ROLE OF BLADDER AND VAGINAL EPITHELIAL CAVEOLAE IN UTIs

女性的尿路感染(UTIs)始于感染微生物附着在阴道和 其次是膀胱上皮细胞。在我们先前的研究中，我们利用原代培养的膀胱上皮细胞(Bec)作为 模型，以确定糖鞘糖脂(GSLS)作为致泌尿系统致病大肠杆菌的附着部位的作用和调节 将这些研究扩展到原代阴道上皮细胞(VEC)培养的新模型。GSLS不是随机发生的 而是被组织成专门的膜域，如小凹，其特征是 富含胆固醇、鞘磷脂、糖脂如神经节苷脂GM1、脂质锚定蛋白和小窝蛋白。 这些结构域涉及多种关键的细胞功能，包括胆固醇的运输， 大分子溶质转运、肿瘤抑制和信号转导。 最近的证据表明，小窝是宿主对附着性尿路致病性E。 在肥大细胞中的大肠杆菌，介导生物体的摄取。我们有初步数据表明小窝蛋白-1存在 在原代培养VEC和BEC单层和自然阴道组织切片中，以及自然阴道上皮细胞 含有GM1，肥大细胞和其他组织中小凹的关键GSL。这项提议的中心假设是 小窝出现在膀胱和阴道上皮中，并参与上皮细胞对附着的初始反应。 泌尿系致病大肠杆菌。我们将追求以下具体目标：(I)最终证明小窝 发生在膀胱和阴道上皮中，我们将从结构和生物化学的角度描述这些结构。 培养的BEC和VEC，包括定义这些细胞中定位于小凹的GSLs；(2)解决以下假设 ，我们将确定小窝蛋白是否与尿路致病大肠杆菌 原代培养的BEC和VEC中的GSLs和/或结合1型甘露糖苷糖蛋白的GSLs 菌毛；(3)研究小窝介导1型菌毛摄取的假说 ，我们将研究这种细菌对培养的原代人BEC的侵袭，检测联合 空泡标志对吞噬细菌的定位及破坏空泡能否预防细菌的研究 摄取；以及(4)使用已建立的膀胱和阴道上皮新器官模型，我们将展示关键发现。 从单层系统，如小凹的存在，细菌入侵，并通过 小窝。这些研究将促进对GSLS和小窝这两个基本细胞成分在 探讨了E。ColiUTI。

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 co- localization 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 ofE. coli UTI.