Structural Analysis of Vibrio cholerae Virulence Gene Regulatory Proteins

描述（由申请人提供）：霍乱弧菌引起致命的流行性腹泻疾病霍乱。其主要毒力因子，毒素协同调节的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通过在tcpPH启动子上的一种新的相互作用来启动级联表达。AphA是一个新的调控家族的成员，而AphB是lysr型激活因子，是最大的转录调控家族之一。一旦表达，TcpP/TcpH与同源跨膜激活剂ToxR/ToxS合作激活toxT启动子。然后，一种arac型调节剂ToxT直接激活主要毒力因子的启动子。这些不同启动子的转录激活仅在对某些环境刺激的反应中发生。其中一种刺激是细胞密度，它通过群体感应系统调节因子HapR抑制aphA启动子的表达来影响毒力级联反应。本课题的长期目标是了解毒力基因调控的分子基础，从而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控蛋白如何在其同源启动子上起作用以控制基因表达，并最终了解它们如何受到环境刺激的影响。通过与具有结构生物学、毒力基因调控和发病机制等专业知识的实验室的合作，我们获得了分辨率为2.2 a的AphA晶体结构。它的结构揭示了一个带翼螺旋DNA结合域和一个拓扑独特的二聚化域的存在。Aim 1的重点是获得高分辨率的结构(1)AphA及其同源结合位点，(2)单独和同源结合位点存在的AphB，以及(3)AphA和AphB与DNA组成三元配合物。此外，特定的结构预测将使用定点诱变进行测试。目标2和目标3侧重于在缺乏和存在其结合位点的情况下获得ToxT和HapR的高分辨率结构，并且还将进行诱变以测试结构预测。这项拟议的工作将大大增加我们对这些蛋白质如何调节毒力基因表达的理解，以促进识别干扰其功能的新分子，并可能作为新的抗毒药物。

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. 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 cognate promoters to control gene expression and, ultimately, how they are influenced by environmental stimuli. Through a collaborative effort of laboratories with expertise in structural biology, virulence gene regulation and pathogenesis, we have obtained the crystal structure of AphA at 2.2 A resolution. Its structure reveals the presence of a winged-helix DNA binding domain and a topologically unique dimerization domain. Aim 1 focuses on obtaining high resolution structures of (1) AphA with its cognate binding site, (2) AphB alone and in the presence of its cognate binding site, and (3) AphA and AphB together in a ternary complex with DNA. In addition, specific structural predictions will be tested using site-directed mutagenesis. Aims 2 and 3 focus on obtaining high resolution structures of ToxT and HapR in the absence and presence of their binding sites and mutagenesis will also be carried out to test structural predictions. This proposed work will significantly increase our understanding of how these proteins regulate virulence gene expression in order to facilitate efforts to identify new molecules that interfere with their functions and which may serve as novel antivirulence drugs.