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HIV，烧伤伤口，癌症化学疗法或需要机械呼吸机的疾病的人特别容易受到急性感染的影响。即使使用医疗，死亡率仍然很高，抗生素耐药性也越来越普遍。此外，PA长期将患有囊性纤维化患者的患者延伸，从而导致严重的肺部损伤和死亡。由于越来越多地报道了多药抗性分离株，因此对新型细菌治疗靶标的鉴定越来越重要。 该项目的长期目标是了解如何在PA的信号传导水平上启动感染过程，以便我们开发疗法以防止疾病发作。 PA通过使用毒力因子（例如IV型菌毛（TFP））与宿主细胞相连，并使用III型分泌系统（T3SS）向宿主注入宿主的毒性效应蛋白。 TFP和T3SS的产生受环状AMP合成途径的控制，该途径在感染过程中被激活。恩格尔实验室（Engel Lab）发现了一种新型基因FIML，这对于包括TFP和T3S的许多毒力因子的生产和功能很重要，因为它调节了细胞中的环状AMP水平。我们建议确定FIML调节细胞中环状AMP水平的机制。 我们发现FIML以及TFP和T3SS都在细菌中局部局限。我们提出了以下假设，即FIML的极性定位通过调节环状AMP合成蛋白CyAB的定位，从而导致环状AMP和下游成分的空间限制产生。我们将通过1）通过全面和免费的方法来检验我们的假设。我们将使用共沉淀实验和体外蛋白质测定法测试FIML是否与CyAB和其他候选蛋白相互作用。我们还将执行一个遗传屏幕，以找到未知的互动玩家。 2）我们还将确定FIML亚细胞定位所需的因素，以及是否需要FIML才能适当地细胞局部定位已知的毒力因子和调节剂（包括TFP和T3SS）。该途径中的FIML和其他蛋白质可以作为新的和有前途的治疗靶标，以帮助减轻人类患有细菌感染的人。 公共卫生相关性：铜绿假单胞菌的细菌病原体是医院获得感染的主要原因，并引起致命疾病囊性纤维化。我们建议阐明细菌如何在与人类接触时引起疾病的产生的机制。