Regulation of motility and organelle assembly at the Pseudomonas aeruginosa pole

铜绿假单胞菌是一种人类的革兰氏阴性细菌病原体，可急性感染易感患者，导致侵袭性、组织破坏性感染，以及长期定植于患有潜在肺部疾病的患者，如囊性纤维化患者。铜绿假单胞菌的两个极表面细胞器-IV型菌毛和鞭毛-在感染和定植所需的宿主-病原体和病原体-病原体相互作用中发挥着重要作用。这两个表面细胞器都是生物膜的启动和发展所必需的，生物膜是一个结构化的多细胞细菌群落，使铜绿假单胞菌能够持续地在非生物和生物环境中定居，包括囊性纤维化的呼吸道环境。 我们已经鉴定了一种铜绿假单胞菌蛋白FimX，它是菌毛组装、对宿主细胞的黏附和毒力以及厌氧生物膜形成所必需的。FimX是双-(3‘-5’)-环状鸟苷单磷酸(c-di-GMP)的磷酸二酯酶，是第二信使，参与许多与运动、定植和毒力相关的细菌系统的转录后控制。基因组学已经发现了2000多种细菌蛋白质，这些蛋白质含有预测合成或分解c-di-GMP的结构域，以及预测与c-di-GMP结合并将这种信号转导到特定表型的蛋白质。然而，我们对个别的二鸟苷酸环化酶、c-di-GMP磷酸二酯酶和c-di-GMP结合蛋白如何相互作用导致特定的细菌行为的了解还很原始。 在这个应用中，我们将重点关注FimX和一组与其相互作用的蛋白质在毛孔组装的调节中。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代谢酶的活性来响应环境提示，以及(2)通过改变c-di-GMP产生或降解的亚细胞位置来调节和赋予c-di-GMP信号的特异性。通过完成这项提议中概述的实验，我们将扩大我们对铜绿假单胞菌如何在运动和生物膜形成过程中调节菌毛和鞭毛的组装的理解，这些行为对细菌在致病过程中的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与所有原核生物中依赖c-di-GMP的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往往很困难：这些细菌对大多数抗生素具有高度抗药性，并以生物膜的形式生长，无法通过抗菌剂或人类免疫反应有效地根除。这些实验的完成将推动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，使其能够定植和感染人类宿主。我们预计，这些知识将使我们能够开发能够扰乱这些适应行为并限制假单胞菌导致人类疾病的能力的治疗剂。

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.