Bacteria-Epithelial Cell Cytoskeleton Interaction

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，预示着支气管扩张的更糟糕的结局。在临床上，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。气道上皮细胞的顶膜是与生物体接触的初始位置，通过与细胞骨架支架蛋白相连接的高度有序的顶膜蛋白阵列发挥重要的屏障作用。本研究的重点是了解P. aeruginosa接触依赖性外毒素在早期细菌-上皮细胞相互作用过程中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，并可被铜绿假单胞菌III型外毒素ExoS破坏。在铜绿假单胞菌感染原代培养分化的气道上皮细胞后，我们观察到foxj1表达下降，随后上皮细胞表型发生变化，与foxj1缺失状态一致。其特征是细胞骨架支架ERM家族和pdz结构域NHERF/EBP50蛋白的根尖定位降低，纤毛基体被破坏。我们假设：(1)气道上皮细胞对铜绿假单胞菌ExoS的反应破坏了根尖膜的组织；(2)ExoS的这些影响是通过foxj1表达的缺失和RhoA通路的中断来介导的，从而改变根尖膜的细胞骨架蛋白并损害宿主的防御。该假设将在高保真度小鼠气道上皮细胞原代培养模型和体内感染模型中进行综合测试，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对根尖膜支架蛋白和调节根尖膜组织的分子的作用；2)通过检测气道上皮细胞的特定根尖膜功能，在体外确定ExoS的生理后果；在体内使用基因缺陷小鼠模型来评估宿主防御中上皮细胞特异性缺陷。3)研究在根尖膜通路中增加分子的药理学和遗传学作用，以消除外源性os的影响。本研究将确定铜绿假单胞菌诱导的宿主防御破坏的靶点，并为铜绿假单胞菌呼吸道感染的预防提供新的策略。

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.