Structural Analysis of Vibrio cholerae Virulence Gene Regulatory Proteins

霍乱弧菌引起致命的流行性腹泻！霍乱其主要毒力的表达 毒素共调节菌毛和霍乱毒素，通过涉及几个转录级联发生， 激活蛋白，并作为细菌毒力调节的范例。AphA和AphB启动 通过在tcpPH启动子处的新相互作用来表达级联。AphA是一个新的成员 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和同源跨膜激活剂之间的合作， ToxR/ToxS激活toxT启动子。ToxT是一种AraC型调节因子，然后直接激活启动子 主要的毒力因子。这些不同启动子的转录激活仅发生在 对某些环境刺激的反应。其中一种刺激，细胞密度，影响毒性级联反应 通过群体感应系统调节剂HapR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， 促进发展更好的策略来预防和治疗细菌性疾病。实现这些 这些目标需要了解特定的调节蛋白如何在其同源启动子中发挥作用 来控制基因表达，并最终控制它们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，我们已经获得了2.2 A分辨率的AphA的晶体结构。它的结构揭示了 翼螺旋DNA结合结构域和拓扑学上独特的二聚化结构域的存在。要求1 重点在于获得（1）AphA及其同源结合位点，（2）单独的AphB， 在其同源结合位点的存在下，和（3）AphA和AphB一起与DNA形成三元复合物。 此外，将使用定点诱变检测特定结构预测。目标2和3重点 在缺乏和存在ToxT和HapR结合位点的情况下获得高分辨率结构 还将进行诱变以测试结构预测。这项工作将大大 增加我们对这些蛋白质如何调节毒力基因表达的理解， 努力鉴定干扰其功能的新分子， 抗病毒药物

Vibrio cholerae causes the fatal epidemic diarrhea! 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 DMA 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.