Determining the transcriptional mechanism of c-di-GMP-dependent biofilm gene activation

摘要 生物膜及其持久性构成了一个严重的公共卫生问题，特别是在医院环境中。 生物膜既附着在生物表面，也附着在非生物表面，与各种不同的感染有关。 由于它们的抗菌药敏感性和清除性抗性显著降低。他们不仅是 发现于医疗设备上，如导管、人工关节植入物和假体设备，但生物膜 在囊性纤维化肺和慢性皮肤伤口中也可发现。在大多数细菌物种中， 第二信使c-di-GMP是生物膜形成的中心调节因子，广泛存在于生物膜中。我们 另一些人最近发现，c-di-GMP直接与一组转录因子相互作用。 属于广泛存在的调节生物被膜基因的NTRC样细菌增强子结合蛋白(EBP)家族 表达以及毒力因子、群体感应和动力基因的表达。霍乱弧菌中， 霍乱是威胁生命的疾病的病原体，造成500万例病例和100,000多人死亡 每年，反应调节子VpsR是与c-di-GMP相互作用的主要EBP，以正向调节 生物膜基因在体内的表达部分位于vpsL生物膜基因启动子。尽管EBPs通常会激活RNA 含有交替的S因子s54的聚合酶，在VpsR的关键残基上需要替换 EBP功能提示VpsR的激活机制是新的。此外，该机制通过 C-di-GMP与转录激活剂相互作用直接改变基因表达尚不清楚。使用V。 以霍乱弧菌为模型研究c-di-GMP信号转导和细菌细胞内生物被膜的形成 这项研究的目的是阐明这种c-di-GMP依赖的转录机制。带着我的 前期工作，我已经建立了一个体外系统，第一次展示了EBP不仅可以 与c-di-GMP一起在体外直接激活转录，也与c-di-GMP一起激活VpsR 转录自RNAP，含有初选的S，s70。使用这些条件，我将在目标1中确定 VpsR/c-di-GMP通过评估DNA结合，RNAP募集， 开放复合体的形成和启动子的清除。在目标2中，我将使用遗传和生化工具来 研究蛋白质-DNA和蛋白质-蛋白质的相互作用，以构建一个三维分子图谱 转录复合体。最后，在目标3，我将确定VpsR/c-di-GMP结合口袋使用高- 通过对c-di-GMP结合和转录激活的体外确认进行遗传筛选 以及体内生物膜形成的分析。这项拟议的研究将是第一次利用体外转录 研究确定c-di-GMP如何与转录调控因子相互作用直接改变基因表达。 这一理解不仅将为c-di-GMP依赖的转录激活和 阐明调节生物膜形成的机制过程，但也提供了所需的基础 抗霍乱弧菌和以生物膜为基础的医院感染的新型化学抑制剂的开发。 好了！

Abstract Biofilms and their persistence pose a serious public health concern, particularly in the hospital setting. Adhering to both biotic and abiotic surfaces, biofilms are implicated in a variety of different infections largely due to their significant decrease in antimicrobial susceptibility and clearance resistance. Not only are they found on medical devices, such as catheters, artificial joint implants, and prosthetic devices, but biofilms are also found in cystic fibrosis lungs and chronic cutaneous wounds. In the majority of bacterial species, the highly ubiquitous and important second messenger, c-di-GMP, is a central regulator of biofilm formation. We and others have recently discovered that c-di-GMP directly interacts with a subset of transcription factors belonging to the widespread NtrC-like bacterial enhancer binding protein (EBP) family to modulate biofilm gene expression as well as virulence factor, quorum sensing, and motility gene expression. In Vibrio cholerae, the causative agent of the life-threatening disease cholera responsible for 5 million cases and over 100,000 deaths per year, the response regulator VpsR is the master EBP that interacts with c-di-GMP to positively regulate biofilm gene expression in vivo in part at the vpsL biofilm gene promoter. Although EBPs typically activate RNA polymerase (RNAP) containing the alternate s factor, s54, substitutions at crucial residues in VpsR needed for EBP function have suggested that the mechanism of VpsR activation is novel. Furthermore, the mechanism by which c-di-GMP interacts with transcriptional activators to directly alter gene expression is unknown. Using V. cholerae as a model to study c-di-GMP signaling and biofilm formation in vitro and in the bacterial cell, the goal of the proposed research is to elucidate this c-di-GMP-dependent transcription mechanism. With my preliminary work, I have established an in vitro system to show for the very first time that not only can an EBP together with c-di-GMP directly activate transcription in vitro, but also VpsR together with c-di-GMP activates transcription from RNAP containing the primary s, s70. Using these conditions, I will determine in Aim 1 the specific step by which VpsR/c-di-GMP activates transcription by assessing DNA binding, RNAP recruitment, open complex formation, and promoter clearance. In Aim 2, I will use genetic and biochemical tools to investigate protein-DNA and protein-protein interactions to construct a 3-dimensional molecular map of the transcription complex. Finally, in Aim 3, I will determine the VpsR/c-di-GMP binding pocket using a high- throughput genetic screen followed by in vitro confirmation for c-di-GMP binding and transcription activation and in vivo analysis for biofilm formation. The proposed research will be the first to utilize in vitro transcriptional studies to determine how c-di-GMP interacts with transcriptional regulators to directly change gene expression. This understanding will not only provide a new paradigm in c-di-GMP-dependent transcription activation and elucidate mechanistic processes that regulate biofilm formation, but also provide the foundation needed for the development of novel chemical inhibitors against V. cholerae and biofilm-based nocosomial infections. !