Recruitment of cytoskeletal and signaling proteins to enteropathogenic and enterohemorrhagic Escherichia coli pedestals

Enteropathogenic Escherichia coli (EPEC) is a human pathogen that attaches to intestinal epithelial cells and causes chronic watery diarrhea. A close relative, enterohemorrhagic E. coli (EHEC), causes severe bloody diarrhea and hemolytic-uremic syndrome. Both pathogens insert a protein, Tir, into the host cell plasma membrane where it binds intimin, the outer membrane ligand of EPEC and EHEC. This interaction triggers a cascade of signaling events within the host cell and ultimately leads to the formation of an actin-rich pedestal upon which the pathogen resides. Pedestal formation is critical in mediating EPEC- and EHEC-induced diarrhea, yet very little is known about its composition and organization. In EPEC, pedestal formation requires Tir tyrosine 474 phosphorylation. In EHEC Tir is not tyrosine phosphorylated, yet the pedestals appear similar. The composition of the EPEC and EHEC pedestals was analyzed by examining numerous cytoskeletal, signaling, and adapter proteins. Of the 25 proteins examined, only two, calpactin and CD44, were recruited to the site of bacterial attachment independently of Tir. Several others, including ezrin, talin, gelsolin, and tropomyosin, were recruited to the site of EPEC attachment independently of Tir tyrosine 474 phosphorylation but required Tir in the host membrane. The remaining proteins were recruited to the pedestal in a manner dependent on Tir tyrosine phosphorylation or were not recruited at all. Differences were also found between the EPEC and EHEC pedestals: the adapter proteins Grb2 and CrkII were recruited to the EPEC pedestal but were absent in the EHEC pedestal. These results demonstrate that although EPEC and EHEC recruit similar cytoskeletal proteins, there are also significant differences in pedestal composition.     

Amino acid residues within enterohemorrhagic Escherichia coli O157:H7 Tir involved in phosphorylation, alpha-actinin recruitment, and Nck-independent pedestal formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) adherence to epithelial cells results in the formation of actin pedestals. Pedestal formation requires the bacterial protein Tir, which is inserted into the epithelial cell plasma membrane by the type III secretion system. EPEC and EHEC use different Tir-based mechanisms for pedestal formation, and the EPEC Tir residues required have been well described. In contrast, little is known about the regions of EHEC O157:H7 Tir that are essential for pedestal formation. Additionally, EHEC O157:H7 Tir is serine/threonine phosphorylated, although the residues involved and their role in pedestal formation are not known. In this study, we describe two regions within the carboxy terminus of EHEC O157:H7 Tir that are required for phosphorylation and pedestal formation. Serines 436 and 437 are substrates for protein kinase A phosphorylation, although this is not required to form pedestals. Using a series of internal deletion mutants, we found that amino acids 454 to 463 are required for efficient pedestal formation. Deleting this region resulted in a significant decrease in the recruitment of both filamentous actin and the actin binding protein alpha-actinin. As alpha-actinin binds directly to the EHEC O157:H7 amino terminus, these data suggest that its recruitment is dependent on pedestal formation.     

Interaction of enteropathogenic and Shiga toxin-producing Escherichia coli and porcine intestinal mucosa: role of intimin and Tir in adherence

The ileal in vitro organ culture (IVOC) model using tissues originating from colostrum-deprived newborn piglets has proven to be an effective way to study the attaching and effacing (A/E) phenotype of porcine enteropathogenic Escherichia coli (EPEC) ex vivo. The aim of this study was to investigate the role of intimin subtype and Tir in the adherence of EPEC and Shiga-toxin-producing E. coli (STEC), isolated from different animal species, to porcine intestinal IVOC. Moreover, the role of intimin in Tir-independent adherence of the human EPEC strain E2348/69 was investigated using intimin and Tir-deficient derivatives. Our results demonstrated that A/E E. coli strains (AEEC) from various animal species and humans induce the A/E phenotype in porcine ileal IVOC and that intimin subtype influences intestinal adherence and tropism of AEEC strains. We also showed that a tir mutant of EPEC strain E2348/69 demonstrates close adherence to the epithelial cells of porcine ileal IVOC segments, with microvillous effacement but with no evidence of actin polymerization or pedestal formation, and that intimin seems to be involved in this phenotype. Overall, this study provides further evidence for the existence of one or more host-cell-encoded intimin receptor(s) in the pig gut.     

