From structure to systems: Understanding cyclic di-GMP control of transcription

从结构到系统：了解转录的环状二 GMP 控制

基本信息

批准号：
9102193
负责人：
CHRISTOPHER M WATERS
金额：
$ 39.74万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9102193
关键词：
Acute Affect Agonist Amino Acids Anabolism Antibiotics Bacteria Bacterial Infections Behavior Binding Biochemical Biochemistry Bioinformatics Biological Assay Chemicals Chromatin Loop Collaborations Complex Computer Simulation Coupled DNA Binding DNA-Directed RNA Polymerase Data Development Family Feasibility Studies Fluorescence-Activated Cell Sorting Gene Expression Genes Genetic Genetic Transcription Goals Homologous Protein Human In Vitro Infection Infection prevention Knowledge Libraries Maps Mediating Methods Microbial Biofilms Microbial Genetics Modeling Molecular Molecular Conformation Outcome Pathogenicity Pathway interactions Phenotype Positioning Attribute Process Protein Family Proteins Publications Regulation Regulon Research Roentgen Rays Second Messenger Systems Signal Pathway Signal Transduction Signal Transduction Pathway Stimulus Structure System Technology Testing Therapeutic Intervention Transcription Coactivator Transcriptional Regulation Vibrio cholerae Virulence Virulence Factors base blind cell motility combat deep sequencing enhancer binding protein in vitro Assay in vivo insight mutant novel novel strategies pathogen pathogenic bacteria prevent public health relevance quorum sensing response second messenger structural biology therapeutic target trait transcription factor transcriptome sequencing transcriptomics

项目摘要

DESCRIPTION (provided by applicant): Cyclic di-GMP (c-di-GMP) is a near-ubiquitous, newly appreciated second messenger signal in bacteria that contributes to pathogenicity-promoting behaviors including biofilm formation, motility, virulence factor expression, development, and quorum sensing. Its signaling pathways are thus potentially attractive targets for new approaches to combat biofilm-based or acute infections, but the mechanisms by which it regulates transcription in bacteria are largely unknown. The goal of our research is to elucidate, and thus potentially enable therapeutic targeting of, the mechanisms that mediate c-di-GMP signaling in bacteria by integrating genetic, biochemical, chemical, structural, bioinformatic, and computational approaches. We and others have previously found that a subset of transcription factors belonging to the NtrC-like bacterial enhancer binding protein (EBP) family directly bind and respond to c-di-GMP. EBPs are widespread in bacteria, and regulate fundamental bacterial behaviors including biofilm formation, motility, quorum sensing, and virulence factor expression. We further found that c-di-GMP binds to and inhibits the ability of the Vibrio cholerae 54- dependent EBP FlrA to induce motility. Our preliminary data suggest that c-di-GMP inhibits transcription by locking dimeric FlrA into a conformation incapable of DNA binding, but conversely binds to and activates the 70-dependent V. cholerae EBP VpsR to induce biofilm formation. We hypothesize that c-di-GMP activates transcription by stimulating VpsR oligomerization. In Aims 1 & 2 we will test these hypotheses, using combined in vivo and in vitro genetic and biochemical assays to identify critical structural determinants for this regulation and define the impact of c-di-GMP on transcription factor activity. These studies will be integrated with the elucidation of the X-ray crystal structures of FlrA and VpsR in the presence and absence of c-di-GMP binding to formulate a mechanistic model of c-di-GMP regulation of EBPs. Elucidating these mechanisms will allow us to identify among the thousands of EBPs in diverse bacterial species those that are c-di-GMP-regulated. Preliminary studies generating crystals of purified FlrA proteins, and the identification of c-di-GMP-insensitive, constitutively active FlrA and VpsR mutants support the feasibility of these studies. In Aim 3 we will expand our analysis to identify novel c-di-GMP-dependent transcriptional machinery in V. cholerae and completely define the c-di-GMP-dependent regulatory network. This analysis will fully harness newly developed deep sequencing technologies (TN-seq, RNA-seq, and IPODHR). We will use these data to formulate a computation model of the c-di-GMP regulon in V. cholerae, gaining an appreciation for the global impact of c-di-GMP on this pathogen and uncovering fundamental principles that generally underpin c-di-GMP regulatory networks. Our studies will advance current concepts of the control of bacterial transcriptional initiation, identifying novel targets or development of new antibiotics that are agonists or antagonists of c-di-GMP-mediated regulation in pathogenic bacterial species.

描述（由申请人提供）：环二 GMP (c-di-GMP) 是细菌中近乎普遍存在的、新近被认识的第二信使信号，有助于致病性促进行为，包括生物膜形成、运动、毒力因子表达、发育、因此，它的信号通路是对抗基于生物膜或急性感染的新方法的潜在有吸引力的目标，但它调节细菌转录的机制在很大程度上是未知的。我们研究的目的是通过整合遗传、生化、化学、结构、生物信息学和生物信息学，阐明细菌中介导 c-di-GMP 信号传导的机制，从而有可能实现治疗靶向。我们和其他人之前发现，属于 NtrC 样细菌增强子结合蛋白 (EBP) 家族的转录因子子集直接结合并响应 c-di-GMP，并调节基本细菌。我们进一步发现 c-di-GMP 结合并抑制霍乱弧菌 54 依赖性 EBP 的能力。 FlrA 诱导运动。我们的初步数据表明，c-di-GMP 通过将二聚体 FlrA 锁定为无法结合 DNA 的构象来抑制转录，但相反，它会结合并激活 70 依赖性霍乱弧菌 EBP VpsR 以诱导生物膜形成。我们认为 c-di-GMP 通过刺激 VpsR 寡聚化来激活转录。在目标 1 和 2 中，我们将使用体内组合来测试这些假设。以及体外遗传和生化测定，以确定这种调节的关键结构决定因素和定义 c-di-GMP 对转录因子活性的影响，这些研究将与在存在和不存在 c-di-GMP 结合的情况下阐明 FlrA 和 VpsR 的 X 射线晶体结构相结合，以制定机制模型。阐明这些机制将使我们能够在不同细菌物种的数千个 EBP 中识别出那些受 c-di-GMP 调节的晶体。纯化的 FlrA 蛋白的纯化，以及对 c-di-GMP 不敏感、组成型活性 FlrA 和 VpsR 突变体的鉴定支持了这些研究的可行性。在目标 3 中，我们将扩展我们的分析，以鉴定新的 c-di-GMP 依赖性转录机制。该分析将充分利用新开发的深度测序技术（TN-seq、RNA-seq 和 IPODHR）。使用这些数据制定霍乱弧菌中 c-di-GMP 调节子的计算模型，了解 c-di-GMP 对这种病原体的全球影响，并揭示通常支持 c-di-GMP 监管的基本原理我们的研究将推进当前控制细菌转录起始的概念，确定新的靶点或开发新的抗生素，这些抗生素是病原菌物种中 c-di-GMP 介导的调节的激动剂或拮抗剂。