Vaccination of pregnant dams with intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection

Cattle are important reservoirs of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that cause disease in humans. Both dairy and beef cattle are asymptomatically and sporadically infected with EHEC. Our long-term goal is to develop an effective vaccine to prevent cattle from becoming infected and transmitting EHEC O157:H7 to humans. We used passive immunization of neonatal piglets (as a surrogate model) to determine if antibodies against EHEC O157 adhesin (intimin(O157)) inhibit EHEC colonization. Pregnant swine (dams) with serum anti-intimin titers of < or =100 were vaccinated twice with purified intimin(O157) or sham-vaccinated with sterile buffer. Intimin(O157)-specific antibody titers in colostrum and serum of dams were increased after parenteral vaccination with intimin(O157). Neonatal piglets were allowed to suckle vaccinated or sham-vaccinated dams for up to 8 h before they were inoculated with 10(6) CFU of a Shiga toxin-negative (for humane reasons) strain of EHEC O157:H7. Piglets were necropsied at 2 to 10 days after inoculation, and intestinal samples were collected for determination of bacteriological counts and histopathological analysis. Piglets that ingested colostrum containing intimin(O157)-specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin(O157) as a viable candidate for an EHEC O157:H7 antitransmission vaccine.     

Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatal calves.

Cattle are an important reservoir of Shiga toxin-producing enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains, foodborne pathogens that cause hemorrhagic colitis and hemolytic uremic syndrome in humans. EHEC O157:H7 strains are not pathogenic in calves >3 weeks old. Our objective was to determine if EHEC O157:H7 strains are pathogenic in neonatal calves. Calves <36 h old inoculated with EHEC O157:H7 developed diarrhea and enterocolitis with attaching and effacing (A/E) lesions in both the large and small intestines by 18 h postinoculation. The severity of diarrhea and inflammation, and also the frequency and extent of A/E lesions, increased by 3 days postinoculation. We conclude that EHEC O157:H7 strains are pathogenic in neonatal calves. The neonatal calf model is relevant for studying the pathogenesis of EHEC O157:H7 infections in cattle. It should also be useful for identifying ways to reduce EHEC O157:H7 infections in cattle and thus reduce the risk of EHEC O157:H7 disease in humans.     

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.     

Detection of Intimins α, β, γ, and δ, Four Intimin Derivatives Expressed by Attaching and Effacing Microbial Pathogens

Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated α, β, δ, and γ, respectively.     

Decreased adherence of enterohemorrhagic Escherichia coli to HEp-2 cells in the presence of antibodies that recognize the C-terminal region of intimin

Antiserum raised against intimin from enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain 86-24 has been shown previously by our laboratory to inhibit adherence of this strain to HEp-2 cells. In the present study, we sought to identify the region(s) of intimin important for the effect of anti-intimin antisera on EHEC adherence and to determine whether antisera raised against intimin from an O157:H7 strain could reduce adherence of other strains. Compared to preimmune serum controls, polyclonal sera raised against the histidine-tagged intimin protein RIHisEae (intimin(O157)) or against His-tagged C-terminal fragments of intimin from strain 86-24 reduced adherence of this strain. Furthermore, an antibody fraction purified from the anti-RIHisEae serum that contained antibodies to the C-terminal third of intimin, the putative receptor-binding domain, also reduced adherence of strain 86-24, but a purified fraction containing antibodies to the N-terminal two-thirds of intimin did not inhibit adherence. The polyclonal anti-intimin(O157) serum raised against RIHisEae inhibited, to different degrees, the adherence of another O157:H7 strain, an EHEC O55:H7 strain, one of two independent EHEC O111:NM isolates tested, and one of two EHEC O26:H11 strains tested. Adherence of the other O26:H11 and O111:NM strains and an EPEC O127:H6 strain was not reduced. Finally, immunoblot analysis indicated a correlation between the antigenic divergence in the C-terminal third of intimins from different strains and the capacity of anti-intimin(O157) antiserum to reduce adherence of heterologous strains. Taken together, these data suggest that intimin(O157) could be used as an immunogen to elicit adherence-blocking antibodies against O157:H7 strains and closely-related EHEC.     

