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ABSTRACTEnteropathogenic Escherichia coli(EPEC) is classified as typical (tEPEC) or atypical (aEPEC) based on the presence or absence of the E. coliadherence factor plasmid (pEAF), respectively. The hallmark of EPEC infection is the formation of the attaching and effacing (A/E) lesions on the gut mucosa. We compared the kinetics of A/E lesion formation induced by aEPEC and tEPEC. The examination of infected HEp-2 cells clearly demonstrated delayed A/E lesion formation by aEPEC in comparison to tEPEC. This delay was associated with the expression of locus of enterocyte effacement (LEE)-encoded virulence factors (i.e., intimin and EspD). Indeed, the insertion of a plasmid containing perABC, a transcriptional regulator of virulence factors involved in A/E formation, into aEPEC strains increased and accelerated the formation of A/E lesions. Interestingly, the enhanced expression and translocation of LEE-encoded proteins, such as those expressed in LEE5 (intimin) and LEE4 (EspD), in aEPEC (perABC) was independent of bacterial adhesion. The secretion kinetics of these two proteins representing LEE5 and LEE4 expression correlated with A/E lesion formation. We conclude that the lack of Per in the regulation network of virulence genes is one of the main factors that delay the establishment of A/E lesions induced by aEPEC strains.

ABSTRACTEspC is a non-locus of enterocyte effacement (LEE)-encoded autotransporter produced by enteropathogenic Escherichia coli(EPEC) that is secreted to the extracellular milieu by a type V secretion system and then translocated into epithelial cells by the type III secretion system. Here, we show that this efficient EspC delivery into the cell leads to a cytopathic effect characterized by cell rounding and cell detachment. Thus, EspC is the main protein involved in epithelial cell cytotoxicity detected during EPEC adhesion and pedestal formation assays. The cell detachment phenotype is triggered by cytoskeletal and focal adhesion disruption. EspC-producing EPEC is able to cleave fodrin, paxillin, and focal adhesion kinase (FAK), but these effects are not observed when cells are infected with an espCisogenic mutant. Recovery of these phenotypes by complementing the mutant with the espCgene but not with the espCgene mutated in the serine protease motif highlights the role of the protease activity of EspC in the cell detachment phenotype. In vitroassays using purified proteins showed that EspC, but not EspC with an S256I substitution [EspCS256I], directly cleaves these cytoskeletal and focal adhesion proteins. Kinetics of protein degradation indicated that EspC-producing EPEC first cleaves fodrin (within the 11th and 9th repetitive units at the Q1219 and D938 residues, respectively), and this event sequentially triggers paxillin degradation, FAK dephosphorylation, and FAK degradation. Thus, cytoskeletal and focal adhesion protein cleavage leads to the cell rounding and cell detachment promoted by EspC-producing EPEC.

ABSTRACTFimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli(EAEC) pathogenesis. To date, four fimbriae have been described for EAEC; aggregative adherence fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II fimbriae. Purified AafA-dscprotein, the major subunit of AAF/II fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dscprotein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II fimbriae, AafA-dscprotein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.

ABSTRACTA hallmark of enteroaggregative Escherichia coli(EAEC) infection is a formation of biofilm, which comprises a mucus layer with immersed bacteria in the intestines of patients. While studying the mucinolytic activity of Pic in an in vivosystem, rat ileal loops, we surprisingly found that EAEC induced hypersecretion of mucus, which was accompanied by an increase in the number of mucus-containing goblet cells. Interestingly, an isogenic picmutant (EAEC Δpic) was unable to cause this mucus hypersecretion. Furthermore, purified Pic was also able to induce intestinal mucus hypersecretion, and this effect was abolished when Pic was heat denatured. Site-directed mutagenesis of the serine protease catalytic residue of Pic showed that, unlike the mucinolytic activity, secretagogue activity did not depend on this catalytic serine protease motif. Other pathogens harboring the picgene, such as Shigella flexneriand uropathogenic E. coli(UPEC), also showed results similar to those for EAEC, and construction of isogenic picmutants of S. flexneriand UPEC confirmed this secretagogue activity. Thus, Pic mucinase is responsible for one of the pathophysiologic features of the diarrhea mediated by EAEC and the mucoid diarrhea induced by S. flexneri.

ABSTRACTAt least five proteins are secreted extracellularly by enteropathogenic Escherichia coli(EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the espCgene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E. coli. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E. colieliminated EspC enterotoxin activity. Like the EAF plasmid, the espCpathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.

ABSTRACTThe plasticity region of Helicobacter pyloristrain J99 is a large chromosomal segment containing 33 strain-specific open reading frames (ORFs) with characteristics of a pathogenicity island. To study the diversity of the plasticity region, 22 probes corresponding to 20 ORFs inside the plasticity region and two ORFs on its boundaries were hybridized to genomic DNA isolated from clinical strains of H. pylorifrom patients with gastritis or gastric adenocarcinoma. Highly variable hybridization patterns were observed. The majority of the clinical strains presented a hybridization profile similar to that of J99; thus, these ORFs are not J99 strain specific. No association was found between a particular hybridization pattern and the clinical origin of the strain. Nevertheless, two single ORFs (JHP940 and JHP947) were more likely to be found in gastric cancer strains. They may be new pathogenicity markers. An in vitro expression study of these ORFs was also performed for the J99 strain, under different conditions. Thirteen ORFs were consistently expressed, six were consistently shut off, and three were expressed differentially. Most of the constitutionally expressed genes were located on the 3′ part of the plasticity region. Our results show that the plasticity region, rather than being considered a pathogenicity island per se, should be considered a genomic island, which represents a large fragment of foreign DNA integrated into the genome and not necessarily implicated in the pathogenic capacity of the strain.

ABSTRACTEnteroaggregative Escherichia coli(EAEC) strains have been shown to adhere to human intestinal tissue in an in vitro organ culture (IVOC) model, and certain strains manifest mucosal toxicity. We have recently described the EAEC plasmid-encoded toxin (Pet), a member of a specific serine protease subclass of the autotransporter proteins. When injected into rat ileal loops, Pet both elicited fluid accumulation and had cytotoxic effects on the mucosa. Furthermore, the Pet protein caused rises in short circuit current from rat jejunal tissue mounted in a Ussing chamber and rounding of intestinal epithelial cells in culture. We therefore hypothesized that the mucosal pathology induced by EAEC strains in the IVOC model was related to expression of the Pet protein. Here, we have examined the effects of EAEC strain 042 and its isogenic petmutant in the IVOC model. 042-infected colonic explants exhibited dilation of crypt openings, increased cell rounding, development of prominent intercrypt crevices, and absence of apical mucus plugs. Colonic tissue incubated with the petmutant exhibited significantly fewer mucosal abnormalities both subjectively and as quantitated morphometrically by measurement of crypt aperture diameter. Mucosal effects were restored upon complementation of the petmutation intrans. Interestingly, we found that the ability of 042 to damage T84 cells was not dependent upon Pet. The data suggest that the Pet toxin is active on the human intestinal mucosa but that EAEC may have other mechanisms of eliciting mucosal damage.