Bacteria-Epithelial Cell Cytoskeleton Interaction

描述（由申请方提供）：铜绿假单胞菌是危及生命的院内呼吸道感染的一种日益增加的原因，并预示着支气管扩张症的预后更差。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，并且通过与细胞骨架支架蛋白连接的高度组织化的顶膜蛋白阵列来发挥重要的屏障作用。 拟议研究的重点是了解早期细菌-上皮细胞相互作用期间铜绿假单胞菌接触依赖性外毒素对气道上皮细胞顶端膜蛋白的分子要求和细胞后果。 我们已经发现，宿主防御所需的顶端膜组织依赖于叉头因子foxj 1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。铜绿假单胞菌感染原代培养分化的气道上皮细胞后，我们观察到foxj 1表达减少，随后上皮细胞表型发生变化，与foxj 1缺陷状态一致。 其特征在于细胞骨架支架ERM家族和PDZ结构域NHERF/EBP 50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的反应破坏了顶端膜组织，（2）ExoS的这些作用是通过foxj 1表达的丧失和RhoA通路的中断介导的，以改变顶端膜的细胞骨架蛋白并损害宿主防御。该假设将使用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的综合方法进行测试，以揭示感染发病机制中的细胞和分子事件。具体目的是1）表征铜绿假单胞菌ExoS对顶端膜支架蛋白和调节顶端膜组织的分子的作用，2）通过测定气道上皮细胞的特异性顶端膜功能在体外确定ExoS的生理后果，并使用遗传缺陷小鼠模型在体内评估宿主防御中的上皮细胞特异性缺陷，和3）研究顶端膜途径中分子的药理学和遗传增强以消除exoS的作用。拟议的研究将确定铜绿假单胞菌诱导的宿主防御破坏的靶点，并导致预防铜绿假单胞菌气道感染的新策略。

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.