Mechanical fractionation reveals structural requirements for enteropathogenic Escherichia coli Tir insertion into host membranes

Enteropathogenic Escherichia coli (EPEC) inserts its receptor for intimate adherence (Tir) into host cell membranes by using a type III secretion system. Detergents are frequently used to fractionate infected host cells to investigate bacterial protein delivery into mammalian cells. In this study, we found that the Triton X-100-soluble membrane fraction from EPEC-infected HeLa cells was contaminated with bacterial proteins. We therefore applied a mechanical method of cell lysis and ultracentrifugation to fractionate infected HeLa cells to investigate the biology and biochemistry of Tir delivery and translocation. This method demonstrates that the translocation of Tir into the host cell membrane requires its transmembrane domains, but not tyrosine phosphorylation or binding to Tir's ligand, intimin.     

Cortactin is necessary for F-actin accumulation in pedestal structures induced by enteropathogenic Escherichia coli infection

Cortactin and the translocated intimin receptor, Tir, interacted with each other in pedestal formation in HeLa cells infected with enteropathogenic Escherichia coli (EPEC). Cortactin is shown to be necessary for organizing actin pedestals in response to EPEC, based on the expression of green fluorescent protein-fused cortactin derivatives in HeLa cells.     

Intimin-dependent binding of enteropathogenic Escherichia coli to host cells triggers novel signaling events, including tyrosine phosphorylation of phospholipase C-gamma1.

Enteropathogenic Escherichia coli (EPEC) interactions with HeLa epithelial cells induced the tyrosine phosphorylation of a host protein of approximately 150 kDa, Hp150. Phosphorylation of this protein band was dependent on the interaction of the EPEC protein intimin with epithelial cell surfaces and was correlated with pedestal formation. Hp150 phosphorylation was specifically inhibited by the addition of cytochalasin D, an inhibitor of actin polymerization, although this appeared to be an indirect effect preventing interaction of intimin with its receptor, tyrosine-phosphorylated Hp90, and thus triggering Hp150 phosphorylation. This suggests the involvement of an actin-based movement of membrane-bound tyrosine-phosphorylated Hp90 to allow its interaction with intimin. Analysis of the tyrosine-phosphorylated Hp150 protein demonstrated that it is heterogeneous in composition, with phospholipase C-gamma1 (PLC-gamma1) being a minor component. Activation of PLC-gamma1 by tyrosine phosphorylation leads to inositol triphosphate and Ca2+ fluxes, events detected following EPEC infection. EPEC also induced tyrosine dephosphorylation of host proteins, including a 240-kDa host protein (Hp240), following EPEC infection. Protein dephosphorylation appears to be a signaling event which occurs independently of intimin. Inhibition of host tyrosine dephosphorylation events by the addition of the tyrosine phosphatase inhibitor sodium vanadate did not prevent actin accumulation beneath the adherent bacteria. We conclude that EPEC induces two sets of signaling events following infection. One set is dependent on EPEC proteins secreted by the type III secretion pathway (EspA and EspB) which induces Hp90 tyrosine phosphorylation and dephosphorylation of host phosphotyrosine proteins. The second set, which is also dependent on the first signaling events, requires intimin interaction with its receptor, tyrosine-phosphorylated Hp90, to trigger Hp150 and PLC-gamma1 tyrosine phosphorylation as well as pedestal formation. Inhibition of pedestal formation by tyrosine kinase inhibitors indicates an important role for tyrosine phosphorylation events during EPEC subversion of host processes.     

