Dissecting mechanisms of actin pedestal formation induced by Enterohemorrhagic E. coli.

About This Grant

SUMMARY Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that breaches the intestinal epithelium and causes outbreaks of bloody diarrhea and hemolytic uremic syndrome. Despite the threat EHEC poses to public health, several gaps remain in our understanding of its unique infectious mechanism. EHEC’s native target is the intestinal epithelium, which primarily consists of polarized enterocytes that display actin-based protrusions known as microvilli on their apical/luminal surface. During infection, EHEC destroys microvilli and attaches firmly to the apical host cell surface. Subsequently, EHEC reorganizes the host cytoskeleton to form dynamic actin- rich structures known as “pedestals,” which are suggested to aid in cell-to-cell spread, enhancing its colonization of the intestine. Notably, EHEC remains extracellular during infection and exploits host cytoplasmic proteins by secreting bacterial effector proteins into the host. The current model for EHEC pathogenesis requires two bacterial secreted effectors, Translocated intimin receptor (Tir) and E. coli secreted protein F in prophage U (EspFU), for pedestal formation. Once translocated into the host cytoplasm, Tir inserts into the plasma membrane through an undefined mechanism and binds to intimin, an EHEC surface protein, via its extracellular domain to initiate bacterial attachment. EspFU then drives actin pedestal assembly by activating Arp2/3, an actin nucleating complex. Actin assembly in turn promotes pedestal motility for efficient bacterial cell-to-cell spread. Despite EspFU being the major driver of actin pedestal assembly and motility, an EHEC strain lacking EspFU (EHECDEspFU) still forms pedestals and colonizes the intestine, suggesting that other pathways, independent of EspFu, remain to be discovered. Interestingly, Tir and EspFU do not bind directly to each other, rather they sequester the host protein Insulin Receptor Tyrosine Kinase Substrate (IRTKS) to form a Tir-IRTKS-EspFU complex. In addition to its scaffolding function, IRTKS has been implicated in outward membrane curvature via its Inverse BAR (I-BAR) domain and more recently in the actin assembly of microvilli via its actin binding Wiskott- Aldrich homology 2 (WH2) domain. How the I-BAR and/or WH2 domains contribute to EHEC infection remains unexplored. My preliminary data shows that overexpression of IRTKS leads to increased bacterial attachment and that the loss of IRTKS results in decreased levels of Tir beneath adherent bacteria. These results suggests that IRTKS contributes to bacterial attachment and to the enrichment of Tir in the apical plasma membrane. Based on published and preliminary data, I hypothesize the I-BAR and WH2 domains of IRTKS promote Tir- mediated EHEC attachment and subsequent actin pedestal assembly, respectively. This project will take advantage of state-of-the-art microscopy and biochemical methods to test this hypothesis by: (Aim 1) defining how IRTKS promotes bacterial attachment, and (Aim 2) determining if IRTKS contributes to actin pedestal assembly and motility independent of EspFU. This work will allow us to build a comprehensive mechanistic model of EHEC pathogenesis and develop our understanding of EHEC’s infectious life cycle.