The novel protein, FimL, regulates virulence in Pseudomonas aeruginosa

新型蛋白质 FimL 可调节铜绿假单胞菌的毒力

• 批准号: 8064387
• 负责人: Yuko F. Inclan
• 金额: $ 3.09万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2011-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8064387
• 关键词: Acute; Adenylate Cyclase; Affect; Antibiotic Resistance; Bacteria; Bacterial Infections; Binding; Biochemistry; Biological Assay; Burn injury; Cell Death; Cells; Cessation of life; Chemotherapy-Oncologic Procedure; Co-Immunoprecipitations; Complex; Cyclic AMP; Cystic Fibrosis; Cytoplasm; Disease; Elements; Flagella; Genes; Genetic; Genetic Screening; Goals; HIV; Human; Hybrids; Immunocompromised Host; Immunoprecipitation; In Vitro; Individual; Infection; Link; Lung; Mammalian Cell; Mechanical Ventilators; Mediating; Medical; Membrane; Microbiology; Multi-Drug Resistance; Nosocomial Infections; Onset of illness; Pathway interactions; Patients; Pilum; Process; Production; Protein Biosynthesis; Proteins; Pseudomonas aeruginosa; Regulation; Reporting; Second Messenger Systems; Sequence Homology; Signal Pathway; Signal Transduction; Signaling Protein; System; Testing; Type II Secretion System Pathway; Type III Secretion System Pathway; Virulence; Virulence Factors; cell type; cofactor; genetic analysis; mortality; mutant; novel; pathogen; prevent; protein-histidine kinase; public health relevance; quorum sensing; research study; second messenger; therapeutic target; therapy development

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa (PA) is an opportunistic pathogen and an important cause of disease in humans. Immunocompromised individuals such as those suffering from HIV, burn wounds, cancer chemotherapy, or illnesses that require mechanical ventilators are particularly vulnerable to acute infection. Even with medical treatment, mortality remains high and antibiotic resistance is increasingly common. In addition, PA chronically colonizes patients afflicted with Cystic Fibrosis, leading to severe pulmonary damage and death. Because multi-drug resistant isolates are increasingly reported, identification of novel bacterial therapeutic targets is increasingly important. The long-term goal of this project is to understand how the infection process is initiated at the signaling level in PA so we can develop therapies to prevent the onset of disease. PA initiates infection by using virulence factors such as the type IV pilus (TFP) to attach to host cells and injects the host with toxic effector proteins using the Type III secretion system (T3SS). TFP and T3SS production is controlled by the cyclic AMP synthesis pathway, which is activated during the infection process. The Engel lab found a novel gene, FimL that is important for the production and function of many virulence factors including TFP and the T3SS because it regulates the level of cyclic AMP in the cell. We propose to determine the mechanism by which FimL regulates cyclic AMP levels in the cell. We have discovered that FimL is polarly localized in the bacteria, as are the TFP and T3SS. We propose the hypothesis that polar localization of FimL leads to spatially restricted production of cyclic AMP and downstream components by regulating the localization of the cyclic AMP synthesis protein, CyaB. We will test our hypothesis by 1) identifying interacting partners of FimL by comprehensive and complimentary approaches. We will test if FimL interacts with CyaB and other candidate proteins using co- immunoprecipitation experiments, and in vitro protein assays. We will also perform a genetic screen to find unknown interacting players. 2) We will also determine which factors are required for subcellular localization of FimL and if FimL is required for proper subcellular localization of known virulence factors and regulators, including the TFP and T3SS. FimL and other proteins in this pathway could serve as new and promising therapeutic targets to aid in alleviating human suffering from bacterial infections. PUBLIC HEALTH RELEVANCE: The bacterial pathogen Pseudomonas aeruginosa is the leading cause of hospital-acquired infections and causes the fatal disease Cystic Fibrosis. We propose to elucidate the mechanism of how the bacteria initiates production of disease causing elements when in contact with humans.

描述（由申请人提供）：铜绿假单胞菌（PA）是一种机会性病原体，是人类疾病的重要原因。免疫功能低下的个体，例如患有艾滋病毒、烧伤、癌症化疗或需要机械呼吸机的疾病的人，特别容易受到急性感染。即使采取药物治疗，死亡率仍然很高，抗生素耐药性也越来越普遍。此外，PA 长期寄生在患有囊性纤维化的患者身上，导致严重的肺部损伤和死亡。由于多重耐药菌株的报道越来越多，新的细菌治疗靶点的鉴定变得越来越重要。 该项目的长期目标是了解感染过程是如何在 PA 信号水平上启动的，以便我们能够开发出预防疾病发作的疗法。 PA 通过使用毒力因子（例如 IV 型菌毛 (TFP)）附着在宿主细胞上来启动感染，并使用 III 型分泌系统 (T3SS) 向宿主注射毒性效应蛋白。 TFP 和 T3SS 的产生由环 AMP 合成途径控制，该途径在感染过程中被激活。 Engel 实验室发现了一种新基因 FimL，它对于包括 TFP 和 T3SS 在内的许多毒力因子的产生和功能很重要，因为它调节细胞中环 AMP 的水平。我们建议确定 FimL 调节细胞中环 AMP 水平的机制。 我们发现 FimL 在细菌中极性定位，TFP 和 T3SS 也是如此。我们提出这样的假设：FimL 的极性定位通过调节环 AMP 合成蛋白 CyaB 的定位，导致环 AMP 和下游成分的空间限制。我们将通过以下方式检验我们的假设：1）通过全面和互补的方法识别 FimL 的交互伙伴。我们将使用免疫共沉淀实验和体外蛋白质测定来测试 FimL 是否与 CyaB 和其他候选蛋白质相互作用。我们还将进行基因筛选来寻找未知的互动玩家。 2) 我们还将确定 FimL 的亚细胞定位需要哪些因子，以及已知毒力因子和调节因子（包括 TFP 和 T3SS）的正确亚细胞定位是否需要 FimL。 FimL 和该途径中的其他蛋白质可以作为新的有前途的治疗靶点，帮助减轻人类遭受细菌感染的痛苦。 公共卫生相关性：细菌病原体铜绿假单胞菌是医院获得性感染的主要原因，并导致致命疾病囊性纤维化。我们建议阐明细菌在与人类接触时如何启动产生致病元素的机制。