receptor for phage 6 and colicin K. Both YaeT and Tsx are conserved in E. coli K-12 and in other members of the Enterobacteriaceae and their role in bacterial adherence merits further investigation. An 8-kDa outer membrane protein has also been implicated in EHEC O157:H7 adherence by using specific antiserum; however, the identity of this protein has yet to be determined (226).
Colonization of mucosal surfaces is expected to be a key stage in EHEC pathogenesis and much progress has been made in the identification and characterization of the bacterial factors mediating adherence to epithelial cells. Screening of EHEC transposon mutants and specific immune sera for effects on adherence to epithelial cells in vitro has provided valuable insights, but the role of candidate adhesins identified by such approaches in intestinal colonization and EHEC virulence is largely unknown. A combination of in vitro and in vivo studies have confirmed the major role played by the locus of enterocyte effacement-encoded proteins in EHEC adherence, and subunit vaccines based on such proteins show promise in controlling EHEC in reservoir hosts. However, it is clear that numerous accessory adhesins contribute to EHEC adherence, mostly without affecting the function of the LEE, and further studies are required to determine how such molecules influence initial adherence, tissue tropism, interbacterial interactions, and intestinal colonization. Few of the non-LEE encoded factors influencing EHEC adherence resemble known adhesins in other bacteria and caution is required in interpreting the role of major cell surface components given the potential for pleiotropic effects.
Sequencing of the genomes of two EHEC O157:H7 strains has accelerated the identification of adhesins; however, some adhesins are not expressed by EHEC O157:H7, despite playing important roles in the host cell interactions of non-O157 EHEC serogroups (e.g., Efa1, Saa, type I and type IV fimbriae). Comparison of the genomes of four EHEC isolates of serotypes O91:H–, O103:H2, O111:H–, and O157:H– using DNA microarrays has indicated that they differ markedly in their virulence gene content (53). Non-O157 EHEC pose an increasing threat to human and animal health and an awareness of the prevalence and function of adhesins of both O157 and non-O157 EHEC will improve our ability to respond to future disease emergencies.
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Module8.3.2.3