Immunological Characterization of Escherichia coli O157:H7 Intimin γ1

Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a(+) expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a(+). Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin typesα (O serogroups 86, 127, and 142), β1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), γ1 (O serogroups 55, 145, and 157), γ2 (O serogroups 111 and 103), and (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intγ1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin γ1-producing E. coli strains such as E. coli O157:H7.     

Enterohemorrhagic Escherichia coli O157:H7 produces Tir, which is translocated to the host cell membrane but is not tyrosine phosphorylated

Intimate attachment to the host cell leading to the formation of attaching and effacing (A/E) lesions is an essential feature of enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. In a related pathogen, enteropathogenic E. coli (EPEC), this activity is dependent upon translocation of the intimin receptor, Tir, which becomes tyrosine phosphorylated within the host cell membrane. In contrast, the accumulation of tyrosine-phosphorylated proteins beneath adherent EHEC bacteria does not occur, leading to questions about whether EHEC uses a Tir-based mechanism for adherence and A/E lesion formation. In this report, we demonstrate that EHEC produces a functional Tir that is inserted into host cell membranes, where it serves as an intimin receptor. However, unlike in EPEC, in EHEC Tir is not tyrosine phosphorylated yet plays a key role in both bacterial adherence to epithelial cells and pedestal formation. EHEC, but not EPEC, was unable to synthesize Tir in Luria-Bertani medium but was able to secrete Tir into M9 medium, suggesting that Tir synthesis and secretion may be regulated differently in these two pathogens. EHEC Tir and EPEC Tir both bind intimin and focus cytoskeletal rearrangements, indicating that tyrosine phosphorylation is not needed for pedestal formation. EHEC and EPEC intimins are functionally interchangeable, but EHEC Tir shows a much greater affinity for EHEC intimin than for EPEC intimin. These findings highlight some of the differences and similarities between EHEC and EPEC virulence mechanisms, which can be exploited to further define the molecular basis of pedestal formation.     

Bovine colostrum contains immunoglobulin G antibodies against intimin, EspA, and EspB and inhibits hemolytic activity mediated by the type three secretion system of attaching and effacing Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is the main cause of hemolytic-uremic syndrome, an endemic disease in Argentina which had an incidence in 2005 of 13.9 cases per 100,000 children younger than 5 years old. Cattle appear to be a major reservoir of EHEC, and a serological response to EHEC antigens has been demonstrated in natural and experimental infections. In the current study, antibodies against proteins implicated in EHEC's ability to form attaching and effacing lesions, some of which are exported to the host cell via a type three secretion system (TTSS), were identified in bovine colostrum by Western blot analysis. Twenty-seven (77.0%) of the 35 samples examined contained immunoglobulin G (IgG) antibodies against the three proteins assayed in this study: EspA, EspB, and the carboxy-terminal 280 amino acids of γ-intimin, an intimin subtype associated mainly with O157:H7 and O145:H- serotypes. Every colostrum sample was able to inhibit, in a range between 45.9 and 96.7%, the TTSS-mediated hemolytic activity of attaching and effacing E. coli. The inhibitory effect was partially mediated by IgG and lactoferrin. In conclusion, we found that early colostrum from cows contains antibodies, lactoferrin, and other unidentified substances that impair TTSS function in attaching and effacing E. coli strains. Bovine colostrum might act by reducing EHEC colonization in newborn calves and could be used as a prophylactic measure to protect non-breast-fed children against EHEC infection in an area of endemicity.     

Tissue tropism of enteropathogenic Escherichia coli strains belonging to the O55 serogroup

