Proteomic approach to identify mediators of PI3K activation by P. aeruginosa

描述(由申请人提供)：微生物病原体克服正常高度极化的宿主粘膜上皮屏障的能力是发病机制中早期和关键的一步。这对铜绿假单胞菌(PA)等机会性病原体尤其关键，PA是人类最致命的机会性病原体之一。在上皮损伤和细胞极性丧失的情况下，PA可以有效地定植于粘膜表面，造成进一步的损伤，并进行扩散。我们最近发现，PA与培养上皮细胞根尖(AP)表面的结合诱导了一个细胞重编程过程，在这个过程中，局部质膜从根尖(AP)膜转变为基底侧(BL)膜，产生了一个有利于细菌定植和进入上皮细胞的微环境。我们已经通过大量证据证明，磷脂酰肌醇3-激酶(PI3K)的激活是这一过程中的关键步骤。一个尚未解决的非常重要的问题是，在结合时激活的信号通路是什么，导致细菌摄取和创造这个新的微环境。PI3K适合于接受蛋白酪氨酸激酶或酪氨酸磷酸化蛋白的调节输入。已知PA感染上皮细胞可引起宿主蛋白酪氨酸磷酸化状态的改变。我们假设PA感染上皮细胞引起酪氨酸磷酸化信号级联，导致PI3K激活。我们的长期目标是了解PA如何与宿主细胞相互作用并导致疾病。我们的短期目标是剖析PA在AP进入上皮细胞过程中将AP转化为BL膜的信号通路。我们概述了两个特定的目标来剖析PA结合和PI3K激活之间的信号通路(S)。在目标1中，我们采用候选蛋白质的方法，探索特定的宿主受体和非受体酪氨酸激酶在PA介导的PI3K激活和由此导致的AP膜变化中的作用。这些候选基因中的许多都是在父母资助中进行的RNAi筛查中确定的。在目标2中，我们概述了两种互补的方法来识别宿主蛋白，其酪氨酸磷酸化变化响应于PA与极化MDCK细胞AP表面的结合。这些研究将为PA与宿主细胞上皮附着后引发的内化过程提供一个机械性的见解。他们将确定细菌利用来致病的宿主因素。在分子水平上阐明PA-宿主细胞的相互作用将为开发新药打开大门，这些新药不是针对致病菌本身，而是针对它用来入侵和致病的关键机制。这项研究将主要在乌拉圭的蒙得维的亚巴斯德研究所与Arlinet Kierbel合作进行，作为NIH第号补助金的延伸。R01AI065902。7.公共卫生相关性：铜绿假单胞菌是人类最致命的机会性病原体之一，是各种急性和慢性感染的病原体。我们正在研究这种细菌如何通过颠覆宿主细胞机制来感染上皮细胞。与假单胞菌感染相关的新的识别宿主细胞蛋白可能被用作新的药物靶点。

DESCRIPTION (provided by applicant): The ability of microbial pathogens to overcome the normally highly polarized host mucosal epithelial barrier is an early and critical step in pathogenesis. This is particularly critical for opportunistic pathogens such as Pseudomonas aeruginosa (PA), one of the most virulent opportunistic pathogens of man. In the setting of epithelial injury and loss of cell polarity PA can effectively colonize the mucosal surfaces, cause further damage, and disseminate. We have recently discovered that binding of PA to the apical (AP) surface of cultured epithelial cells induces a cell reprogramming process in which the local plasma membrane transforms from apical (AP) to basolateral (BL) membrane, generating a microenvironment that facilitates bacterial colonization and entry into epithelial cells. We have demonstrated through a large body of evidence that activation of Phosphoinositide 3-kinase (PI3K) is a key step in this process. A very important question that has not been addressed is what is the signaling pathway that is activated upon binding that results in bacterial uptake and creation of this new microenvironment. PI3Ks are adapted to receive regulatory input from protein tyrosine kinases or tyrosine phosphorylated proteins. It is known that PA infection of epithelial cells induces changes in the tyrosine phosphorylation status of host proteins. We hypothesize that PA infection of epithelial cells causes a tyrosine phosphorylation signaling cascade leading to activation of PI3K. Our long-term goal is to understand how PA interacts with host cells and causes disease. Our short-term goal is to dissect the signaling pathway that PA utilizes to transform AP into BL membrane during AP entry into epithelial cells. We outline two specific aims to dissect the signaling pathway(s) between binding of PA and activation of PI3K. In aim 1, we take a candidate protein approach and explore the role of specific host receptor and non-receptor tyrosine kinases in PA-mediated activation of PI3K and resultant changes in the AP membrane. Many of these candidate genes were identified in the RNAi screen that was carried out in the parent grant. In aim 2, we outline two complementary approaches to identify host proteins whose tyrosine phosphorylation changes in response to PA binding to the AP surface of polarized MDCK cells. These studies will provide a mechanistic insight into the process of internalization triggered by PA after attachment to the host cell epithelium. They will identify host factors that the bacteria exploit to cause disease. The elucidation of PA-host cell interactions at the molecular level will open the door for the development of new drugs that target not the pathogenic bacteria itself but the key mechanisms that it uses to invade and cause disease. This research will be done primarily in Uruguay at Institut Pasteur de Montevideo in collaboration with Arlinet Kierbel, as an extension of NIH Grant No. R01AI065902. 7. Public Health Relevance: Pseudomonas aeruginosa is one of the most virulent opportunistic pathogens of man and is the causative agent of a variety of acute and chronic infections. We are investigating how this bacterium infects epithelial cells by subverting the host cell machinery. New identify host cell proteins involved in Pseudomonas infection can potentially be used as novel drug targets.