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

 描述（由申请人提供）：环状二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，有助于致病性促进行为，包括生物膜形成、运动性、毒力因子表达、发育和群体感应。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靶点，但它调节细菌转录的机制在很大程度上是未知的。我们研究的目标是通过整合遗传、生物化学、化学、结构、生物信息学和生物信息学，阐明介导细菌中c-di-GMP信号传导的机制，从而潜在地实现治疗靶向。 计算方法。我们和其他人以前已经发现，属于NtrC样细菌增强子结合蛋白（EBP）家族的转录因子的一个子集直接结合并响应于c-di-GMP。EBP广泛存在于细菌中，并调节细菌的基本行为，包括生物膜形成、运动性、群体感应和毒力因子表达。我们进一步发现，c-di-GMP结合并抑制霍乱弧菌54依赖性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FlrA锁定为不能结合DNA的构象来抑制转录，但相反地结合并激活依赖于EBP 70的霍乱弧菌EBP VpsR以诱导生物膜形成。我们假设c-di-GMP通过刺激VpsR寡聚化激活转录。在目标1和2中，我们将测试这些假设，使用组合的体内和体外遗传和生化测定来鉴定这种调节的关键结构决定因素， 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FlrA和VpsR的X射线晶体结构的阐明相结合，以制定EBP的c-di-GMP调节的机制模型。阐明这些机制将使我们能够在不同细菌物种中的数千种EBP中鉴定出那些受c-di-GMP调节的EBP。初步研究产生纯化的FlrA蛋白的晶体，并鉴定c-di-GMP不敏感的，组成型活性的FlrA和VpsR突变体支持这些研究的可行性。在目标3中，我们将扩大我们的分析，以确定新的c-di-GMP依赖的转录机制在霍乱弧菌和完全定义的c-di-GMP依赖的调控网络。该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调节网络的基本原则。我们的研究将推进目前的概念控制细菌转录起始，确定新的目标或开发新的抗生素，是激动剂或拮抗剂的c-di-GMP介导的调节致病性细菌物种。

 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.