Mechanisms of Enteropathogenic E. coli Effects on Polarity and Tight Junctions

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DESCRIPTION (provided by applicant): Enteropathogenic E. coli (EPEC) is an important human bacterial pathogen that infects primarily infants in developing countries causing significant morbidity and mortality from diarrhea. EPEC pathogenesis is dependent on the injection of several bacterial effector molecules into host cells by a type III secretion system. EPEC effectors have a multitude of host cell targets. While recent studies focused on single effector proteins have revealed intriguing biochemical data, they have not shed light on the mechanisms by which downstream intestinal physiology is perturbed. Several EPEC effectors contribute to tight junction (TJ) disruption but the mechanisms and cooperative interactions are not known. TJ formation and maintenance in intestinal epithelial cells is dependent on physical and functional interactions between the Par and Crumbs polarity complexes. EPEC effectors may interfere with this process. The long-term goal of this proposal is to enhance our understanding of how multiple EPEC effectors coordinate to perturb intestinal epithelial function, particularly TJ regulation. Recognition of the interdependent relationships between EPEC effectors underscores the need for novel and comprehensive investigative approaches to define the complex mechanisms underlying EPEC pathogenesis. The objective of this proposal is to elucidate the mechanisms by which specific EPEC effectors cooperate to perturb key host intestinal epithelial physiologic endpoints that underlie pathogenesis. The central hypothesis is that EPEC effectors target, in a coordinated fashion, the Par and Crumbs polarity complexes ultimately disrupting TJ structure and barrier function. In vitro infection and transfection models as well as in vivo models will be used to address the hypothesis. Molecular, cellular, microbiological, and physiological approaches will be coupled with state-of-the-art microscopic techniques to achieve the stated goals. The rationale for the proposed research is that the identification of mechanisms by which pathogenic bacterial proteins hijack host cell regulatory controls to alter physiology will guide the development of novel strategies aimed at preserving host functions. The realization that bacterial effectors cooperate highlights the need for expanded efforts toward holistic approaches that examine the interplay between multiple effectors in executing a common goal. The following Specific Aims will address the hypothesis: 1. Define the mechanisms by which Map, EspG, and EspF cooperate to disrupt Par polarity complex. 2. Determine if Map, EspG and EspF disrupt TJ structure/function by targeting cell polarity complexes. 3. Investigate the interdependence of key EPEC effector molecules on disruption of epithelial polarity complexes and TJs using a multicellular and in vivo model of infection.