Dataset Information

Translocated intimin receptors (Tir) of Shiga-toxigenic Escherichia coli isolates belonging to serogroups O26, O111, and O157 react with sera from patients with hemolytic-uremic syndrome and exhibit marked sequence heterogeneity.

ABSTRACT: The capacity to form attaching and effacing (A/E) lesions on the surfaces of enterocytes is an important virulence trait of several enteric pathogens, including enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC). Formation of such lesions depends upon an interaction between a bacterial outer membrane protein (intimin) and a bacterially encoded receptor protein (Tir) which is exported from the bacterium and translocated into the host cell membrane. Intimin, Tir, and several other proteins necessary for generation of A/E lesions are encoded on a chromosomal pathogenicity island termed the locus for enterocyte effacement (LEE). Reports of sequence heterogeneity and antigenic variation in the region of intimin believed to be responsible for receptor binding raise the possibility that the receptor itself is also heterogeneous. We have examined this by cloning and sequencing tir genes from three different STEC strains belonging to serogroups O26, O111, and O157. The deduced amino acid sequences for the Tir homologues from these strains varied markedly, exhibiting only 65.4, 80.2, and 56.7% identity, respectively, to that recently reported for EPEC Tir. STEC Tir is also highly immunogenic in humans. Western blots of E. coli DH5alpha expressing the various STEC tir genes cloned in pBluescript [but not E. coli DH5alpha(pBluescript)] reacted strongly with convalescent sera from patients with hemolytic-uremic syndrome (HUS) caused by known LEE-positive STEC. Moreover, no reaction was seen when the various clone lysates were probed with serum from a patient with HUS caused by a LEE-negative STEC or with serum from a healthy individual. Covariation of exposed epitopes on both intimin and Tir may be a means whereby STEC avoid host immune responses without compromising adhesin-receptor interaction.

SUBMITTER: Paton AW

PROVIDER: S-EPMC108704 | biostudies-literature | 1998 Nov

REPOSITORIES: biostudies-literature

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Translocated intimin receptors (Tir) of Shiga-toxigenic Escherichia coli isolates belonging to serogroups O26, O111, and O157 react with sera from patients with hemolytic-uremic syndrome and exhibit marked sequence heterogeneity.

Paton A W AW Manning P A PA Woodrow M C MC Paton J C JC

Infection and immunity 19981101 11

The capacity to form attaching and effacing (A/E) lesions on the surfaces of enterocytes is an important virulence trait of several enteric pathogens, including enteropathogenic Escherichia coli (EPEC) and Shiga-toxigenic E. coli (STEC). Formation of such lesions depends upon an interaction between a bacterial outer membrane protein (intimin) and a bacterially encoded receptor protein (Tir) which is exported from the bacterium and translocated into the host cell membrane. Intimin, Tir, and sever ...[more]

PMID: 9784578

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Project description:Western blot analysis was used to assess the reactivity of convalescent-phase sera from patients who were associated with an outbreak of hemolytic-uremic syndrome (HUS) caused by fermented sausage contaminated with Shiga toxin-producing Escherichia coli (STEC). The predominant STEC isolated from HUS patients belonged to serotype O111:H-, and reactivity to O111:H- whole-cell lysates, treated or untreated with proteinase K, was examined. As expected, all five serum samples demonstrated a marked anti-lipopolysaccharide response, but several protein bands were also immunoreactive, particularly one with an apparent size of 94 kDa. One convalescent-phase serum sample was subsequently used to screen an O111:H- cosmid bank and 2 of 900 cosmid clones were found to be positive, both of which contained a similar DNA insert. Western blot analysis of one of these clones identified three major immunoreactive protein bands of approximately 94, 70, and 50 kDa. An immune response to the three proteins was detectable with all five convalescent-phase serum samples but not with healthy human serum. Immunoreactive 94- and 50-kDa species were produced by a deletion derivative of the cosmid containing a 7-kb STEC DNA insert. Sequence analysis of this region indicated that it is part of the locus for enterocyte effacement, including the eaeA gene which encodes intimin. The deduced amino acid sequence of the O111:H- intimin was 88.6% identical to intimin from O157:H7 STEC, and the most divergent region was the 200 residues at the carboxyl terminus, which were only 75% identical. Such variation may be antigenically significant as serum from a HUS patient infected only with the O111:H- STEC reacted with intimin from an enteropathogenic E. coli O111 strain, as well as several other eaeA-positive STEC isolates, but not with an eaeA-positive STEC belonging to serotype O157:H-. Sera from two of the other HUS patients also failed to react with intimin from this latter strain. However, intimin from O157:H- STEC did react with serum from a patient infected with both O111:H- and O157:H- STEC.

