Enterohemorrhagic E. coli (EHEC) is a food borne pathogen that causes a range of symptoms including non- bloody diarrhea and hemorrhagic colitis. While the most serious manifestations of EHEC infection are attributed to Shiga-like toxins, other EHEC virulence factors are encoded in a pathogenicity island, the locus of enterocyte effacement (LEE). LEE-encoded proteins include structural components of a type III secretion system (TTSS) and effector proteins delivered into host cells by TTSS. The effector protein Tir (translocated intimin receptor), following translocation into host cells, inserts into the cell membrane and engages the bacterial surface adhesin, intimin. In concert with the non-LEE secreted protein UEspF, Tir recruits the host protein N-WASP, which nucleates actin polymerization resulting in a pedestal-like structure below attached bacteria. Deletion of UespF, but not the LEE-encoded homolog espF, abrogates EHEC pedestal formation. EPEC and EHEC disrupt tight junctions (TJ). In EPEC, this is mediated partly by the LEE-encoded protein EspF, yet deletion of espFEHEC, which displays sequence similarity to espFEPEC, has no effect on EHEC-induced barrier function alteration. Deletion of UespF, however, attenuates the effects of EHEC on TJs, suggesting a direct role for UEspF in TJ disruption or that UEspF-mediated pedestal formation promotes barrier disruption. EspFEHEC and UEspF contain proline-rich domains (PRDs) allowing interaction with host proteins. We have identified SNX9 as a binding partner of EspF and UEspF via PRDs;these proteins also house the 14-3-3 consensus binding sequence. We will investigate the role of these interactions in the disruption of intestinal barrier function. Both pathogens also induce myosin light chain (MLC) phosphorylation, which contributes to barrier disruption. The EHEC effector responsible for increasing MLC phosphorylation has not been identified. The overall hypothesis of this proposal, therefore, is that the roles of UEspF in pedestal formation and TJ disruption are linked and that the full effect of EHEC on host epithelial barrier function requires the concerted action of several effectors.
The Specific Aims that will address this hypothesis are: 1. To define the role of EHEC-induced pedestal formation in intestinal epithelial tight junction (TJ) disruption using in vitro and in vivo models. 2. To characterize the host cell interaction partners of EspF/UEspF and define their roles in EHEC-induced barrier function alteration. 3. To identify the signaling pathway(s) responsible for EHEC-induced phosphorylation of myosin light chain (MLC) and the effector(s) involved.
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