Adapting to a changing environment: How surface contact induces virulence factor production in Pseudomonas aeruginosa

适应不断变化的环境：表面接触如何诱导铜绿假单胞菌产生毒力因子

• 批准号: 9403170
• 负责人: Joanne N. Engel
• 金额: $ 39.63万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9403170
• 关键词: Acute; Adenylate Cyclase; Amoeba genus; Antibiotic Resistance; Bacteria; Bacterial Adhesins; Binding; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Biophysics; Cells; Chemicals; Chemotaxis; Chronic; Complex; Coupling; Cues; Data; Defense Mechanisms; Development; Environment; Epithelial; Event; Fimbriae Proteins; Fluorescence Resonance Energy Transfer; Frequencies; Gene Activation; Gene Expression; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Grant; Homologous Gene; Human; Image; Infection; Integral Membrane Protein; Lead; Link; Lung; Mechanics; Mediating; Membrane; Microbial Biofilms; Modeling; Modification; Molecular Conformation; Multi-Drug Resistance; Nosocomial Infections; Pathway interactions; Periodicity; Phosphorylation; Phylogenetic Analysis; Physiological; Pilum; Polysaccharides; Production; Property; Proteins; Proteomics; Pseudomonas aeruginosa; Scaffolding Protein; Second Messenger Systems; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Solid; Source; Stretching; Surface; Swimming; System; Technology; Testing; Therapeutic; Transcription Coactivator; Transmembrane Domain; Up-Regulation; Virulence; Virulence Factors; cell motility; cystic fibrosis patients; experience; experimental study; extracellular; fluorescence imaging; in vivo; mathematical ability; monolayer; monomer; neutrophil; novel therapeutic intervention; pathogen; periplasm; prevent; resistant strain; response; ventilator-associated pneumonia

Project summary Pseudomonas aeruginosa (PA) is a versatile opportunistic pathogen that is a leading cause of hospital- acquired infections. PA antibiotic resistance continues to explode, making development of new therapeutic approaches a critical need. One largely unexplored therapeutic venue is the uncommonly large number of sensing systems that PA has evolved. These signal transduction pathways allow PA to rapidly adapt to a wide variety of environments, such as transitioning from swimming to surface-associated states. Through genetic screens, we and others have identified three systems in PA—the type IV pilus (TFP), the Chp chemosensory system (a complex chemosensory system), and the second messenger 3', 5'-cyclic monophosphate (cAMP) and its allosterically regulated protein binding partner, Vfr-- that are required for upregulation of virulence factors upon surface binding. Activation of the Chp system by TFP retraction leads to a cascade of phosphorylation events that leads to the activation of a membrane-bound adenylate cyclase (CyaB), the primary enzymatic source of cAMP. cAMP binds to a transcriptional activator (Vfr) to induce transcription of >200 genes, many of which are involved in virulence in humans and in causing acute lung damage. We have discovered that in response to surface binding and retraction, the TFP functions as a mechanotransducer to activate the Chp phosphorelay, which in turn increases the activity of the transmembrane adenylate cyclase CyaB. Using multiple approaches, we have uncovered interactions between various components of this system that identify two key signal integrating hubs. We propose 3 specific aims to test and further refine our model and that will deepen our understanding of TFP-Chp-CyaB mechanochemical signaling pathway. Aim 1. Test the hypothesis that PilJ serves as a central integrator of MCS by coordinately regulating the mechanical input signal (altered pilin monomers) with activation of the Chp phosphorelay and with activation of CyaB. We will use genetic screens, in vivo assays of physiologic function, in vivo biochemistry, and live cell fluorescence imaging including FRET to define the (A) PilA/PilJ/CyaB/ and (B) PilJ/PilH interaction landscapes. Aim 2. Test the hypothesis that FimV/FimL/PilG hub links TFP function to the Chp/CyaB system. We will (A) define the FimL/PilG interaction landscape and (B) use Phos-tag technology 16 to examine PilG and PilH phosphorylation in vivo during MCS. Aim 3. Define key spatial and temporal properties of TFP-Chp-CyaB mechanochemical signal transduction during biotic biofilm formation on polarized lung epithelial monolayers. We will (A) Determine the contribution of TFP-Chp-CyaB MCS during biotic biofilm formation and (B) Determine the temporal and spatial dynamics of the surface-activated gene expression during biotic biofilm formation.

项目摘要 铜绿假单胞菌（PA）是一种多功能的机会致病菌，是医院感染的主要原因。 获得性感染PA抗生素耐药性持续爆发，使得新的治疗药物的开发 接近一个关键的需求。一个很大程度上未开发的治疗场所是不寻常的大量 PA已经进化的传感系统。这些信号转导通路使PA能够迅速适应广泛的 各种环境，如从游泳过渡到表面相关状态。通过基因 筛选，我们和其他人已经确定了PA中的三个系统-IV型菌毛（TFP），Chp化学感受器 系统（一个复杂的化学感受系统）和第二信使3 '，5'-环磷酸（cAMP） 及其变构调节蛋白结合伴侣Vfr--这是上调毒力所必需的 表面结合的因素。通过TFP收缩激活Chp系统导致级联反应， 磷酸化事件导致膜结合腺苷酸环化酶（CyaB）的活化， cAMP的主要酶源。cAMP与转录激活因子（Vfr）结合以诱导 超过200个基因，其中许多涉及人类的毒力和引起急性肺损伤。我们有 发现在响应表面结合和收缩时，TFP作为机械传感器发挥作用， 激活Chp磷酸化中继，进而增加跨膜腺苷酸环化酶的活性 CyaB.使用多种方法，我们已经发现了这个系统的各个组件之间的相互作用 识别出两个关键的信号整合中心。我们提出了3个具体目标来测试和进一步完善我们的模型 这将加深我们对TFP-Chp-CyaB机械化学信号通路的理解。目标1.测试 假设PilJ作为MCS的中心整合者，通过协调调节 机械输入信号（改变的菌毛蛋白单体）与Chp磷酸化继电器的激活和与 CyaB的激活。我们将使用遗传筛选、体内生理功能测定、体内生物化学， 以及活细胞荧光成像，包括FRET以确定（A）PilA/PilJ/Cya B/和（B）PilJ/PilH相互作用 的风景.目标2.检验FimV/FimL/PilG中心将TFP功能与Chp/CyaB连接的假设 系统我们将（A）定义FimL/PilG交互景观，并且（B）使用Phos标签技术16来 检查MCS期间体内PilG和PilH磷酸化。目标3。定义关键的空间和时间 生物被膜形成过程中TFP-Chp-CyaB机械化学信号转导的特性 极化的肺上皮单层。我们将（A）确定TFP-Chp-CyaB MCS的贡献， 生物生物膜形成和（B）确定表面活化基因的时间和空间动态 在生物膜形成期间表达。