The established intimin receptor Tir and the putative eucaryotic intimin receptors nucleolin and beta1 integrin localize at or near the site of enterohemorrhagic Escherichia coli O157:H7 adherence to enterocytes in vivo

For enterohemorrhagic Escherichia coli (EHEC) O157:H7 to adhere tightly to the intestinal epithelium and produce attach and efface (A/E) lesions, the organism must express the adhesin intimin and insert the bacterially encoded translocated intimin receptor Tir into the plasma membrane of the host enterocyte. Additionally, some reports based on tissue culture experiments indicate that intimin has affinity for the eucaryotic proteins nucleolin and beta1 integrin. To address the potential biological relevance of these eucaryotic proteins in the infection process in vivo, we sought to compare the proximity of Tir, nucleolin, and beta1 integrin to regions of EHEC O157:H7 attachment in intestinal sections from three different inoculated animals: piglets, neonatal calves, and mice. Piglets and neonatal calves were chosen because intimin-mediated adherence of EHEC O157:H7 and subsequent A/E lesion formation occur at high levels in these animals. Mice were selected because of their ease of manipulation but only after we first demonstrated that in competition with the normal mouse gut flora, an EHEC O157:H7 strain with a nonpolar deletion in the intimin gene was cleared faster than strains that produced wild-type or hybrid intimin. In all three animal species, we noted immunostained Tir beneath and stained nucleolin closely associated with adherent bacteria in intestinal sections. We also observed immunostained beta1 integrin clustered at locations of bacterial adherence in porcine and bovine tissue. These findings indicate that nucleolin and beta1 integrin are present on the luminal surface of intestinal epithelia and are potentially accessible as receptors for intimin during EHEC O157:H7 infection.     

TccP2 of O157:H7 and non-O157 enterohemorrhagic Escherichia coli (EHEC): challenging the dogma of EHEC-induced actin polymerization

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir(EHEC O157) is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF(U), which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, beta-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 DeltatccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.     

The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli

Regulation of virulence gene expression in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2, LEE3, and LEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2, orf10, rorf10, orf19, and espF, indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. delta ler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.     

Enterohemorrhagic Escherichia coli Colonization of Human Colonic Epithelium In Vitro and Ex Vivo

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by using in vitro organ culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect the in vivo situation.     

Antibody against the carboxyl terminus of intimin alpha reduces enteropathogenic Escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization

The C-terminal third of intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of intimin alpha.     

Intimin-specific immune responses prevent bacterial colonization by the attaching-effacing pathogen Citrobacter rodentium

The formation of attaching and effacing (A/E) lesions on gut enterocytes is central to the pathogenesis of enterohemorrhagic (EHEC) Escherichia coli, enteropathogenic E. coli (EPEC), and the rodent pathogen Citrobacter rodentium. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Here we show that the LEE-encoded proteins EspA, EspB, Tir, and intimin are the targets of long-lived humoral immune responses in C. rodentium-infected mice. Mice infected with C. rodentium developed robust acquired immunity and were resistant to reinfection with wild-type C. rodentium or a C. rodentium derivative, DBS255(pCVD438), which expressed intimin derived from EPEC strain E2348/69. The receptor-binding domain of intimin polypeptides is located within the carboxy-terminal 280 amino acids (Int280). Mucosal and systemic vaccination regimens using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280alpha from EPEC strain E2348/69. Mice vaccinated subcutaneously with Int280alpha, in the absence of adjuvant, were significantly more resistant to oral challenge with DBS255(pCVD438) but not with wild-type C. rodentium. This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of intimin (Int388-667) as a vaccine. These results show that anti-intimin immune responses can modulate the outcome of a C. rodentium infection and support the use of intimin as a component of a type-specific EPEC or EHEC vaccine.     

