Structural Analysis of Vibrio cholerae Virulence Gene Regulatory Proteins

霍乱弧菌导致致命的流行性腹泻！霍乱。其主要毒力的表达 毒素共同调节的菌毛和霍乱毒素等因子通过转录级联反应发生，涉及几个 激活蛋白，并作为调节细菌毒力的范例。APHA和AphB启动子 在tcpPH启动子上通过一种新的相互作用来表达级联蛋白。APHA是一个新的 AphB是一种LysR型激活物，是最大的转录调控家族之一。 一旦表达，TCPP/TcpH与同源跨膜激活剂之间的协同作用 ToxR/ToxS激活ToxT启动子。ToxT是一种AraC型调节子，然后直接激活启动子 主要的致病因子。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。一种这样的刺激，细胞密度，影响毒力级联反应。 通过群体感应系统调节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，以便 促进制定更好的预防和治疗细菌性疾病的战略。实现这些目标 Goals要求了解特定的调控蛋白在其同源启动子中的功能。 以控制基因的表达，并最终控制它们如何受到环境刺激的影响。通过一个 拥有结构生物学、毒力基因调控和 发病机制，我们在2.2A分辨率下获得了APHA的晶体结构。它的结构揭示了 存在带翼的螺旋DNA结合域和拓扑上唯一的二聚化结构域。目标1 重点是获得(1)APHA及其同源结合部位，(2)AphB单独和 (3)APHA和AphB一起与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和目标3焦点 ToxT和HapR在没有和存在结合部位的情况下获得高分辨结构的研究 还将进行突变试验，以检验结构预测。这项拟议的工作将显著 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 努力确定干扰其功能的新分子，并可能作为新的 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.