Four enteropathogenic Escherichia coli (EPEC) strains belonging to the O55 serogroup (G21 and G30 [both O55:H6], G35 [O55:H-], and G58 [O55:H7]) were tested for their tissue tropism by using human intestinal in vitro organ culture. Strains showed restricted adhesion with attaching-and-effacing activity to follicle-associated epithelium of Peyer's patches, with no apparent adhesion to duodenum or colon. G35 and G58 express intimin gamma and show a similar tropism to intimin gamma-expressing enterohemorrhagic E. coli (EHEC) O157:H7. However, strains G21 and G30 were unusual because they expressed intimin alpha and had a restricted tissue tropism of intimin gamma phenotype. The amino acid sequence of the carboxy-terminal 280 amino acids of intimin from G21 was determined. Comparison with the prototype intimin alpha from strain E2348/69 (O127:H6) showed a single amino acid difference (corresponding to Val907 and Ala907 in the whole intimins). This mutation was reproduced by site-directed mutagenesis in an intimin alpha plasmid template, pCVD438, with the hypothesis that it may induce a change in tropism. However, when the mutated plasmid was placed in both EPEC and EHEC backgrounds, duodenal adhesion in a manner similar to strain E2348/69 was evident upon in vitro organ culture. Thus, additional factor(s) unrelated to intimin exist in the O55:H6 genome that influence human intestinal tissue tropism.     

The role of intimin in the adherence of enterohaemorrhagic Escherichia coli (EHEC) O157: H7 to HEp-2 tissue culture cells and to bovine gut explant tissues

Intimin, an outer membrane protein encoded by eaeA, is a key determinant for the formation of attaching and effacing (AE) lesions by enterohaemorrhagic Escherichia coli (EHEC). To investigate the role of intimin in adherence, the eaeA gene was insertionally inactivated in three EHEC O157:H7 strains of diverse origin. The absence or presence of intimin did not correlate with the extent of adhesion of mutant or wild-type O157:H7 in tissue culture and neonatal calf gut tissue explant adherence assays. Adherence of the eaeA mutants to HEp-2 cells was diffuse with no evidence of intimate attachment whereas wild-type bacteria formed microcolonies and AE lesions. Intimin-independent adherence to neonatal calf gut explants was demonstrated by eaeA mutants and wild-type strains which adhered in the greatest numbers to colon but least well to rumen tissue. These results confirm that intimin is necessary for intimate attachment and that additional adherence factors are involved in intimin-independent adherence.     

Expression and characterization of the eaeA gene product of Escherichia coli serotype O157:H7.

In enteropathogenic Escherichia coli, the eaeA gene produces a 94-kDa outer membrane protein called intimin which has been shown to be necessary but not sufficient to produce the attaching-and-effacing lesion. The purpose of this study was to characterize the intimin specified by the eaeA allele of the enterohemorrhagic E. coli (EHEC) serotype O157:H7 strain CL8 and to determine its role in adherence. The carboxyl-terminal 266 amino acids of the CL8 intimin were expressed as a protein fusion with glutathione S-transferase, which was used to raise antiserum in rabbits. The antiserum reacted in Western immunoblots with a 97-kDa outer membrane protein of EHEC strains of serogroups O5, O26, O111, and O157 and enteropathogenic E. coli strains of serogroups O55 and O127. Surface labelling of CL8 with 125I showed that intimin was surface exposed. An eaeA insertional inactivation mutant of CL8 was produced and was designated CL8-KO1. Total adherence of CL8-KO1 to HEp-2 cells was not significantly different from that of CL8, but CL8-KO1 gave a negative result in the fluorescent actin staining test. The eaeA gene expressed alone in E. coli HB101 also gave a negative fluorescent actin staining test result. The eaeA gene of CL8 was able to complement the eaeA deletion mutation in CVD206. We conclude that the product of the EHEC eaeA gene is a 97-kDa surface-exposed protein and propose that it be designated intiminO157. Sherman et al. described a 94-kDa outer membrane protein which played an important role in adherence of E. coli O157:H7 (Infect. Immun. 59:890-899, 1991). Western immunoblotting and indirect fluorescent antibody studies showed that the protein described by Sherman et al. is not intimin.     

Plant cell-based intimin vaccine given orally to mice primed with intimin reduces time of Escherichia coli O157:H7 shedding in feces

Intimin is the primary adhesin of Escherichia coli O157:H7, the most common infectious cause of bloody diarrhea in the United States and the leading cause of acute kidney failure in children who develop hemolytic uremic syndrome. Cattle are the primary reservoir of E. coli O157:H7. Indeed, most cases of E. coli O157:H7 infection in the United States occur after ingestion of contaminated undercooked hamburger or produce that had contact with bovine manure. Because intimin is required for persistent colonization of neonatal calves and adult cattle, we hypothesized that an intimin-based vaccination strategy in calves would reduce colonization of cattle with E. coli O157:H7. To test this concept in a small-animal model, we developed transgenic tobacco plant cells that express the carboxy-terminal host cell-binding domain of E. coli O157:H7 intimin. Mice were either immunized intraperitoneally with intimin expressed from the plant cells, fed transgenic plant cells, or both. Here we show that these mice generated an intimin-specific mucosal immune response when primed parenterally and then boosted orally and also exhibited a reduced duration of E. coli O157:H7 fecal shedding after challenge.     