Project description:BackgroundShiga-toxin producing Escherichia coli (STEC) have emerged as important foodborne pathogens, among which seven serogroups (O26, O45, O103, O111, O121, O145, O157) are most frequently implicated in human infection. The aim was to determine if a light scattering sensor can be used to rapidly identify the colonies of STEC serogroups on selective agar plates.Methodology/principal findingsInitially, a total of 37 STEC strains representing seven serovars were grown on four different selective agar media, including sorbitol MacConkey (SMAC), Rainbow Agar O157, BBL CHROMagarO157, and R&F E. coli O157:H7, as well as nonselective Brain Heart Infusion agar. The colonies were scanned by an automated light scattering sensor, known as BARDOT (BActerial Rapid Detection using Optical scattering Technology), to acquire scatter patterns of STEC serogroups, and the scatter patterns were analyzed using an image classifier. Among all of the selective media tested, both SMAC and Rainbow provided the best differentiation results allowing multi-class classification of all serovars with an average accuracy of more than 90% after 10-12 h of growth, even though the colony appearance was indistinguishable at that early stage of growth. SMAC was chosen for exhaustive scatter image library development, and 36 additional strains of O157:H7 and 11 non-O157 serovars were examined, with each serogroup producing unique differential scatter patterns. Colony scatter images were also tested with samples derived from pure and mixed cultures, as well as experimentally inoculated food samples. BARDOT accurately detected O157 and O26 serovars from a mixed culture and also from inoculated lettuce and ground beef (10-h broth enrichment +12-h on-plate incubation) in the presence of natural background microbiota in less than 24 h.ConclusionsBARDOT could potentially be used as a screening tool during isolation of the most important STEC serovars on selective agar plates from food samples in less than 24 h.

Project description:Cattle are a reservoir for Shiga toxin-producing Escherichia coli (STEC), zoonotic pathogens that cause serious clinical disease. Scotland has a higher incidence of STEC infection in the human population than the European average. The aim of this study was to investigate the prevalence and epidemiology of non-O157 serogroups O26, O103, O111, and O145 and Shiga toxin gene carriage in Scottish cattle. Fecal samples (n = 2783) were collected from 110 herds in 2014 and 2015 and screened by real-time PCR. Herd-level prevalence (95% confidence interval [CI]) for O103, O26, and O145 was estimated as 0.71 (0.62, 0.79), 0.43 (0.34, 0.52), and 0.23 (0.16, 0.32), respectively. Only two herds were positive for O111. Shiga toxin prevalence was high in both herds and pats, particularly for stx2 (herd level: 0.99; 95% CI: 0.94, 1.0). O26 bacterial strains were isolated from 36 herds on culture. Fifteen herds yielded O26 stx-positive isolates that additionally harbored the intimin gene; six of these herds shed highly pathogenic stx2-positive strains. Multiple serogroups were detected in herds and pats, with only 25 herds negative for all serogroups. Despite overlap in detection, regional and seasonal effects were observed. Higher herd prevalence for O26, O103, and stx1 occurred in the South West, and this region was significant for stx2 at the pat level (P = 0.015). Significant seasonal variation was observed for O145 prevalence, with the highest prevalence in autumn (P = 0.032). Negative herds were associated with Central Scotland and winter. Herds positive for all serogroups were associated with autumn and larger herd size and were not housed at sampling.IMPORTANCE Cattle are reservoirs for Shiga toxin-producing Escherichia coli (STEC), bacteria shed in animal feces. Humans are infected through consumption of contaminated food or water and by direct contact, resulting in serious disease and kidney failure in the most vulnerable. The contribution of non-O157 serogroups to STEC illness was underestimated for many years due to the lack of specific tests. Recently, non-O157 human cases have increased, with O26 STEC of particular note. It is therefore vital to investigate the level and composition of non-O157 in the cattle reservoir and to compare them historically and by the clinical situation. In this study, we found cattle prevalence high for toxin, as well as for O103 and O26 serogroups. Pathogenic O26 STEC were isolated from 14% of study herds, with toxin subtypes similar to those seen in Scottish clinical cases. This study highlights the current risk to public health from non-O157 STEC in Scottish cattle.

Project description:Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

Dataset Information

Translocated intimin receptors (Tir) of Shiga-toxigenic Escherichia coli isolates belonging to serogroups O26, O111, and O157 react with sera from patients with hemolytic-uremic syndrome and exhibit marked sequence heterogeneity.

Publications

Translocated intimin receptors (Tir) of Shiga-toxigenic Escherichia coli isolates belonging to serogroups O26, O111, and O157 react with sera from patients with hemolytic-uremic syndrome and exhibit marked sequence heterogeneity.

Similar Datasets

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