Structural Analysis of Vibrio cholerae Virulence Gene Regulatory Proteins

霍乱弧菌毒力基因调控蛋白的结构分析

• 批准号: 8293819
• 负责人: Fredrick Jon Kull
• 金额: $ 46.22万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-15 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293819
• 关键词: Affect; Affinity; Bacterial Infections; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Carbon; Cell Density; Cessation of life; Characteristics; Cholera; Cholera Toxin; Collaborations; Complex; Crystallography; DNA; DNA Binding; DNA Sequence; Data; Development; Dimerization; Disease; Docking; Enteral; Epidemic; Family; Family member; Fatty Acids; Foundations; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Homologous Gene; Homology Modeling; Indium; Investigation; Knowledge; Laboratories; Lead; Ligand Binding; Ligands; Methods; Molecular; Monounsaturated Fatty Acids; Mutation; Nature; Operon; Pathogenesis; Pathogenicity; Pharmaceutical Preparations; Pilum; Process; Proteins; Regulation; Research Design; Resolution; Role; Site-Directed Mutagenesis; Staging; Stimulus; Structure; Structure-Activity Relationship; System; Toxin; Transcriptional Activation; Vibrio cholerae; Virulence; Virulence Factors; World Health Organization; base; chemical genetics; dimer; disorder control; genetic regulatory protein; improved; inhibitor/antagonist; insight; member; mutant; novel; novel therapeutics; palmitoleic acid; pathogenic bacteria; prevent; promoter; protein function; quorum sensing; response; small molecule; structural biology; therapeutic development

DESCRIPTION (provided by applicant): Vibrio cholerae causes the fatal epidemic diarrheal disease cholera. The expression of its primary virulence factors, toxin-coregulated pilus and cholera toxin, occurs via a transcriptional cascade involving several activator proteins and serves as a paradigm for the regulation of bacterial virulence. AphA and AphB initiate the expression of the cascade by a novel interaction at the tcpPH promoter. AphA is a member of a new regulator family and AphB is a LysR-type activator, one of the largest transcriptional regulatory families. Once expressed, cooperation between TcpP/TcpH and the homologous transmembrane activators ToxR/ToxS activates the toxT promoter. ToxT, an AraC-type regulator, then directly activates the promoters of the primary virulence factors in a fatty acid dependent manner. Transcriptional activation at these various promoters occurs only in response to certain environmental stimuli. One such stimulus, cell density, influences the virulence cascade through the quorum sensing system regulator HapR which represses the expression of the aphA promoter. The long term goals of this proposal are to understand the molecular basis of virulence gene regulation so as to facilitate the development of better strategies to prevent and cure bacterial diseases. Achieving these goals requires an understanding of how the specific regulatory proteins function at their promoters to control gene expression and, ultimately, how they are influenced by environmental stimuli. Through a collaborative effort involving laboratories with expertise in structural biology, virulence gene regulation, and pathogenesis, we have solved crystal structures of AphA, AphB, HapR, and ToxT. This proposal will build upon this structural data, as well as our functional results, in ordr to continue to elucidate the detailed mechanisms required for regulation of the V. cholerae virulence genes. In Aim 1, we propose to determine the crystal structures of AphA, AphB, and HapR in complex with their respective DNA binding sites, allowing us to observe the structural changes that take place upon DNA binding. In Aim 2, we plan to characterize the ligand binding pockets of AphB and HapR. As the natural ligands for these proteins are not known, we plan to identify small molecule ligands for HapR and AphB, and then visualize the structural changes induced in the proteins by ligand binding. Aim 3 carries on our investigation of the mechanism by which fatty acid binding regulates the activity of ToxT, the master regulator of virulence gene expression in V. cholerae. In addition to crystallography, we will utilize structure based site directed mutagenesis, biochemical activity assays, biophysical characterization assays, spectroscopic characterization of binding and conformational change, and computational and NMR based methods for identifying ligands. These studies will greatly clarify the mechanistic and structural roles of proteins involved in the regulation of bacterial virulence gene expression. Such knowledge will facilitate the identification of molecules interfering with regulatory cascades, and could lead to the development of novel therapeutics. PUBLIC HEALTH RELEVANCE: According to the World Health Organization, each year there are an estimated 3 to 5 million cases of cholera, resulting in over 100,000 deaths. Cholera is caused by Vibrio cholerae, a pathogenic bacterium that utilizes a highly regulated transcriptional cascade to produce its two major virulence genes. These studies are designed to investigate the structural and functional characteristics of these regulatory proteins, and could lead to the development of therapeutics to treat or prevent cholera, as well as other enteric bacterial infections.

描述（由申请人提供）：霍乱弧菌引起致命的流行性腹泻病霍乱。其主要毒力因子（毒素共调节菌毛和霍乱毒素）的表达通过涉及几种激活蛋白的转录级联发生，并作为细菌毒力调节的范例。 AphA 和 AphB 通过 tcpPH 启动子处的新型相互作用启动级联的表达。 AphA 是新调节家族的成员，AphB 是 LysR 型激活剂，是最大的转录调节家族之一。一旦表达，TcpP/TcpH 与同源跨膜激活剂 ToxR/ToxS 之间的合作会激活 toxT 启动子。 ToxT 是一种 AraC 型调节因子，然后以脂肪酸依赖性方式直接激活初级毒力因子的启动子。这些不同启动子的转录激活仅在响应某些环境刺激时发生。其中一种刺激物，细胞密度，通过群体感应系统调节器 HapR 影响毒力级联，HapR 抑制 aphA 启动子的表达。该提案的长期目标是了解毒力基因调控的分子基础，以促进制定更好的策略来预防和治疗细菌性疾病。实现这些目标需要了解特定调节蛋白如何在其启动子上发挥作用以控制基因表达，以及最终如何受到环境刺激的影响。通过具有结构生物学、毒力基因调控和发病机制专业知识的实验室的共同努力，我们解决了 AphA、AphB、HapR 和 ToxT 的晶体结构。该提案将建立在这些结构数据以及我们的功能结果的基础上，以便继续阐明霍乱弧菌毒力基因调节所需的详细机制。在目标 1 中，我们建议确定 AphA、AphB 和 HapR 与其各自 DNA 结合位点复合物的晶体结构，使我们能够观察 DNA 结合时发生的结构变化。在目标 2 中，我们计划表征 AphB 和 HapR 的配体结合口袋。由于这些蛋白质的天然配体尚不清楚，我们计划鉴定 HapR 和 AphB 的小分子配体，然后可视化配体结合引起的蛋白质结构变化。目标 3 继续研究脂肪酸结合调节 ToxT 活性的机制，ToxT 是霍乱弧菌毒力基因表达的主要调节因子。除了晶体学之外，我们还将利用基于结构的定点诱变、生化活性测定、生物物理表征测定、结合和构象变化的光谱表征以及基于计算和核磁共振的方法来识别配体。这些研究将极大地阐明参与细菌毒力基因表达调节的蛋白质的机制和结构作用。 这些知识将有助于识别干扰调控级联的分子，并可能导致新疗法的开发。 公共卫生相关性：据世界卫生组织统计，每年估计有 3 至 500 万霍乱病例，导致超过 10 万人死亡。霍乱是由霍乱弧菌引起的，霍乱弧菌是一种病原菌，利用高度调控的转录级联产生其两个主要毒力基因。这些研究旨在研究这些调节蛋白的结构和功能特征，并可能导致开发治疗或预防霍乱以及其他肠道细菌感染的疗法。