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Sensing, Signaling and the Role of Aer-2-Regulated Chemosensory Systems

Aer-2 调节的化学感应系统的传感、信号传导和作用

基本信息

批准号：
9899254
负责人：
Kylie Jane Watts
金额：
$ 28.04万
依托单位：
LOMA LINDA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-05-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9899254
关键词：
Address Affinity Bacteria Basic Science Behavioral Behavioral Assay Binding Biochemical Biological Assay Biological Process Caenorhabditis elegans Chemoreceptors Chemotaxis Collaborations Complex Crystallization Data Defect Disease Disulfides Drug Targeting Environment Escherichia coli Foundations Gases Gene Expression Genetic Genomic Library Goals Heme Hemeproteins In Vitro Individual Infection Intervention Joints Knowledge Ligands Lung Masks Mediating Medical Membrane Methods Microbial Biofilms Mission Modeling Molecular Conformation Molecular Sieve Chromatography Morbidity - disease rate Mutagenesis National Institute of General Medical Sciences Nature Nematoda Nosocomial Infections Output Pathogenesis Pathway interactions Phenotype Phosphorylation Prokaryotic Cells Proteins Pseudomonas aeruginosa Publications Receptor Signaling Research Design Role Sensory Sensory Receptors Signal Transduction Site Structure Surface Swimming System Tertiary Protein Structure Time Two-Hybrid System Techniques Universities Virulence antimicrobial drug base cell motility crosslink cystic fibrosis patients dimer experience experimental study in vivo insight monomer mortality mutant novel pathogen pathogenic bacteria prototype public health relevance receptor response sensor sound structured data yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Bacterial pathogens seek, tolerate and escape different environments by changing swimming direction (chemotaxis), surface motility, gene expression, biofilm formation, and the course of infection. Responses are species-specific but all are modulated by chemosensory systems composed of sensory receptors (chemoreceptors) and effector proteins. One such chemosensory system is regulated by a chemoreceptor called Aer2, which senses O2 via a PAS sensing domain that uses novel methods to coordinate heme and stabilize O2-binding. PAS signals are transduced directly through contiguous poly-HAMP domains. Aer2- regulated systems occur in a number of pathogens, including Pseudomonas aeruginosa, where the system is called Che2. P. aeruginosa is an important opportunistic pathogen, a significant cause of hospital-acquired infection, and a major cause of both morbidity and mortality in cystic fibrosis patients. The long-term goals of this project are to characterize the Aer2-regulated Che2 pathway in P. aeruginosa, from gas sensing to cellular response, and to determine the exact role of that response during infection. These goals align with the NIGMS mission to support basic research that increases our understanding of biological processes and lays a foundation for medical advances. In this initial project period, Aer2 sensing, signaling, an output responses will be characterized. Experiments in Specific Aim 1 will define unique Aer2 sensing and signaling mechanisms. A combination of mutagenesis, behavioral assays and ligand affinity experiments will be used to characterize Aer2 PAS sensing and signal initiation. To determine whether current, non-physiologic structures represent conformational changes that occur in vivo, PAS structures will be solved in both signal-on and signal-off states. Postulated PAS dimer-to-monomer transitions during Aer2 signaling will be assessed by disulfide crosslinking, surface accessibility, bacterial two-hybrid and size exclusion chromatography. To assess HAMP conformational inversions along the Aer2 poly-HAMP chain, signaling defects will be transposed among HAMP domains, and evaluated both behaviorally and for their influence on PAS quaternary structure. Experiments in Specific Aim 2 will assess the virulence of Aer2 and Che2 mutants in an in vivo Caenorhabditis elegans slow-kill assay. Effects on P. aeruginosa virulence will be compared with in vitro phosphorylation assays using Che2 protein components. The role of the Che2 chemosensory system in P. aeruginosa will be uncovered by identifying downstream interacting partners of the Che2 effector protein, CheY2; candidates will be isolated by in vitro pull-down assays, and in vivo bacterial two-hybrid assays with a cloned P. aeruginosa genomic library. Overall, these studies will use an experimentally accessible system to impact our understanding of how Aer2 receptors sense and signal; it will also provide insights into PAS/poly-HAMP signaling mechanisms that will be applicable to many other proteins. Lastly, this study will help define the role of Aer2-regulated chemosensory systems and clarify how that role impacts bacterial pathogenesis.

描述（由申请人提供）：细菌病原体通过改变游动方向（趋化性）、表面运动性、基因表达、生物膜形成和感染过程来寻找、耐受和逃离不同的环境。反应是物种特异性的，但都是由感觉受体（化学感受器）和效应蛋白组成的化学感受系统调制。一个这样的化学感受系统是由称为Aer 2的化学感受器调节的，Aer 2通过PAS感测结构域来感测O2，PAS感测结构域使用新的方法来协调血红素并稳定O2结合。PAS信号直接通过连续的多聚HAMP结构域转导。Aer 2调节系统存在于许多病原体中，包括铜绿假单胞菌，其中该系统被称为Che 2。铜绿假单胞菌是一种重要的条件致病菌，是引起医院获得性感染的重要原因，也是囊性纤维化患者发病率和死亡率的主要原因。该项目的长期目标是表征铜绿假单胞菌中Aer 2调节的Che 2途径，从气体传感到细胞反应，并确定该反应在感染期间的确切作用。这些目标与NIGMS的使命相一致，即支持基础研究，增加我们对生物过程的理解，并为医学进步奠定基础。在这个项目的初始阶段，Aer 2传感，信号，输出响应将被表征。具体目标1中的实验将定义独特的Aer 2传感和信号传导机制。将使用诱变、行为测定和配体亲和力实验的组合来表征Aer 2 PAS传感和信号起始。为了确定当前的非生理结构是否代表体内发生的构象变化，PAS结构将在信号开启和信号关闭状态下求解。将通过二硫键交联、表面可及性、细菌双杂交和分子排阻色谱法评估Aer 2信号传导期间假定的PAS二聚体至单体转换。为了评估沿着Aer 2聚HAMP链的HAMP构象反转，将在HAMP结构域之间转置信号传导缺陷，并在行为上和其对PAS四级结构的影响两者上进行评估。特定目标2中的实验将在体内秀丽隐杆线虫慢杀试验中评估Aer 2和Che 2突变体的毒力。对铜绿假单胞菌毒力的影响将与使用Che 2蛋白组分的体外磷酸化测定进行比较。将通过鉴定Che 2效应蛋白CheY 2的下游相互作用伴侣来揭示Che 2化学传感系统在铜绿假单胞菌中的作用;将通过体外下拉测定和具有克隆的铜绿假单胞菌基因组文库的体内细菌双杂交测定来分离候选物。总的来说，这些研究将使用一个实验可访问的系统来影响我们对Aer 2受体如何感知和信号传导的理解;它还将提供对PAS/poly-HAMP信号传导机制的见解，这些机制将适用于许多其他蛋白质。最后，这项研究将有助于确定Aer 2调节的化学感受系统的作用，并阐明该作用如何影响细菌的发病机制。