ROLE OF BLADDER AND VAGINAL EPITHELIAL CAVEOLAE IN UTIs

描述（由申请人提供）：女性尿路感染（uti）始于感染微生物附着于阴道和随后的膀胱上皮。在我们之前的研究中，我们利用原代膀胱上皮细胞（BEC）培养作为模型来定义鞘糖脂（GSLs）作为尿路致病菌大肠杆菌附着位点的作用和调控，后来将这些研究扩展到原代阴道上皮细胞（VEC）培养的新模型。GSLs不是随机出现在细胞膜上，而是被组织成特殊的膜结构域，如小窝，其特征是胆固醇、鞘磷脂、糖脂如神经节苷脂GM1、脂质锚定蛋白和小窝蛋白富集。这些结构域涉及多种关键的细胞功能，包括胆固醇转运、大分子溶质转运、肿瘤抑制和信号转导。

DESCRIPTION (provided by applicant): Urinary tract infections (UTIs) in women begin with the attachment of infecting microorganisms to vaginal and subsequently to bladder epithelium. In our prior studies, we utilized primary bladder epithelial cell (BEC) cultures as a model to define the role and regulation of glycosphingolipids (GSLs) as attachment sites for uropathogenic E. coli, later extending these studies to a new model of primary vaginal epithelial cell (VEC) cultures. GSLs do not occur randomly in cell membranes, but instead are organized into specialized membrane domains such as caveolae, characterized by enrichment in cholesterol, sphingomyelin, glycolipids such as ganglioside GM1, lipid-anchored proteins, and caveolin. These domains are involved in a wide variety of key cellular functions, including transport of cholesterol, macromolecular solute transport, tumor suppression, and signal transduction. Recent evidence demonstrates that caveolae are key molecules in the initial host response to attaching uropathogenic E. coli in mast cells, mediating the uptake of organisms. We have preliminary data demonstrating that caveolin-1 is present in primary cultured VEC and BEC monolayers and native vaginal tissue sections, and that native vaginal epithelial cells contain GM1, a key GSL of caveolae in mast cells and other tissues. The central hypothesis of this proposal is that caveolae occur in bladder and vaginal epithelium and participate in the initial epithelial responses to attachment of uropathogenic E. coli. We will pursue the following specific aims: (I) To conclusively demonstrate that caveolae occur in bladder and vaginal epithelium, we will structurally and biochemically characterize these structures in primary cultured BEC and VEC, including defining GSLs localized to caveolae in these cells; (2) To address the hypothesis that caveolae contain key receptor molecules for uropathogenic E. coli, we will determine if caveolin co-localizes with the globoseries GSLs in primary cultured BEC and VEC and/or with mannosylated glycoproteins that bind Type 1 fimbriated E. coli; (3) To investigate the hypothesis that caveolae mediate uptake of Type 1 fimbriated E. coli into urogenital epithelium, we will investigate invasion of cultured primary human BEC by this organism, testing for colocalization of engulfed bacteria with caveolar markers and investigating if disruption of caveolae prevents bacterial uptake; and (4) using established neo-organ models of bladder and vaginal epithelium, we will demonstrate key findings from the monolayer systems, such as the presence of caveolae, bacterial invasion, and mediation of this process by caveolae. These studies will advance knowledge of the role of GSLs and caveolae, fundamental cellular components, in the pathogenesis of E. coli UTI.