Differential modulation by Ca2+ of type III secretion of diffusely adhering enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) strains are a common cause of persistent diarrhea among infants, primarily in developing countries. The pathogenicity of EPEC is associated with the expression and secretion of bacterial proteins encoded by the chromosomal locus of enterocyte effacement (LEE). The LEE-encoded type III-secreted proteins EspA, EspB, and EspD are part of a molecular syringe, which is used by EPEC to translocate effector proteins directly into the cytoplasm of host cells. The type III-secreted translocated intimin receptor (Tir) protein is thought to be delivered by an Esp-dependent mechanism into the host cell, and this is followed by insertion into the host plasma membrane, where the protein serves as the receptor for intimin, an afimbrial bacterial adhesin. Type III secretion is subject to environmental regulation, and secretion can be induced in vitro by growing bacteria in cell culture medium. In this study we found that Ca(2+) is involved in the regulation of type III secretion both in classical locally adherent EPEC and in atypical diffusely adherent EPEC. Interestingly, we observed contrasting secretion of Esp proteins and Tir in response to Ca(2+). While the secretion of Tir is clearly enhanced and the protein is integrated into HeLa membranes under calcium chelation conditions, Esp secretion is strongly reduced under these conditions. These data suggest that under Ca(2+)-depleted conditions Tir might be secreted into the medium and integrated into host membranes by an Esp-independent mechanism, without the need for a functional type III translocation machinery.     

Role for CD2AP and Other Endocytosis-Associated Proteins in Enteropathogenic Escherichia coli Pedestal Formation

Enteropathogenic Escherichia coli (EPEC) strains are extracellular pathogens that generate actin-rich structures (pedestals) beneath the adherent bacteria as part of their virulence strategy. Pedestals are hallmarks of EPEC infections, and their efficient formation in vitro routinely requires phosphorylation of the EPEC effector protein Tir at tyrosine 474 (Y474). This phosphorylation results in the recruitment and direct attachment of the host adaptor protein Nck to Tir at Y474, which is utilized for actin nucleation through a downstream N-WASP-Arp2/3-based mechanism. Recently, the endocytic protein clathrin was demonstrated to be involved in EPEC pedestal formation. Here we examine the organization of clathrin in pedestals and report that CD2AP, an endocytosis-associated and cortactin-binding protein, is a novel and important component of EPEC pedestal formation that also utilizes Y474 phosphorylation of EPEC Tir. We also demonstrate the successive recruitment of Nck and then clathrin prior to actin polymerization at pedestals during the Nck-dependent pathway of pedestal formation. This study further demonstrates that endocytic proteins are key components of EPEC pedestals and suggests a novel endocytosis subversion strategy employed by these extracellular bacteria.The extracellular bacterial pathogen enteropathogenic Escherichia coli (EPEC) causes serious diarrheal disease in humans and is a prevalent microbe involved in childhood mortality in the developing world. This microbe is part of a larger family of bacteria called the attaching and effacing (A/E) pathogens that also includes the human-specific pathogen enterohemorrhagic E. coli (EHEC) and the murine disease-causing bacterium Citrobacter rodentium. These bacteria attach to intestinal epithelial cells and use a type III secretion system to directly deliver effector proteins from the bacterial cytosol into the cytoplasm of host cells. Among other functions, these effectors harness the host cell''s cytoskeleton (4) to generate actin-rich pedestals that are hallmarks of virulence for this class of pathogens (14). One of these effectors, the translocated intimin receptor (Tir), is key to pedestal formation. Following translocation into the host cell, Tir becomes embedded in the host cell plasma membrane, where its extracellular domain acts to firmly anchor the pathogen to the epithelial cell. In cultured cells, the intracellular cytoplasmic domain of EPEC Tir can become phosphorylated at tyrosine 474 (Y474) (6), where it recruits the adaptor protein Nck (7). These events all occur prior to actin filament polymerization beneath the attached bacteria via an N-WASP- and Arp2/3-based mechanism (7, 11). Although this is the prominent strategy used by EPEC to recruit actin to pedestals, a Y474-independent strategy also exists, but it occurs at a much lower frequency. During such instances, EPEC Tir becomes phosphorylated at Y454 and actin recruitment is independent of Nck (1).Previous work highlighted a role for clathrin during some bacterial infections (19, 20). Although the role of clathrin during enteropathogenic E. coli infections was not investigated until recently (20), the finding of clathrin at the tips of EPEC pedestals, coupled with the discovery of dynamin-2, another protein known to be involved in endocytosis, associated within the actin stalk of EPEC pedestals (18), suggests a possible role for additional endocytosis-associated proteins and indicates that a unique mechanism is employed by EPEC to remain extracellular despite the presence of these endocytic components. Other proteins, including the actin-associated protein cortactin, are also prominent at these structures. Cortactin is found throughout EPEC pedestals as well as pedestals formed by other attaching and effacing pathogens (2, 3). Thus, in order to further examine other endocytosis-associated proteins during EPEC pedestal formation, we opted to immunolocalize the endocytosis-related protein CD2AP (CD-2-associated protein) during these infections. CD2AP is a clathrin-associated endocytosis protein that directly associates with cortactin in other systems (12, 17).We discovered that CD2AP is present at EPEC pedestals and is a crucial component for their formation. Through the use of various host cell modification strategies, we subsequently explored the recruitment of the endocytosis-associated proteins at EPEC pedestals and found that during Nck-dependent pedestal formation, EPEC sequentially recruits Nck, clathrin, cortactin, and then CD2AP at the pedestal tip prior to the actin filament polymerization machinery at these sites.     

