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

鉴定铜绿假单胞菌 PI3K 激活介质的蛋白质组学方法

• 批准号: 7587371
• 负责人: Joanne N. Engel
• 金额: $ 3.79万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7587371
• 关键词: 1-Phosphatidylinositol 4-Kinase; Actins; Acute; Adaptor Signaling Protein; Address; Animal Disease Models; Anti-Bacterial Agents; Apical; Arts; Bacteria; Binding; Biological; Candidate Disease Gene; Cell Communication; Cell Polarity; Cell membrane; Cells; Cellular biology; Chronic; Collaborations; Communities; Cytoskeleton; Developed Countries; Developing Countries; Development; Disease; Drug Delivery Systems; E-Cadherin; Epithelial; Epithelial Cells; Epithelium; Faculty; Family; Funding; Gene Silencing; Genes; Genetic; Genetic Screening; Goals; Grant; Guanosine Triphosphate Phosphohydrolases; Health; Human; Infection; Injury; Institutes; Integration Host Factors; International; Invaded; Journals; Lead; MDCK cell; Mammalian Cell; Mediating; Mediator of activation protein; Membrane; Methods; Microbiology; Molecular; Nature; PDGFRB gene; Paper; Parents; Pathogenesis; Pharmaceutical Preparations; Phosphatidylinositols; Phosphotransferases; Phosphotyrosine; Positioning Attribute; Principal Investigator; Process; Protein Kinase; Protein Tyrosine Kinase; Proteins; Proteome; Proteomics; Pseudomonas Infections; Pseudomonas aeruginosa; Publishing; RNA Interference; Receptor Protein-Tyrosine Kinases; Research; Research Design; Research Personnel; Resistance; Rho-associated kinase; Role; STI571; Science; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Surface; Technology; Testing; Therapeutic; Tyrosine; Tyrosine Phosphorylation; United States National Institutes of Health; Uruguay; Virulent; Work; Wound Healing; antimicrobial; apical membrane; base; basolateral membrane; genome-wide; human disease; innovation; insight; interest; man; microbial; novel; p21 activated kinase; parent grant; pathogen; pathogenic bacteria; phosphatidylinositol 3,4,5-triphosphate; polarized cell; post-doctoral training; prevent; public health relevance; receptor; response; rho; src-Family Kinases; uptake

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.

描述（由申请人提供）：微生物病原体克服通常高度极化的宿主粘膜上皮屏障的能力是发病机理的早期和关键步骤。这对于机会性病原体（例如铜绿假单胞菌（PA））尤其重要，这是人类最有毒的机会病原体之一。在上皮损伤和细胞极性丧失的情况下，PA可以有效地定居粘膜表面，导致进一步的损害并传播。我们最近发现，PA与培养的上皮细胞的顶端（AP）结合诱导细胞重编程过程，其中局部质膜从根尖（AP）转化为基底外侧（BL）膜，产生微环境，从而促进细菌共同化和进入上皮细胞。我们已经通过大量证据证明，磷酸肌醇3-激酶（PI3K）的激活是此过程的关键步骤。尚未解决的一个非常重要的问题是，在结合后激活的信号传导途径是什么导致细菌摄取并创造了这种新的微环境。 PI3K适应从蛋白酪氨酸激酶或酪氨酸磷酸化蛋白质中接收调节输入。众所周知，上皮细胞的PA感染会诱导宿主蛋白的酪氨酸磷酸化状态的变化。我们假设上皮细胞的PA感染会导致酪氨酸磷酸化信号传导级联反应导致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引起疾病。我们的短期目标是剖析PA在进入上皮细胞期间将AP转化为BL膜的信号传导途径。我们概述了两个特定目的，旨在在PA的结合和PI3K的激活之间剖析信号通路。在AIM 1中，我们采用候选蛋白方法，并探索特定宿主受体和非受体酪氨酸激酶在PA介导的PI3K激活中的作用以及AP膜的结果变化。这些候选基因中的许多是在父母赠款中进行的RNAi屏幕上鉴定的。在AIM 2中，我们概述了两种互补方法，以鉴定宿主蛋白的酪氨酸磷酸化变化，以响应PA与极化MDCK细胞的AP表面结合。这些研究将提供对宿主细胞上皮附着后PA触发的内在化过程的机械洞察力。他们将确定细菌利用引起疾病的宿主因素。在分子水平上阐明Pa-host细胞相互作用将为开发而不是致病性细菌本身而是其用于侵袭和引起疾病的关键机制的新药物打开大门。这项研究将主要在与Arlinet Kierbel合作的乌拉圭在乌拉圭进行，作为NIH赠款号R01AI065902的扩展。 7。公共卫生相关性：铜绿假单胞菌是人类最有毒的机会病原体之一，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。参与假单胞菌感染的新鉴定宿主细胞蛋白可以可能用作新的药物靶标。