Immune mediated exocytosis of intravesicular UPEC from bladder cells

描述(由申请人提供)：尿路感染(UTIs)仍然是人类第二常见的细菌感染。大多数这些感染是由尿路致病性大肠杆菌(UPEC)引起的，它可以进入膀胱上皮细胞，并在细胞内持续存在，在那里它们无法被免疫系统和抗生素获得。值得注意的是，最近对受感染的膀胱上皮细胞进行的体内和体外研究表明，这些细胞具有强大的能力，能够在不损失细胞活力的情况下排出大量感染UPEC的细胞。这些观察表明，BECs具有强大的感知细胞内细菌和启动细菌胞吐的能力。他们还提出，如果了解了潜在的机制，就有可能利用针对这一途径的药物来增强这一细胞防御机制。到目前为止，利用生化和分子手段，我们已经确定了负责识别细胞内UPEC的关键分子是免疫监视分子Toll样受体(TLR)4。我们还鉴定了细菌胞吐作用的几个关键介质，其中包括胞外复合体a Rab GTPase、Rab11和SNARE复合体的成分。此外，我们还发现，在细菌通过质膜的挤出过程中，亚细胞微区被称为小凹。在这里，我们建议研究这些不同的信号组件、通路和亚细胞微生物体是如何整合和实现细菌驱逐的。我们计划在以下特定目标上证实和推广这一观察结果：(I)阐明TLR4是如何在BEC中检测到小泡内UPEC的。(2)确定囊泡运输的细胞机制如何调节细菌胞吐作用。(Iii)研究SNARE蛋白在囊泡内UPEC对接和放电中的作用(IV)。利用蛋白质组挖掘方法鉴定新的细菌排出组分。

DESCRIPTION (provided by applicant): Urinary tract infections (UTIs) continue to be the second most common bacterial infections in man. Most of these infections are caused by uropathogenic E. coli (UPEC) that gain access into bladder epithelial cells and persist intracellularly, where they are inaccessible to the immune system and also antibiotics. Remarkably, recent in vivo and in vitro studies on infected bladder epithelial cells have revealed that these cells have a powerful capacity to exocytose large proportions of infecting UPEC without loss of cell viability. These observations point to a powerful capacity of BECs to sense intracellular bacteria and initiate bacterial exocytosis. They also suggest that if the underlying mechanism were understood, it may be possible to utilize drugs targeting this pathway to boost this cellular defense mechanism. To date, using biochemical and molecular approaches, we have determined that a key molecule responsible for recognizing intracellular UPEC is the imunosurveillance molecule, Toll like Receptor (TLR) 4. We have also identified several key mediators of bacterial exocytosis, which include components of the exocyst complex a Rab GTPase, Rab11 and the SNARE complex. Additionally, we have implicated subcellular microdomains called caveolae in this exocytic process, in the extrusion of bacteria through the plasma membrane. Here we propose to examine how these various signaling components, pathways and subcellular microdomians integrate and achieve bacterial expulsion. We plan to confirm and extend this observation in the following specific aims: (i) Elucidate how intravesicular UPEC are detected by TLR4 in BECs. (ii) Determine how cellular machinery for vesicle trafficking mediates bacterial exocytosis. (iii) Investigate the role of SNARE proteins to docking and discharge of intravesicular UPEC (IV). Identify novel bacterial expulsion components using proteome mining approach.