Caveolae Mediated Bacterial Uptake in the Urinary Tract

描述(申请人提供)：泌尿系致病性大肠埃希菌(UPEC)占尿路感染(UTIs)的80%以上。目前已知，膀胱上皮细胞(BECs)的侵袭是尿路感染发生的关键步骤。我们已经证明，UPEC的侵袭局限于BEG膜上不同的细胞实体，宽泛地称为小凹。这些小窝由胆固醇、脂类和精选蛋白质组成，如小窝蛋白-1。我们假设UPEC进入BECs是一个高度动态的事件，因此介导细菌进入的宿主细胞蛋白可能是选择性地招募到小窝中的蛋白质，或者在接触UPEC后在这些微域中被激活。我们的初步研究表明，两种蛋白质，Flotillin-1和Annexin-11，被招募到小窝中，而第三种蛋白质，小窝蛋白-1，在暴露于UPEC后发生酪氨酸磷酸化。从对BEC的蛋白质组分析中发现的另一个发现是，已知相当大比例的空泡成分受cAMP调节，cAMP是一种主要的第二信使，通过触发作为腔膜储存库的BEG囊泡的内吞和胞吐来调节膀胱腔表面积。这一发现揭示了小窝介导的细菌进入BECs和BECs排尿后对腔膜的规律性内吞作用之间可能的相似之处。这项研究建议的目标是延伸和扩大这一调查路线。因此，我们建议：)阐明Flotillin-1、Annexin-11和Caveolin-1促进细菌进入3EC的机制。(2)利用蛋白质组挖掘技术扩大对UPEC侵袭BECs所必需的空泡成分的鉴定。(3)研究cAMP对UPEC侵袭BECs的调节作用，以及细胞内cAMP调节剂在抗实验性UTIs中的作用。我们相信，识别细菌进入的空泡决定因素的系统方法将提供在入侵过程中发生的动态分子相互作用的全面图景，并产生可能作为药物治疗靶点的候选蛋白质。

DESCRIPTION (provided by applicant): Uropathogenic E.coli (UPEC) account for over 80% of urinary tract infections (UTIs). It is now known invasion of the bladder epithelial cells (BECs) is a critical initiating step in UTIs. We have demonstrated that UPEC invasion is localized to distinct cellular entities in the BEG membrane loosely- termed caveolae. These caveolae are comprised of lateral assemblies of cholesterol, lipids and select proteins such as caveolin-1. We hypothesized that the entry of UPEC into BECs is a highly dynamic event and therefore the host-cell proteins that mediate bacterial entry could be proteins that are selectively recruited to caveolae or become activated within these microdomains following exposure to UPEC. Our initial studies have revealed that two proteins, flotillin-1 and annexin-ll, were recruited to caveolae whereas the third, caveolin-1, became tyrosine-phosphorylated following exposure to UPEC. Another finding emerging from proteome analysis of BECs is that a significant proportion of the proteins identified as caveolar components are known to be regulated by cAMP, a major second messenger implicated in regulating luminal surface area of the bladder by triggering endo- and exocytosis of BEG vesicles which serve as repositories of luminal membranes. This finding, revealed possible parallels between caveolae mediated bacterial entry nto BECs and regular endocytosis of luminal membrane by BECs following voiding of urine. The goal of this research proposal is to extend and expand this line of investigation. Therefore, we propose to: ) Elucidate the mechanism by which flotillin-1, annexin-ll, and caveolin-1 contribute to bacterial entry into 3ECs. (2) Use proteome mining approaches to extend the identification of caveolar components essential for UPEC invasion of BECs.(3) Investigate the regulatory role of cAMP on UPEC invasion of BECs and the mpact of modulators of intracellular cAMP in conferring protection against experimental UTIs. We believe that a systematic approach to the identification of caveolar determinants of bacterial entry will provide a comprehensive picture of the dynamic molecular interactions occurring in the invasion process and also yield candidate proteins that could potentially serve as targets for drug therapy.