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：
8887427
负责人：
CHRISTOPHER M WATERS
金额：
$ 42.71万
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/8887427
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（通过应用程序提供）：环状DI-GMP（C-DI-GMP）是细菌中几乎普遍，新近欣赏的第二信号信号，有助于致病性促进行为，包括生物膜形成，运动性，病毒因子因子，发展，发育，发育和Quorum敏感性。因此，它的信号通路是对抗生物膜或急性感染的新方法的潜在有吸引力的目标，但是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，生物化学，化学，结构，生化和生化和生化，并有可能实现介导细菌中C-DI-GMP信号传导的机制的治疗靶向。计算方法。我们和其他人以前已经发现，属于NTRC样细菌增强蛋白（EBP）家族的转录因子的子集直接结合并响应C-DI-GMP。 EBP在细菌中广泛宽度，并调节基本细菌行为，包括生物膜形成，运动性，群体敏感性和病毒因子表达。我们进一步发现，C-DI-GMP与纤维状霍乱54-依赖性EBP FLRA的能力结合并抑制了诱导运动的能力。我们的初步数据表明，C-DI-GMP通过将二聚体FLRA锁定成DNA结合的构象来抑制转录，但相反，与70依赖性V. cholerae EBP VPSR结合并激活了诱导生物膜的形成。我们假设C-DI-GMP通过刺激VPSR寡聚化激活转录。在AIMS 1和2中，我们将使用体内和体外遗传和生化测定的合并来检验这些假设，以确定该调节和定义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，以制定EBP的C-DI-GMP调节的机械模型。阐明这些机制将使我们能够在潜水细菌中成千上万的EBP中识别那些受C-DI-GMP调节的EBP。产生纯化FLRA蛋白晶体的初步研究，以及C-DI-GMP不敏感的组成型活性FLRA和VPSR突变体的鉴定支持这些研究的可行性。在AIM 3中，我们将扩展我们的分析，以识别V.霍乱中的新型C-DI-GMP依赖性转录机械，并完全定义C-DI-GMP依赖性调节网络。该分析将充分利用新开发的深层测序技术（TN-Seq，RNA-Seq和iPodhr）。我们将使用这些数据来形成V.霍乱中的C-DI-GMP调节的计算模型，从而对C-DI-GMP对这种病原体的全球影响产生了欣赏，并发现了通常支持C-DI-GMP调节网络的基本原理。我们的研究将推动当前控制细菌转录起始的概念，确定新的靶标或开发新的抗生素，这些抗生素是致病细菌中C-DI-GMP介导的调节的激动剂或拮抗剂。