ROLE OF BLADDER AND VAGINAL EPITHELIAL CAVEOLAE IN UTIs

描述（由申请方提供）：女性尿路感染（UTI）开始是感染微生物附着于阴道，随后附着于膀胱上皮。在我们之前的研究中，我们利用原代膀胱上皮细胞（BEC）培养物作为模型来确定鞘糖脂（GSL）作为尿路致病性大肠杆菌附着位点的作用和调节。大肠杆菌，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。GSL不是随机出现在细胞膜中，而是组织成专门的膜结构域，如小窝，其特征在于富含胆固醇、鞘磷脂、糖脂如神经节苷脂GM 1、脂质锚定蛋白和小窝蛋白。这些结构域参与多种关键细胞功能，包括胆固醇转运、大分子溶质转运、肿瘤抑制和信号转导。 最近的证据表明，窖是宿主对尿路致病性大肠杆菌的初始反应的关键分子。肥大细胞中的大肠杆菌，介导生物体的摄取。我们有初步的数据表明，小窝蛋白-1存在于原代培养的VEC和BEC单层和天然阴道组织切片，天然阴道上皮细胞含有GM 1，肥大细胞和其他组织中小窝的关键GSL。该假说的中心假设是，小窝发生在膀胱和阴道上皮中，并参与上皮对致尿病性大肠杆菌附着的初始反应。杆菌我们将追求以下具体目标：（1）为了最终证明小窝发生在膀胱和阴道上皮中，我们将在原代培养的BEC和VEC中对这些结构进行结构和生化表征，包括定义这些细胞中定位于小窝的GSL;（2）为了解决小窝包含尿路致病性E.大肠杆菌中，我们将确定窖蛋白是否与原代培养的BEC和VEC中的球状系列GSL和/或与结合1型菌毛大肠杆菌的甘露糖基化糖蛋白共定位。（3）探讨Caveolae介导1型菌毛大肠杆菌摄取的假说。大肠杆菌进入泌尿生殖道上皮，我们将研究这种微生物对培养的原代人BEC的侵袭，测试吞噬细菌与小窝标记物的共定位，并研究小窝的破坏是否阻止细菌摄取;以及（4）使用已建立的膀胱和阴道上皮的新器官模型，我们将证明来自单层系统的关键发现，例如小窝的存在，细菌侵入，并通过小凹来调节这一过程。这些研究将进一步了解GSL和小窝，基本的细胞成分，在发病机制中的作用。coli UTI。

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.