Surface-layer protein A (SlpA) is a major contributor to host-cell adherence of Clostridium difficile.

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.

Enteropathogenic E. coli non-LEE encoded effectors NleH1 and NleH2 attenuate NF-κB activation.

Enteric bacterial pathogens have evolved sophisticated strategies to evade host immune defences. Some pathogens deliver anti-inflammatory effector molecules into the host cell cytoplasm via a type III secretion system (T3SS). Enteropathogenic Escherichia coli (EPEC) inhibits inflammation by an undefined, T3SS-dependent mechanism. Two proteins encoded outside of the EPEC locus of enterocyte effacement (LEE) pathogenicity island, non-LEE-encoded effector H1 (NleH1) and H2 (NleH2), display sequence similarity to Shigella flexneri OspG, which inhibits activation of the pro-inflammatory transcription factor NF-κB. We hypothesized that the anti-inflammatory effects of EPEC were mediated by NleH1 and NleH2. In this study, we examined the effect of NleH1/H2 on the NF-κB pathway. We show that NleH1/H2 are secreted via the T3SS and that transfection of cells with plasmids harbouring nleH1 or nleH2 decreased IKK-ß-induced NF-κB activity and attenuated TNF-α-induced degradation of phospho-IκBα by preventing ubiquitination. Serum KC levels were higher in mice infected with ΔnleH1H2 than those infected with WT EPEC, indicating that NleH1/H2 dampen pro-inflammatory cytokine expression. ΔnleH1H2 was cleared more rapidly than WT EPEC while complementation of ΔnleH1H2 with either NleH1 or NleH2 prolonged colonization. Together, these data show that NleH1 and NleH2 function to dampen host inflammation and facilitate EPEC colonization during pathogenesis.

Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner.

Shiga toxin (Stx) is implicated in the development of hemorrhagic colitis and hemolytic-uremic syndrome, but early symptoms of enterohemorrhagic Escherichia coli (EHEC) infection such as nonbloody diarrhea may be Stx independent. In this study, we defined the effects of EHEC, in the absence of Stx, on the intestinal epithelium using a murine model. EHEC colonization of intestines from two groups of antibiotic-free and streptomycin-treated C57Bl/6J mice were characterized and compared. EHEC colonized the cecum and colon more efficiently than the ileum in both groups; however, greater amounts of tissue-associated EHEC were detected in streptomycin-pretreated mice. Imaging of intestinal tissues of mice infected with bioluminescent EHEC further confirmed tight association of the bacteria with the cecum and colon. Greater numbers of EHEC were also cultured from stool samples obtained from streptomycin-pretreated mice, as compared with those that received no antibiotics. Transmission electron microscopy shows that EHEC infection leads to microvillous effacement of mouse colonocytes. Hematoxylin and eosin staining of the colonic tissues of infected mice revealed a slight increase in the number of lamina propria polymorphonuclear leukocytes. Transmucosal electrical resistance, a measure of epithelial barrier function, was reduced in the colonic tissues of infected animals. Increased mucosal permeability to 4- kDa FITC-dextran was also observed in the colonic tissues of infected mice. Immunofluorescence microscopy showed that EHEC infection resulted in redistribution of the tight junction (TJ) proteins occludin and claudin-3 and increased the expression of claudin-2, whereas ZO-1 localization remained unaltered. Quantitative real-time PCR showed that EHEC altered mRNA transcription of OCLN, CLDN2, and CLDN3. Most notably, claudin-2 expression was significantly increased and correlated with increased intestinal permeability. Our data indicate that C57Bl/6J mice serve as an in vivo model to study the physiological effects of EHEC infection on the intestinal epithelium and suggest that altered transcription of TJ proteins has a role in the increase in intestinal permeability.

Enteropathogenic E. coli-induced barrier function alteration is not a consequence of host cell apoptosis.

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that perturbs intestinal epithelial function. Many of the alterations in the host cells are mediated by effector molecules that are secreted directly into epithelial cells by the EPEC type III secretion system. The secreted effector molecule EspF plays a key role in redistributing tight junction proteins and altering epithelial barrier function. EspF has also been shown to localize to mitochondria and trigger membrane depolarization and eventual host cell death. The relationship, if any, between EspF-induced host cell death and epithelial barrier disruption is presently not known. Site-directed mutation of leucine 16 (L16E) of EspF impairs both mitochondrial localization and consequent host cell death. Although the mutation lies within a region critical for type III secretion, EspF(L16E) is secreted efficiently from EPEC. Despite its inability to promote cell death, EspF(L16E) was not impaired for tight junction alteration or barrier disruption. Consistent with this, the pan-caspase inhibitor Q-VD-OPH, despite reducing EPEC-induced host cell death, had no effect on infection-mediated barrier function alteration. Thus EPEC alters the epithelial barrier independent of its ability to induce host cell death.

Enteropathogenic Escherichia coli-induced epidermal growth factor receptor activation contributes to physiological alterations in intestinal epithelial cells.

The diarrheagenic pathogen enteropathogenic Escherichia coli (EPEC) is responsible for significant infant mortality and morbidity, particularly in developing countries. EPEC pathogenesis relies on a type III secretion system-mediated transfer of virulence effectors into host cells. EPEC modulates host cell survival and inflammation, although the proximal signaling pathways have not been well defined. We therefore examined the effect of EPEC on the epidermal growth factor receptor (EGFR), a known upstream activator of both the prosurvival phosphoinositide 3-kinase/Akt and proinflammatory mitogen-activated protein (MAP) kinase pathways. EPEC induced the autophosphorylation of EGFR in intestinal epithelial cells within 15 min postinfection, with maximal phosphorylation being observed at 4 h. Filter-sterilized supernatants of EPEC cultures also stimulated EGFR phosphorylation, suggesting that a secreted component(s) contributes to this activity. EPEC-induced EGFR phosphorylation was blocked by the pharmacological inhibitor tyrphostin AG1478, as well as by EGFR-neutralizing antibodies. Inhibition of EGFR phosphorylation by AG1478 had no effect on bacterial adherence, actin recruitment to sites of attachment, or EPEC-induced epithelial barrier function alteration. EPEC-mediated Akt phosphorylation, however, was inhibited by both AG1478 and EGFR-neutralizing antibodies. Correspondingly, inhibition of EGFR activation increased the apoptosis/necrosis of infected epithelial cells. Inhibition of EGFR phosphorylation also curtailed EPEC-induced ERK1/2 (MAP kinase) phosphorylation and, correspondingly, the production of the proinflammatory cytokine interleukin-8 by infected epithelial cells. Our studies suggest that EGFR is a key proximal signaling molecule during EPEC pathogenesis.