ROLE OF BLADDER AND VAGINAL EPITHELIAL CAVEOLAE IN UTIs

膀胱和阴道上皮细胞小窝在尿路感染中的作用

• 批准号: 7257245
• 负责人: Ann E Stapleton
• 金额: $ 16.95万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7257245
• 关键词: Address; Adherence; Antibodies; Bacteria; Binding; Biological Assay; Bladder; Caveolae; Caveolins; Cell membrane; Cell physiology; Cells; Cholesterol; Collaborations; Cytokeratin; Data; Disruption; Electron Microscopy; Epithelial; Epithelial Cells; Epithelium; Escherichia coli; Estrogens; Fractionation; Ganglioside GM1; Genitourinary system; Gentamicins; Globosides; Glycolipids; Glycoproteins; Glycosphingolipids; Human; Immune response; Knowledge; Lipids; Localized; Mediating; Mediation; Membrane; Microscopy; Modeling; Organ Model; Organism; Pathogenesis; Pattern; Process; Regulation; Role; Signal Transduction; Site; Sphingomyelins; Staining method; Stains; Structure; Subcellular Fractions; System; Techniques; Testing; Tissue Engineering; Tissues; Toxin; Tumor Suppression; Urinary tract infection; Uropathogenic E. coli; Vagina; Woman; caveolin 1; mast cell; microorganism; monolayer; prevent; receptor; research study; response; solute; uptake

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.

女性的尿路感染（UTI）开始是感染性微生物附着在阴道上， 随后是膀胱上皮。在我们之前的研究中，我们利用原代膀胱上皮细胞（BEC）培养物作为一种免疫抑制剂。 模型，以确定的作用和调节的鞘糖脂（GSL）作为附着位点的尿路致病性大肠杆菌。大肠杆菌，稍后 将这些研究扩展到原代阴道上皮细胞（VEC）培养的新模型。GSL不是随机发生的 在细胞膜中，而是被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，包括胆固醇的转运， 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。我们有初步数据证明存在小窝蛋白-1 在原代培养VEC和BEC单层和天然阴道组织切片以及天然阴道上皮细胞中 含有GM 1，这是肥大细胞和其他组织中小窝的关键GSL。这一提议的核心假设是， 小窝发生在膀胱和阴道上皮中，参与上皮对附着的初始反应 尿致病性E.杆菌我们将追求以下具体目标：（一）最终证明， 发生在膀胱和阴道上皮，我们将在结构和生物化学上表征这些结构， 培养的BEC和VEC，包括定义这些细胞中定位于小窝的GSL;（2）为了解决以下假设， 小窝含有致病性E.大肠杆菌，我们将确定是否caveolin共定位与 在原代培养的BEC和VEC中的球状系列GSL和/或与结合1型的甘露糖基化糖蛋白 流苏E.（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，我们将研究这种生物体对培养的原代人BEC的侵袭，检测共- 用小窝标记物定位被吞噬的细菌，并研究小窝的破坏是否阻止细菌 摄取;（4）使用已建立的膀胱和阴道上皮新器官模型，我们将证明关键发现 从单层系统，如小窝的存在，细菌入侵，并介导这一过程， 小窝这些研究将促进对GSL和小窝（基本细胞成分）在以下方面作用的认识： 对E. coli UTI。