Structural Analysis of Vibrio cholerae Virulence Gene Regulatory Proteins

描述(申请人提供)：霍乱弧菌导致致命的流行性腹泻疾病霍乱。其主要毒力因子毒素共调节的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，并作为调节细菌毒力的范例。APHA和AphB通过在tcpPH启动子上的一种新的相互作用启动级联蛋白的表达。APHA是一个新的调控家族的成员，AphB是一个LysR类型的激活物，是最大的转录调控家族之一。一旦表达，TCPP/TcpH和同源跨膜激活剂ToxR/ToxS之间的合作就激活了oxT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只发生在对某些环境刺激的反应中。其中一个这样的刺激，细胞密度，通过群体感应系统调节因子HapR影响毒力级联反应，抑制APHA启动子的表达。这项建议的长期目标是了解毒力基因调控的分子基础，以促进开发更好的预防和治疗细菌疾病的策略。要实现这些目标，需要了解特定的调控蛋白在其同源启动子上如何发挥作用来控制基因表达，并最终了解它们是如何受到环境刺激的影响的。通过在结构生物学、毒力基因调控和致病机理方面的专业实验室的共同努力，我们已经获得了2.2A分辨率的APHA的晶体结构。它的结构揭示了一个带翼的螺旋DNA结合域和一个拓扑上唯一的二聚化结构域的存在。目的1获得(1)APHA及其同源结合部位，(2)AphB单独存在及存在其同源结合部位的高分辨结构，(3)APHA和AphB与DNA形成三元络合物。此外，将使用定点突变来测试特定的结构预测。目标2和3的重点是在没有和存在结合位点的情况下获得ToxT和HapR的高分辨结构，并将进行突变以验证结构预测。这项拟议的工作将极大地提高我们对这些蛋白如何调控毒力基因表达的理解，以便于努力寻找干扰其功能的新分子，并将其作为新型抗毒力药物。

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.