Role of intimin-tir interactions and the tir-cytoskeleton coupling protein in the colonization of calves and lambs by Escherichia coli O157:H7

Intimin facilitates intestinal colonization by enterohemorrhagic Escherichia coli O157:H7; however, the importance of intimin binding to its translocated receptor (Tir) as opposed to cellular coreceptors is unknown. The intimin-Tir interaction is needed for optimal actin assembly under adherent bacteria in vitro, a process which requires the Tir-cytoskeleton coupling protein (TccP/EspF(U)) in E. coli O157:H7. Here we report that E. coli O157:H7 tir mutants are at least as attenuated as isogenic eae mutants in calves and lambs, implying that the role of intimin in the colonization of reservoir hosts can be explained largely by its binding to Tir. Mutation of tccP uncoupled actin assembly from the intimin-Tir-mediated adherence of E. coli O157:H7 in vitro but did not impair intestinal colonization in calves and lambs, implying that pedestal formation may not be necessary for persistence. However, an E. coli O157:H7 tccP mutant induced typical attaching and effacing lesions in a bovine ligated ileal loop model of infection, suggesting that TccP-independent mechanisms of actin assembly may operate in vivo.     

Mechanism of action of EPEC type III effector molecules

Enteropathogenic E. coli (EPEC) is a prototypic member of the family of related 'attaching and effacing (A/E)' pathogens that induce diarrhoeal disease, especially to the young that can be fatal, of a wide range of mammalian species. Disease is correlated with the loss of absorptive gut epithelial microvilli and the reorganisation of host cytoskeletal proteins into pedestal-like structures beneath the adherent bacteria. These phenotypes are dependent on a pathogenicity island (LEE; Locus of Enterocyte Effacement) encoding a type III secretion system, secreted proteins, chaperone molecules, regulatory proteins and the bacterial outer membrane protein intimin. The type III secretion apparatus directs the transfer of specific proteins across the bacterial envelope, with a subset (EPEC secreted proteins - EspA, EspB and EspD) functioning to transfer effector proteins into host cells. These effector molecules subvert cellular processes that undoubtedly benefit the pathogen and contribute to disease. Three LEE-encoded EPEC effector molecules have so far been identified with one, Tir (Translocated intimin receptor), being transferred into host cells where it is modified by host kinases and becomes inserted into the plasma membrane to orchestrate cytoskeletal rearrangements linked to disease. This activity is dependent on its interaction with intimin and on tyrosine phosphorylation, with Tir-intimin interaction essential for virulence. A second effector Map, Mitochondrial-associated protein, is targeted to mitochondria where it has membrane-potential disrupting activity. The third, EspF disrupts intestinal barrier function and can induce host cell death by unknown mechanisms. Recent data relating to the mechanism by which Tir and Map function within host cells is discussed.     

