Uropathogenic E.coli (UPEC) account for over 80% of urinary tract infections (UTIs). It is now known invasion of the bladder epithelial cells (BECs) is a critical initiating step in UTIs. We have demonstrated that UPEC invasion is localized to distinct cellular entities in the BEG membrane loosely- termed caveolae. These caveolae are comprised of lateral assemblies of cholesterol, lipids and select proteins such as caveolin-1. We hypothesized that the entry of UPEC into BECs is a highly dynamic event and therefore the host-cell proteins that mediate bacterial entry could be proteins that are selectively recruited to caveolae or become activated within these microdomains following exposure to UPEC. Our initial studies have revealed that two proteins, flotillin-1 and annexin-ll, were recruited to caveolae whereas the third, caveolin-1, became tyrosine-phosphorylated following exposure to UPEC. Another finding emerging from proteome analysis of BECs is that a significant proportion of the proteins identified as caveolar components are known to be regulated by cAMP, a major second messenger implicated in regulating luminal surface area of the bladder by triggering endo- and exocytosis of BEG vesicles which serve as repositories of luminal membranes. This finding, revealed possible parallels between caveolae mediated bacterial entry nto BECs and regular endocytosis of luminal membrane by BECs following voiding of urine. The goal of this research proposal is to extend and expand this line of investigation. Therefore, we propose to: ) Elucidate the mechanism by which flotillin-1, annexin-ll, and caveolin-1 contribute to bacterial entry into 3ECs. (2) Use proteome mining approaches to extend the identification of caveolar components essential for UPEC invasion of BECs.(3) Investigate the regulatory role of cAMP on UPEC invasion of BECs and the mpact of modulators of intracellular cAMP in conferring protection against experimental UTIs. We believe that a systematic approach to the identification of caveolar determinants of bacterial entry will provide a comprehensive picture of the dynamic molecular interactions occurring in the invasion process and also yield candidate proteins that could potentially serve as targets for drug therapy.
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