Structural Analysis of Bacterial Virulence Regulators

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。它的两个主要毒力因子，毒素协同调节菌毛和霍乱毒素的表达，通过涉及几个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在tcpPH启动子上通过一种尚未被理解的协同相互作用启动级联表达。AphA是一个新的未知调节家族的成员，而AphB是最大的转录调节家族之一的lysr型激活剂。一旦表达，同源跨膜调控因子TcpP/TcpH和ToxR/ToxS之间的合作激活了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。这种调控在细菌病原体中很普遍，只允许在适当的生物生态位中进行生产性感染。本课题工作的长期目标是了解环境刺激下这种调控的分子基础，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激活因子本身如何启动基因表达，以及它们如何受到特定环境刺激的影响。通过与结构生物学、毒力基因调控和发病机制等方面的专业实验室的合作，本研究旨在探索霍乱弧菌胞质毒力基因调控蛋白AphA、AphB和ToxT在其同源启动子上的结构/功能关系。具体而言，我们建议获得(1)AphA的高分辨率结构；(2) AphB;(3)结合位点缺失和存在的弓形虫。结合正在进行的突变研究，提出的工作将大大增加我们对这些蛋白质如何激活毒力基因表达的理解，将作为其他细菌病原体中这些调节蛋白家族成员的模型，并将推进鉴定可能作为新疗法的分子的努力。

DESCRIPTION (provided by applicant): Vibrio cholerae causes the frequently fatal epidemic diarrheal disease cholera. The expression of its two 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 an as yet not understood synergistic interaction at the tcpPH promoter. AphA is a member of a new and uncharacterized regulator family and AphB is a LysR-type activator, one of the largest transcriptional regulatory families. Once expressed, cooperation between the homologous transmembrane regulators TcpP/TcpH and 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. Such regulation is widespread among bacterial pathogens and allows productive infections to be mounted only in the appropriate biological niches. The long term goals of the work in this proposal are to understand the molecular basis for this regulation by environmental stimuli, so as to facilitate the development of better strategies to prevent and cure bacterial diseases. Achieving these goals requires an understanding of how the activators themselves function to initiate gene expression and, ultimately, how they are influenced by particular environmental stimuli. Through a collaborative effort of laboratories with expertise in structural biology, virulence gene regulation and pathogenesis, this proposal aims to explore the structure/function relationships of the three cytoplasmic vinflence gene regulator proteins in V. cholerae, AphA, AphB and ToxT, at their cognate promoters. Specifically, we propose to obtain high resolution structures of (1) AphA; (2) AphB; and (3) ToxT in the absence and presence of their binding sites. In combination with ongoing mutational studies, the proposed work will significantly increase our understanding of how these proteins activate virulence gene expression, will serve as models for these regulatory protein family members in other bacterial pathogens, and will advance efforts to identify molecules that may function as novel therapeutics.