The aim of the new guidelines was to achieve a high diagnostic accuracy and to reduce the burden for patients and their families. The performance of these guidelines in clinical practice should be evaluated prospectively.

SummaryEnteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide. Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion. Although EPEC was the first E. coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled. During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular. Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.

SummarySubversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections. Both pathogens translocate the trans -membrane receptor protein -translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton. While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir EPEC requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir EHEC is not phosphorylated and utilizes an unidentified linker. Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Ncklike coupling activity following translocation into host cells. A tccP mutant did not affect Tir translocation and focusing but failed to recruit a a a a -actinin, Arp3, N-WASP and actin to the site of bacterial adhesion.When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line. TccP has a similar biological activity on infected human intestinal explants ex vivo . Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro . These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.

Many bacterial pathogens utilize a type III secretion system to deliver multiple effector proteins into host cells. Here we found that the type III effectors, NleE from enteropathogenic E. coli (EPEC) and OspZ from Shigella, blocked translocation of the p65 subunit of the transcription factor, NF-κB, to the host cell nucleus. NF-κB inhibition by NleE was associated with decreased IL-8 expression in EPEC-infected intestinal epithelial cells. Ectopically expressed NleE also blocked nuclear translocation of p65 and c-Rel, but not p50 or STAT1/2. NleE homologues from other attaching and effacing pathogens as well OspZ from Shigella flexneri 6 and Shigella boydii, also inhibited NF-κB activation and p65 nuclear import; however, a truncated form of OspZ from S. flexneri 2a that carries a 36 amino acid deletion at the C-terminus had no inhibitory activity. We determined that the C-termini of NleE and full length OspZ were functionally interchangeable and identified a six amino acid motif, IDSY(M/I)K, that was important for both NleE- and OspZ-mediated inhibition of NF-κB activity. We also established that NleB, encoded directly upstream from NleE, suppressed NF-κB activation. Whereas NleE inhibited both TNFα and IL-1β stimulated p65 nuclear translocation and IκB degradation, NleB inhibited the TNFα pathway only. Neither NleE nor NleB inhibited AP-1 activation, suggesting that the modulatory activity of the effectors was specific for NF-κB signaling. Overall our data show that EPEC and Shigella have evolved similar T3SS-dependent means to manipulate host inflammatory pathways by interfering with the activation of selected host transcriptional regulators.

Enteroaggregative Escherichia coli (EAEC) has been implicated as an agent of pediatric diarrhea in the developing world. We have shown previously that EAEC adheres to HEp-2 cells by virtue of a plasmid-encoded fimbrial adhesin designated aggregative adherence fimbria I (AAF/I), the genes for which have been cloned and sequenced. However, not all EAEC strains express AAF/I. Using TnphoA mutagenesis, we have characterized a novel fimbria (designated AAF/II) which mediates HEp-2 adherence of the human-pathogenic strain 042. AAF/II is 5 nm in diameter and does not bind AAF/I antiserum, as determined by immunogold transmission electron microscopy. TnphoA identified a gene (designated aafA) which bears significant homology to aggA, the fimbrial subunit of AAF/I (25% identity and 47% similarity at the amino acid level). When hyperexpressed and purified by polyhistidine tagging, the AafA protein assembled into 5-nm-diameter filaments which bound anti-AAF/II antiserum. The cloned aafA gene complemented a mutation in the aggA gene to confer fimbrial expression from the AAF/I gene cluster, manifesting phenotypes characteristic of AAF/II but not AAF/I. The aafA mutant did not adhere to human intestinal tissue in culture, suggesting a role for AAF/II in intestinal colonization. By using DNA probes for AAF/I and AAF/II derived from fimbrial biosynthesis genes, we show that AAF/I and AAF/II are each found in only a minority of EAEC strains, suggesting that still more EAEC adhesins exist. Our data suggest that AAF adhesins represent a new family of fimbrial adhesins which mediate aggregative adherence in EAEC.

Summary Curli are adhesive fimbriae of Escherichia coli and Salmonella enterica. Expression of curli (csgA) and cellulose (bcsA) is co-activated by the transcriptional activator CsgD. In this study, we investigated the contribution of curli and cellulose to the adhesive properties of enterohemorragic (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) O127:H6. While single mutations in csgA, csgD, or bcsA in EPEC and EHEC had no dramatic effect on cell adherence, double csgAbcsA mutants were significantly less adherent than the single mutants or wild-type strains to human colonic HT-29 epithelial cells or to cow colon tissue in vitro. Over-expression of csgD (carried on plasmid pCP994) in a csgD mutant, but not in the single csgA or bscA mutants, led to significant increase in adherence and biofilm formation in EPEC and EHEC, suggesting that synchronized over-production of curli and cellulose enhances bacterial adherence. In line with this finding, csgD transcription was activated significantly in the presence of cultured epithelial cells as compared to growth in tissue culture medium. Analysis of the influence of virulence and global regulators in the production of curli in EPEC identified Fis (factor for inversion stimulation) as a, heretofore unrecognized, negative transcriptional regulator of csgA expression. An EPEC E2348/69Δfis produced abundant amounts of curli whereas a double fiscsgD mutant yielded no detectable curli production. Our data suggest that curli and cellulose act in concert to favor host colonization, biofilm formation, and survival in different environments.

Enteroaggregative Escherichia coli (EAEC) forms thick biofilms on the intestinal mucosa. Here, we show that most EAEC strains form a biofilm on glass or plastic surfaces when grown in cell culture medium with high sugar and osmolarity. Biofilm‐forming ability in two prototype EAEC strains required aggregative adherence fimbriae (AAF), although many other EAEC strains that do not express AAF also developed biofilms under these conditions. Ten thousand transposon mutants of EAEC strain 042 were isolated, and 100 were found to be deficient in biofilm formation. Of these, 93 were either deficient in in vitro growth or mapped to genes known to be required for AAF/II expression. Of the seven remaining insertions, five mapped to one of two unsuspected loci. Two insertions involved the E. coli chromosomal fis gene, a DNA‐binding protein that is involved in growth phase‐dependent regulation. Using reverse transcription–polymerase chain reaction (RT–PCR), we determined that the effect of fis was at the level of transcription of the AAF/II activator aggR. Biofilm formation also required the product of the yafK gene, which is predicted to encode a secreted 28 kDa protein. The yafK product is required for transcription of AAF/II‐encoding genes. Our data do not suggest a role for type 1 fimbriae or motility in biofilm formation. EAEC appears to form a novel biofilm, which may be mediated solely by AAF and may reflect its interactions with the intestinal mucosa.

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