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

确定 c-di-GMP 依赖性生物膜基因激活的转录机制

• 批准号: 9395522
• 负责人: Meng-Lun Hsieh
• 金额: $ 4.04万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-16 至 2019-08-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9395522
• 关键词: 3-Dimensional; Affect; Amino Acids; Animal Model; Antimicrobial susceptibility; Bacteria; Bacterial Infections; Binding; Biochemical; Biochemistry; Biological Assay; Catheters; Cell Cycle Regulation; Cells; Cessation of life; Chemicals; Cholera; Chronic; Complex; Coupled; Cutaneous; Cystic Fibrosis; DNA; DNA Binding; DNA Footprint; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Developing Countries; Development; Dimerization; Disease; Disease Outbreaks; Exhibits; Family; Foundations; Gene Activation; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Guanosine Monophosphate; Haiti; Hospitals; In Vitro; Industry; Infection; Joint Prosthesis; Knowledge; Laboratories; Life; Maps; Mediating; Medical Device; Mentorship; Michigan; Microbial Biofilms; Microbial Genetics; Modeling; Molecular; Outcome; Periodicity; Positioning Attribute; Process; Prosthesis; Protein Family; Proteins; Public Health; Pulmonary Fibrosis; Recruitment Activity; Regulation; Regulatory Pathway; Research; Resistance; Role; Second Messenger Systems; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Site; Structure; Surface; System; Testing; Time; Transcription Coactivator; Transcription Initiation; Transcription Initiation Site; Transcriptional Activation; Transcriptional Regulation; United States National Institutes of Health; Universities; Vibrio cholerae; Virulence; Virulence Factors; Water; Work; base; biochemical tools; cell motility; chronic wound; dimer; enhancer binding protein; experimental study; healing; implantable device; in vivo; inhibitor/antagonist; insight; new therapeutic target; novel; pathogen; prevent; promoter; protein function; protein protein interaction; quorum sensing; response; transcription factor; transmission process; yeast two hybrid system

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. !

抽象的 生物膜及其持续存在构成了严重的公共卫生问题，特别是在医院环境中。 生物膜粘附在生物和非生物表面，很大程度上与多种不同的感染有关 由于它们的抗菌敏感性和清除耐药性显着降低。他们不仅是 生物膜存在于医疗设备中，例如导管、人工关节植入物和假肢装置，但生物膜 也见于囊性纤维化肺和慢性皮肤伤口。在大多数细菌物种中， c-di-GMP 是高度普遍存在且重要的第二信使，是生物膜形成的核心调节因子。我们 等人最近发现 c-di-GMP 直接与转录因子的子集相互作用 属于广泛存在的 NtrC 样细菌增强子结合蛋白 (EBP) 家族，可调节生物膜基因 表达以及毒力因子、群体感应和运动基因表达。在霍乱弧菌中， 致命疾病霍乱的病原体，导致 500 万病例和 10 万人死亡 每年，响应调节器 VpsR 是与 c-di-GMP 相互作用以积极调节的主 EBP 生物膜基因在体内的表达部分位于vpsL生物膜基因启动子处。尽管 EBP 通常会激活 RNA 含有替代 s 因子 s54 的聚合酶 (RNAP)，在 VpsR 中的关键残基处进行取代 EBP功能表明VpsR激活机制是新颖的。此外，该机制通过 哪种 c-di-GMP 与转录激活剂相互作用以直接改变基因表达尚不清楚。使用V。 霍乱作为模型来研究体外和细菌细胞中的 c-di-GMP 信号传导和生物膜形成，目标 拟议研究的目的是阐明这种 c-di-GMP 依赖性转录机制。与我的 前期工作中，我建立了一个体外系统，首次证明 EBP 不仅可以 与 c-di-GMP 一起直接在体外激活转录，而且 VpsR 与 c-di-GMP 一起激活 从含有初级 s、s70 的 RNAP 转录。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、 开放复合物形成和启动子清除。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-蛋白质相互作用，构建 3 维分子图谱 转录复合物。最后，在目标 3 中，我将使用高通量测定 VpsR/c-di-GMP 结合口袋。 通量遗传筛选，然后体外确认 c-di-GMP 结合和转录激活 以及生物膜形成的体内分析。拟议的研究将是第一个利用体外转录的研究 研究确定 c-di-GMP 如何与转录调节因子相互作用以直接改变基因表达。 这种理解不仅将为 c-di-GMP 依赖性转录激活提供新的范例， 阐明调节生物膜形成的机制过程，同时也为生物膜形成提供所需的基础 开发针对霍乱弧菌和基于生物膜的医院感染的新型化学抑制剂。 ！