Caveolae Mediated Bacterial Uptake in the Urinary Tract

描述（由申请人提供）：尿路致病性大肠杆菌（UPEC）占尿路感染（uti）的80%以上。现在已知膀胱上皮细胞（BECs）的侵袭是尿路感染的关键起始步骤。我们已经证明，UPEC的侵袭局限于BEG膜中不同的细胞实体，这些细胞实体被松散地称为小泡。这些小窝由胆固醇、脂质和精选蛋白质（如小窝蛋白-1）组成。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质可能是在暴露于UPEC后选择性地招募到小泡或在这些微域内被激活的蛋白质。我们的初步研究表明，两种蛋白，flotilin -1和annexin- 1，被招募到小窝中，而第三种蛋白，caveolin-1，在暴露于UPEC后被酪氨酸磷酸化。BECs蛋白质组学分析的另一个发现是，被鉴定为腔泡成分的很大一部分蛋白质已知受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囊泡的内分泌和胞外分泌，参与调节膀胱腔表面积。这一发现揭示了小泡介导的细菌进入BECs与BECs在排尿后定期内吞管膜之间可能存在的相似之处。本研究计划的目标是扩展和扩大这条调查线。因此，我们建议：)阐明flotilin -1、annexin- 1和caveolin-1参与细菌进入3ECs的机制。(2)利用蛋白质组挖掘方法扩展对UPEC入侵BECs所必需的空洞成分的识别。(3)探讨cAMP对UPEC侵袭BECs的调控作用，以及细胞内cAMP调节剂对实验性uti的保护作用。我们相信，系统地鉴定细菌进入的空洞决定因素将提供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.