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

• 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
• 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

 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)：c-di-GMP(c-di-GMP)是细菌中一种几乎普遍存在的新发现的第二信使信号，它有助于促进致病行为，包括生物膜的形成、运动、毒力因子的表达、发育和群体感应。因此，它的信号通路可能成为对抗生物被膜或急性感染的新方法的潜在靶点，但它调节细菌转录的机制在很大程度上尚不清楚。我们研究的目标是阐明，并因此有可能实现治疗靶向，通过整合遗传，生化，化学，结构，生物信息学和 计算方法。我们和其他人之前已经发现，属于NTRC样细菌增强子结合蛋白(EBP)家族的转录因子的子集直接结合并响应c-di-GMP。EBPs广泛存在于细菌中，调节细菌的基本行为，包括生物膜的形成、运动性、群体感应和毒力因子的表达。我们进一步发现c-di-GMP结合并抑制依赖霍乱弧菌54的EBP Flra诱导运动的能力。我们的初步数据表明，c-di-GMP通过将二聚体fla锁定在不能与结合的构象来抑制转录，但反过来又与依赖于dna 70的霍乱弧菌EBP VpsR结合并激活，以诱导生物膜的形成。我们假设c-di-GMP通过刺激VpsR寡聚来激活转录。在目标1和目标2中，我们将测试这些假设，使用体内和体外遗传和生化分析相结合的方法来确定这一调控的关键结构决定因素和 确定c-di-GMP对转录因子活性的影响。这些研究将与阐明c-di-GMP结合和不存在c-di-GMP结合时Flra和VpsR的X射线晶体结构相结合，以建立c-di-GMP调控EBPs的机制模型。阐明这些机制将使我们能够在不同细菌物种的数千个EBPs中识别那些受c-di-GMP调控的EBPs。纯化的Flra蛋白晶体的初步研究，以及c-di-GMP不敏感的、成分活性的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，以鉴定霍乱弧菌中依赖c-di-GMP的新的转录机制，并完整地定义依赖c-di-GMP的调控网络。这项分析将充分利用新开发的深度测序技术(TN-SEQ、RNA-SEQ和IPODHR)。我们将使用这些数据来建立霍乱弧菌c-di-GMP调节子的计算模型，从而了解c-di-GMP对这种病原体的全球影响，并揭示一般支撑c-di-GMP调控网络的基本原理。我们的研究将推进当前控制细菌转录启动的概念，识别新的靶点或开发新的抗生素，这些抗生素是c-di-GMP介导的致病细菌物种调节的激动剂或拮抗剂。