Human response to Escherichia coli O157:H7 infection: antibodies to secreted virulence factors

Vaccination has been proposed for the prevention of disease due to enterohemorrhagic Escherichia coli (EHEC), but the immune response following human infection, including the choice of potential antigens, has not been well characterized. To study this, sera were obtained from five pediatric patients with acute diarrhea caused by E. coli O157:H7 0, 8, and 60 days after hospitalization. These sera were used to examine the immune response to four different EHEC virulence factors: Tir (translocated intimin receptor, which is inserted into the host cell membrane), intimin (bacterial outer membrane protein which binds to Tir), EspA (secreted protein which forms filamentous structures on EHEC surface), and EspB (inserted into the host membrane and cytoplasm). The response to O157:H7 lipopolysaccharide was also examined. Sera were assayed against purified recombinant proteins using immunoblot analysis and by enzyme-linked immunosorbent assay to determine the sera's titers to each of the antigens in all patients. We found that there was little reaction to EspA, EspB, and intimin in the acute-phase sera, although there was some reactivity to Tir. By day 8, titers of antibody to all four virulence factors were present in all patients, with a very strong response against Tir (up to a titer of 1:256,000), especially in hemolytic-uremic syndrome patients, and lesser strong responses to the other three antigens. The titer to the antigens 60 days after hospitalization was decreased but was still highest for Tir. These results suggest that there is a strong immune response to Tir, and to a lesser extent to the other three virulence factors, following EHEC disease, indicating that these bacterial molecules are potential vaccine candidates for preventing EHEC disease. They also suggest that bacterial virulence factors that are inserted into host cells during infection by type III secretion systems (Tir or EspB) are still recognized by the host immune response.     

Identification of a family of intimins common to Escherichia coli causing attaching-effacing lesions in rabbits, humans, and swine.

Intimin, an outer membrane protein encoded by eaeA that mediates close attachment of enteropathogenic bacteria to apical surfaces of epithelial cells, is required for formation of the attaching-effacing lesions and for full pathogenesis of the bacteria. Analysis of the eaeA sequence indicates that there is a high degree of homology at the N termini but less at the C termini of intimins. Antisera specific for the C-terminal third of RDEC-1 intimin, used to screen outer membrane proteins from 50 rabbit enteropathogenic Escherichia coli (EPEC), human EPEC, and human enterohemorrhagic E. coli (EHEC) strains, identified cross-reactive intimins from 24 isolates. Sequence analysis of the eaeA genes from human EPEC O111 and EHEC O26 isolates indicates that their intimins have C termini nearly identical to that of RDEC-1 intimin. Our results suggest that there are at least three families of related intimins and that the presence of intimin similar to that of RDEC-1 is not restricted by serogroup or host specificity.     

Enteropathogenic Escherichia coli and Vaccinia Virus Do Not Require the Family of WASP-Interacting Proteins for Pathogen-Induced Actin Assembly

The human pathogens enteropathogenic Escherichia coli (EPEC) and vaccinia virus trigger actin assembly in host cells by activating the host adaptor Nck and the actin nucleation promoter neural Wiskott-Aldrich syndrome protein (N-WASP). EPEC translocates effector molecules into host cells via type III secretion, and the interaction between the translocated intimin receptor (Tir) and the bacterial membrane protein intimin stimulates Nck and N-WASP recruitment, leading to the formation of actin pedestals beneath adherent bacteria. Vaccinia virus also recruits Nck and N-WASP to generate actin tails that promote cell-to-cell spread of the virus. In addition to Nck and N-WASP, WASP-interacting protein (WIP) localizes to vaccinia virus tails, and inhibition of actin tail formation upon ectopic expression of WIP mutants led to the suggestion that WIP is required for this process. Similar studies of WIP mutants, however, did not affect the ability of EPEC to form actin pedestals, arguing against an essential role for WIP in EPEC-induced actin assembly. In this study, we demonstrate that Nck and N-WASP are normally recruited by vaccinia virus and EPEC in the absence of WIP, and neither WIP nor the WIP family members CR16 and WIRE/WICH are essential for pathogen induced actin assembly. In addition, although Nck binds EPEC Tir directly, N-WASP is required for its localization during pedestal formation. Overall, these data highlight similar pathogenic strategies shared by EPEC and vaccinia virus by demonstrating a requirement for both Nck and N-WASP, but not WIP or WIP family members in pathogen-induced actin assembly.