ROLE OF BLADDER AND VAGINAL EPITHELIAL CAVEOLAE IN UTIs

描述(由申请人提供)：女性的尿路感染(UTIs)始于感染微生物附着在阴道上，然后附着到膀胱上皮。在我们以前的研究中，我们利用原代膀胱上皮细胞(BEC)培养作为模型来确定糖鞘糖脂(GSLS)作为泌尿系致病大肠杆菌的附着部位的作用和调节，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。这些结构域参与多种关键的细胞功能，包括胆固醇运输、大分子溶质运输、肿瘤抑制和信号转导。 最近的证据表明，小窝是最初宿主对肥大细胞中附着致尿路病原性大肠杆菌的反应的关键分子，介导了生物体的摄取。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单层和自然阴道组织切片中，天然阴道上皮细胞含有GM1，这是肥大细胞和其他组织中小窝的关键GSL。这一建议的中心假设是，凹陷发生在膀胱和阴道上皮中，并参与了上皮细胞对致尿系致病性大肠杆菌附着的初始反应。我们将追求以下具体目标：(I)为了最终证明小凹存在于膀胱和阴道上皮，我们将在原代培养的BEC和VEC中对这些结构进行结构和生物化学表征，包括定义定位于这些细胞中的小凹的GSLS；(2)为了解决小窝含有致尿系大肠杆菌关键受体分子的假设，我们将确定小窝是否与原代培养的BEC和VEC中的球状GSLS和/或与结合1型菌毛的甘露糖化糖蛋白共定位；(3)为了研究小窝介导1型菌毛大肠杆菌吸收进入泌尿生殖道上皮的假设，我们将研究这种微生物对培养的原代人BEC的入侵，测试被吞噬的细菌与小窝标记的共存，并调查小窝的破坏是否阻止细菌的吸收；以及(4)使用已建立的膀胱和阴道上皮的新器官模型，我们将演示单层系统中的关键发现，例如小窝的存在、细菌的入侵以及小窝对这一过程的调节。这些研究将促进对GSLS和小窝这两种基本细胞成分在大肠杆菌UTI发病机制中的作用的了解。

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.