Bacteria-Epithelial Cell Cytoskeleton Interaction

细菌-上皮细胞细胞骨架相互作用

基本信息

批准号：
6921030
负责人：
Steven Brody
金额：
$ 34.43万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-15 至 2009-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6921030
关键词：
Pseudomonas aeruginosa SDS polyacrylamide gel electrophoresis apical membrane bacteria infection mechanism biological signal transduction cell cell interaction confocal scanning microscopy cytoskeletal proteins cytoskeleton gene expression genetically modified animals host organism interaction laboratory mouse protein localization respiratory epithelium western blottings

项目摘要

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an increasing cause of life-threatening nosocomial respiratory infection and portends a worse outcome in bronchiectasis. Clinically, the host response and outcome is dependent on the initial stages of P. aeruginosa infection however, the molecular events that occur as a result of early bacteria-epithelial cell interactions remain unclear. The apical membrane of airway epithelial cells is the initial site of contact with organisms and plays an essential barrier role achieved through a highly organized array of apical membrane proteins linked to cytoskeletal scaffolding proteins. The focus of the proposed studies is to understand the molecular requirements and cellular consequences of P. aeruginosa contact-dependent exotoxin on airway epithelial cell apical membrane proteins during early bacteria-epithelial cell interaction. We have found that apical membrane organization required for host defense is dependent on forkhead factor foxj1 and Rho-mediated activity and can be disrupted by P. aeruginosa type III exotoxin ExoS. Following P. aeruginosa infection of primary culture differentiated airway epithelial cells, we observed a decrease in foxj1 expression followed by a change in epithelial cell phenotype consistent with the foxj1 deficient state. This was characterized by decreased apical localization of cytoskeletal scaffolding ERM family and PDZ-domain NHERF/EBP50 proteins and disrupted cilia basal bodies. We hypothesize that (1) the airway epithelial cell responses to P. aeruginosa ExoS disrupt apical membrane organization and (2) these effects of ExoS are mediated by loss of foxj1 expression and interruption of RhoA pathways to alter the cytoskeletal proteins of the apical membrane and impair host defense. The hypothesis will be tested using an integrated approach in a high fidelity mouse airway epithelial cell primary culture model and in vivo infection models to reveal cellular and molecular events in infection pathogenesis. Specific Aims are to 1) characterize the role of P. aeruginosa ExoS on apical membrane scaffolding proteins and molecules that regulate apical membrane organization, 2) determine the physiologic consequences of ExoS in vitro by assay of specific apical membrane functions of airway epithelial cells and in vivo using genetically deficient mouse models to evaluate epithelial cell-specific defects in host defense, and 3) investigate pharmacologic and genetic augmentation of molecules in apical membrane pathways to abrogate effects of exoS. The proposed studies will identify targets of P. aeruginosa-induced disruption of host defense and lead to new strategies for prevention of P. aeruginosa airways infection.

描述（由申请人提供）：铜绿假单胞菌是危及生命的院内呼吸道感染的一个日益增加的原因，并且预示着支气管扩张的更糟糕的结果。临床上，宿主反应和结果取决于铜绿假单胞菌感染的初始阶段，然而，由于早期细菌-上皮细胞相互作用而发生的分子事件仍不清楚。气道上皮细胞的顶膜是与生物体接触的初始部位，并通过与细胞骨架支架蛋白连接的高度组织的顶膜蛋白阵列发挥重要的屏障作用。拟议研究的重点是了解早期细菌-上皮细胞相互作用过程中铜绿假单胞菌接触依赖性外毒素对气道上皮细胞顶膜蛋白的分子需求和细胞后果。我们发现宿主防御所需的顶膜组织依赖于叉头因子foxj1和Rho介导的活性，并且可以被铜绿假单胞菌III型外毒素ExoS破坏。原代培养分化的气道上皮细胞在铜绿假单胞菌感染后，我们观察到foxj1表达下降，随后上皮细胞表型发生与foxj1缺陷状态一致的变化。其特征是细胞骨架支架 ERM 家族和 PDZ 结构域 NHERF/EBP50 蛋白的顶端定位减少以及纤毛基体破坏。我们假设 (1) 气道上皮细胞对铜绿假单胞菌 ExoS 的反应破坏了顶膜组织，(2) ExoS 的这些影响是通过 Foxj1 表达的丧失和 RhoA 途径的中断来介导的，从而改变了顶膜的细胞骨架蛋白和损害宿主防御。该假设将在高保真小鼠气道上皮细胞原代培养模型和体内感染模型中使用集成方法进行测试，以揭示感染发病机制中的细胞和分子事件。具体目标是 1) 表征铜绿假单胞菌 ExoS 对调节顶膜组织的顶膜支架蛋白和分子的作用，2) 通过测定气道上皮细胞的特定顶膜功能来确定 ExoS 的体外生理后果，并在体内使用遗传缺陷小鼠模型来评估宿主防御中上皮细胞特异性缺陷，以及3）研究顶膜途径中分子的药理学和遗传增强消除 exoS 的影响。拟议的研究将确定铜绿假单胞菌引起的宿主防御破坏的目标，并产生预防铜绿假单胞菌气道感染的新策略。