Identification of a novel fimbrial gene cluster related to long polar fimbriae in locus of enterocyte effacement-negative strains of enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne cause of bloody diarrhea and the hemolytic-uremic syndrome (HUS) in humans. Most strains of EHEC belong to a group of bacterial pathogens that cause distinctive lesions on the host intestine termed attaching-and-effacing (A/E) lesions. A/E strains of EHEC, including the predominant serotype, O157:H7, are responsible for the majority of HUS outbreaks worldwide. However, several serotypes of EHEC are not A/E pathogens because they lack the locus of enterocyte effacement (LEE) pathogenicity island. Nevertheless, such strains have been associated with sporadic cases and small outbreaks of hemorrhagic colitis and HUS. Of these LEE-negative organisms, O113:H21 is one of the most commonly isolated EHEC serotypes in many regions. Clinical isolates of LEE-negative EHEC typically express Shiga toxin 2 and carry an approximately 90-kb plasmid that encodes EHEC hemolysin, but in the absence of LEE, little is known about the way in which these pathogens colonize the host intestine. In this study we describe the identification of a novel fimbrial gene cluster related to long polar fimbriae in EHEC O113:H21. This chromosomal region comprises four open reading frames, lpfA to lfpD, and has the same location in the EHEC O113:H21 genome as O island 154 in the prototype EHEC O157:H7 strain, EDL933. In a survey of EHEC of other serotypes, homologues of lpfA(O113) were found in 26 of 28 LEE-negative and 8 of 11 non-O157:H7 LEE-positive EHEC strains. Deletion of the putative major fimbrial subunit gene, lpfA, from EHEC O113:H21 resulted in decreased adherence of this strain to epithelial cells, suggesting that lpf(O113) may function as an adhesin in LEE-negative isolates of EHEC.     

Intimin facilitates colonization by Escherichia coli O157:H7 in adult ruminants

We compared the magnitude and duration of fecal shedding of wild-type Escherichia coli O157:H7 to that of an isogenic intimin mutant in young adult cattle and sheep. In both ruminant species, wild-type E. coli O157:H7 was shed in greater numbers and for a longer duration than was the intimin mutant.     

The Cloned Locus of Enterocyte Effacement from Enterohemorrhagic Escherichia coli O157:H7 Is Unable To Confer the Attaching and Effacing Phenotype upon E. coli K-12

The locus of enterocyte effacement (LEE) pathogenicity island of enterohemorrhagic Escherichia coli (EHEC) O157:H7 possesses the same genes in identical order and orientation as the LEE of enteropathogenic E. coli (EPEC) O127:H6 but is unable to form attaching and effacing (A/E) lesions or to secrete Esp proteins when it is cloned in an E. coli K-12 background. The A/E phenotype could not be restored by trans complementation with a variety of cloned EPEC LEE fragments, suggesting functional and/or regulatory differences between the LEE pathogenicity islands of EPEC O127:H6 and EHEC O157:H7.     

Differential virulence of clinical and bovine-biased enterohemorrhagic Escherichia coli O157:H7 genotypes in piglet and Dutch belted rabbit models

