Structural Analysis of Bacterial Virulence Regulators

描述（由申请人提供）：霍乱弧菌引起经常致命的流行性霍乱。它的两个主要毒力因子，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通过在tcpPH启动子处尚未理解的协同相互作用启动级联的表达。AphA是一个新的和未表征的调节因子家族的成员，AphB是一个LysR型激活因子，是最大的转录调节因子家族之一。一旦表达，同源跨膜调节子TcpP/TcpH和ToxR/ToxS之间的合作激活toxT启动子。ToxT是一种AraC型调节因子，然后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响应某些环境刺激时发生。这种调节在细菌病原体中广泛存在，并允许生产性感染仅在适当的生物小生境中进行。这项工作的长期目标是了解环境刺激对这种调节的分子基础，以促进开发更好的预防和治疗细菌性疾病的策略。实现这些目标需要了解激活剂本身如何启动基因表达，以及最终如何受到特定环境刺激的影响。通过在结构生物学、毒力基因调控和致病机理方面具有专业知识的实验室的合作努力，该提议旨在探索霍乱弧菌中的三种细胞质毒力基因调控蛋白AphA、AphB和ToxT在其同源启动子处的结构/功能关系。具体而言，我们建议获得（1）AphA;（2）AphB;和（3）ToxT在其结合位点的存在和不存在下的高分辨率结构。结合正在进行的突变研究，拟议的工作将显着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，并将推动鉴定可能作为新型治疗剂的分子的努力。

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.