Regulation of motility and organelle assembly at the Pseudomonas aeruginosa pole

铜绿假单胞菌是人类的革兰氏阴性细菌病原体，其可以急性感染易感患者以引起侵入性、组织破坏性感染，以及慢性定殖具有潜在肺部疾病的患者，例如患有囊性纤维化的个体。铜绿假单胞菌表面的两种极性细胞器IV型皮利和鞭毛在宿主-病原体和病原体-病原体相互作用中起着重要作用。这两种表面细胞器都是生物膜的起始和发展所必需的，生物膜是结构化的多细胞细菌群落，允许铜绿假单胞菌持续地定殖在非生物和生物环境中，包括囊性纤维化气道的环境。 我们已经表征了铜绿假单胞菌蛋白FimX，其是菌毛组装、粘附宿主细胞和对宿主细胞的毒力以及厌氧生物膜形成所需的。FimX是双-（3 '-5'）-环状二聚鸟苷单磷酸（c-di-GMP）的磷酸二酯酶，其是涉及与运动性、定殖和毒力相关的许多细菌系统的转录后控制的第二信使。基因组学已经揭示了超过2000种细菌蛋白质，其含有预测合成或分解c-di-GMP的结构域，以及预测结合c-di-GMP并将该信号转化为特定表型的蛋白质。然而，我们对单个二鸟苷酸环化酶、c-di-GMP磷酸二酯酶和c-di-GMP结合蛋白如何相互作用以产生特定细菌行为的理解是相当原始的。 在这个应用程序中，我们将专注于FimX和一组蛋白质，与它相互作用的菌毛组装的调节。我们特别感兴趣的是研究我们发现的蛋白质-蛋白质相互作用如何通过（1）改变c-di-GMP代谢酶对环境信号的反应活性和（2）改变c-di-GMP产生或降解的亚细胞位置来调节和赋予c-di-GMP信号传导特异性。通过完成本提案中概述的实验，我们将扩大我们对铜绿假单胞菌在运动和生物膜形成过程中如何调节皮利和鞭毛组装的理解，这些行为对致病过程中细菌定植和持久性至关重要。然而，这项工作也具有更广泛的意义，因为它将使我们能够解决所有原核生物中与c-di-GMP依赖性信号通路相关的一般问题。 相关性：铜绿假单胞菌感染的治疗通常很困难：这些细菌对大多数抗生素具有高度耐药性，并以生物膜的形式生长，无法通过抗菌剂或人体免疫反应有效根除。这些实验的完成将使我们朝着我们的长期目标前进，即了解环境线索如何指导铜绿假单胞菌的行为，使其能够殖民和感染人类宿主。我们预计，这些知识将使我们能够开发出治疗药物，可以破坏这些适应性行为，并限制假单胞菌引起人类疾病的能力。

Pseudomonas aeruginosa is a Gram-negative bacterial pathogen of humans that can acutely infect susceptible patients to cause invasive, tissue-destructive infections, as well as chronically colonize patients with underlying lung disease, such as individuals with Cystic Fibrosis. Two polar surface organelles of P. aeruginosa—Type IV pili and flagella—play important roles in host-pathogen and pathogen-pathogen interactions required for infection and colonization. Both surface organelles are required for the initiation and development of biofilms, the structured multicellular bacterial communities that allow P. aeruginosa to persistently colonize abiotic and biotic environments, including that of the cystic fibrosis airway. We have characterized a P. aeruginosa protein, FimX, which is required for pilus assembly, adherence to and virulence toward host cells, and anaerobic biofilm formation. FimX is a phosphodiesterase for bis-(3’-5’)-cyclic dimeric guanosine monophosphate (c-di-GMP), a second messenger implicated in the post-transcriptional control of many bacterial systems relevant to motility, colonization and virulence. Genomics has revealed over 2000 bacterial proteins containing domains predicted to synthesize or breakdown c-di-GMP, as well as proteins predicted to bind c-di-GMP and transduce this signal into specific phenotypes. However, our understanding of how individual diguanylate cyclases, c-di-GMP phosphodiesterases and c-di-GMP binding proteins interact to bring about specific bacterial behaviors is quite primitive. In this application, we will focus on FimX and a group of proteins that interact with it in the regulation of pilus assembly. We are particularly interested in examining how the protein-protein interactions that we have uncovered regulate and confer specificity upon c-di-GMP signaling by (1) altering the activity of c-di-GMP metabolizing enzymes in response to environmental cues and by (2) changing the subcellular location at which c-di-GMP is produced or degraded. By completing the experiments outlined in this proposal, we will expand our understanding of how P. aeruginosa regulates the assembly of pili and flagella during motility and biofilm formation, behaviors that are crucial to bacterial colonization and persistence during pathogenesis. However this work has broader implications as well, as it will allow us to address general questions relevant to c-di-GMP dependent signaling pathways in all prokaryotes. Relevance: The treatment of P. aeruginosa infections is often difficult: these bacteria are highly resistant to most antibiotics and grow as biofilms that cannot be effectively eradicated by antimicrobials or the human immune response. Completion of these experiments will advance us toward our long-term goal of understanding how environmental cues direct P. aeruginosa behaviors that enable it to colonize and infect human hosts. We anticipate that this knowledge will allow us to develop therapeutic agents that can disrupt these adaptive behaviors and limit the ability of Pseudomonas to cause human disease.