Bacteria-Epithelial Cell Cytoskeleton Interaction

细菌-上皮细胞细胞骨架相互作用

• 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
• 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

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)：铜绿假单胞菌是威胁生命的医院内呼吸道感染的一个日益增加的原因，并预示着更糟糕的结果在支气管扩张。临床上，宿主的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尚不清楚。呼吸道上皮细胞的顶膜是与生物体接触的初始部位，通过与细胞骨架蛋白相连的一系列高度组织化的顶膜蛋白发挥重要的屏障作用。本研究的重点是了解在早期细菌-上皮细胞相互作用过程中，铜绿假单胞菌接触性外毒素对呼吸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。在铜绿假单胞菌感染原代培养的分化的呼吸道上皮细胞后，我们观察到foxj1表达的减少以及与foxj1缺失状态一致的上皮细胞表型的改变。这是以细胞骨架支架ERM家族和PDZ结构域NHERF/EBP50蛋白的顶端定位减少和纤毛基底体破坏为特征的。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的这些作用是通过失去foxj1的表达和阻断RhoA通路来改变顶膜的细胞骨架蛋白而损害宿主防御的。这一假说将在高保真的小鼠呼吸道上皮细胞原代培养模型和体内感染模型中使用综合方法进行验证，以揭示感染发病机制中的细胞和分子事件。其具体目的是：1)确定铜绿假单胞菌EXOS在调节顶膜组织结构的顶膜支架蛋白和分子上的作用；2)通过分析呼吸道上皮细胞特定的顶膜功能来确定EXOS在体外的生理后果；以及在体内利用遗传缺陷的小鼠模型来评估上皮细胞在宿主防御中的特异性缺陷，以及3)研究顶膜通路中分子的药理和遗传增强以消除EXOS的影响。拟议的研究将确定铜绿假单胞菌诱导的宿主防御中断的靶点，并导致预防铜绿假单胞菌呼吸道感染的新策略。