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157) is an important cause of food and waterborne illness in the developed countries. Cattle are a reservoir host of EHEC O157 and a major source of human exposure through contaminated meat products. Shiga toxins (Stxs) are an important pathogenicity trait of EHEC O157. The insertion sites of the Stx-encoding bacteriophages differentiate EHEC O157 isolates into genogroups commonly isolated from cattle but rarely from sick humans (bovine-biased genotypes [BBG]) and those commonly isolated from both cattle and human patients (clinical genotypes [CG]). Since BBG and CG share the cardinal virulence factors of EHEC O157 and are carried by cattle at similar prevalences, the infrequent occurrence of BBG among human disease isolates suggests that they may be less virulent than CG. We compared the virulence potentials of human and bovine isolates of CG and BBG in newborn conventional pig and weaned Dutch Belted rabbit models. CG-challenged piglets experienced severe disease accompanied by early and high mortality compared to BBG-challenged piglets. Similarly, CG-challenged rabbits were likely to develop lesions in kidney and intestine compared with the BBG-challenged rabbits. The CG strains used in this study carried stx2 and produced significantly higher amounts of Stx, whereas the BBG strains carried the stx2c gene variant only. These results suggest that BBG are less virulent than CG and that this difference in virulence potential is associated with the Stx2 subtype(s) carried and/or the amount of Stx produced.     

Immunological characterization of Escherichia coli O157:H7 intimin gamma1.

Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a+ expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a+. Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin types alpha (O serogroups 86, 127, and 142), beta1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), gamma 1 (O serogroups 55, 145, and 157), gamma 2 (O serogroups 111 and 103), and epsilon (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intgamma1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin gamma 1-producing E. coli strains such as E. coli O157:H7.     

Molecular characterization of a serotype O121:H19 clone,a distinct Shiga toxin-producing clone of pathogenic Escherichia coli

Most illnesses caused by Shiga toxin-producing Escherichia coli (STEC) have been attributed to E. coli serotype O157:H7, but non-O157 STEC infections are now increasingly recognized as public health problems worldwide. The O121:H19 serotype is being isolated more frequently from clinical specimens and has been implicated in one waterborne outbreak. We used multilocus virulence gene profiling, a PCR-based assay, to characterize the virulence gene content of 24 isolates of serotype O121:H19 and nonmotile variants. We also performed multilocus enzyme electrophoresis and multilocus sequencing to establish the clonal relatedness of O121 isolates and to elucidate the relationship of O121 to common STEC clones. The 24 isolates were found to represent a single bacterial clone, as there was no allelic variation across 18 enzyme loci among the isolates. The complete nucleotide sequence of the intimin gene differed by four substitutions from that of the epsilon (Int- epsilon ) allele of O103:H2 strain PMK5. The typical O121 virulence gene profile was similar to the profiles of enterohemorrhagic E. coli (EHEC) clones of E. coli: it included a Shiga toxin 2 gene (stx(2)), two genes on the EHEC plasmid (toxB and ehxA), and the gene encoding intimin (eae). Despite the similarities, putative virulence genes distributed on O islands-large chromosomal DNA segments present in the O157:H7 genome-were useful for discriminating among STEC serotypes and the O121:H19 clone had a composite profile that was distinct from the profiles of the other major EHEC clones of pathogenic E. coli. On the basis of sequencing analysis with 13 housekeeping genes, the O121:H19 clone did not fall into any of the four classical EHEC and enteropathogenic E. coli groups but instead was closely related to two eae-negative STEC strains.     

Enterohemorrhagic Escherichia coli O157:H7 requires intimin to colonize the gnotobiotic pig intestine and to adhere to HEp-2 cells.

In a previous study, enterohemorrhagic Escherichia coli (EHEC) O157:H7 with a deletion and insertion in the eaeA gene encoding intimin was used to establish that intimin is required for the organism to attach to and efface microvilli in the piglet intestine (M. S. Donnenberg, S. Tzipori, M. L. McKee, A. D. O'Brien, J. Alroy, and J. B. Kaper, J. Clin. Invest. 92:1418-1424, 1993). However, in the same investigation, a role for intimin in EHEC adherence to HEp-2 cells could not be definitively demonstrated. To analyze the basis for this discrepancy, we constructed an in-frame deletion of eaeA and compared the adherence capacity of this mutant with that of the wild-type strain in vitro and in vivo. We observed a direct correlation between the requisite for intimin in EHEC O157:H7 colonization of the gnotobiotic piglet intestine and adherence of the bacterium to HEp-2 cells. The in vitro-in vivo correlation lends credence to the use of the HEp-2 cell adherence model for further study of the intimin protein.     

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.