Urinary Tract Infections (UTIs) are one of the most common bacterial infections in man, and by far the single most common causative agent of UTIs is Escherichia coli. A critical aspect of bladder infection is bacterial invasion of the bladder epithelium. Recently, we discovered that invasion of human bladder epithelium and subsequent bacterial transcytosis was critically mediated by distinct plasmalemmal cellular entities called caveolae. Type 1 fimbriated uropathogenic E.coli were discovered to invade bladder cells by binding components of cavolae. E.coli invading via caveolae are encased in caveolar membranes and transcytozed without loss of viability. These observations point to caveolae as important portals for entry and infection. Very little is currently known of the caveolar structure within bladder cells, and how it is co-opted by pathogens for entry. It is also not known what keeps E.coli encased in caveolar proteins from fusing with lysosomes following entry. Determining the protein composition of caveolae in bladder epithelial cells before and after bacterial activation could derive valuable information regarding these issues. Here we propose to: (i) Use proteome mining approaches to identify protein composition of caveolae found in human bladder epithelial cells before and after interaction with E.coli. Our focus will be on molecules functioning as (a), receptors (b), signaling molecules and (c), mediators of cytoskeletal rearrangement and trafficking. (ii), Deduce the functions of selected caveolar proteins by specifically suppressing gene expression and examining its effects on the binding, invasion and/or intracellular trafficking activities of the E.coli. These proposed studies involve the utilization of exploratory technology (proteomics), which will complement other NIH funded research currently being undertaken. ? ?
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