Control of virulence in Vibrio cholerae by fatty acids

霍乱弧菌O 1引起致命的流行病霍乱。霍乱弧菌能够引起 人类的疾病依赖于两个主要的毒力因子，毒素共调节菌毛（TCP）， 关键定植因子和霍乱毒素（CT）。这些因子的表达受一个高度依赖的 调节的转录级联，其用作调节细菌毒力的范例。 级联反应的表达是在tcpPH启动子处通过以下相互作用而启动的： 调节剂AphA和AphB。TcpPH和ToxRS是跨膜调节子的同源对， 协同激活toxT启动子的表达。ToxT是一种AraC型调节剂， TCP和CT的表达。毒力级联的转录激活强烈依赖于 来自外部环境的各种刺激。本提案的长期目标是了解 霍乱弧菌毒力基因调控的分子基础，以促进新的 控制其传染性的策略。通过与具有以下专业知识的实验室的合作努力， 结构生物学、毒力基因调控和致病机理等方面，我们发现外源性不饱和脂肪酸 脂肪酸（UFA）是人类胆汁的成分，能够与ToxT结合并损害其毒性。 激活毒力基因表达的能力。UFA结合到ToxT N-末端结构域的配体口袋中 并抑制其二聚化以及与DNA结合的能力。Bicarbonate，它能中和 来自胃，已被证明作为第二个体内信号，与UFA相反， 刺激ToxT并通过未知机制增强其DNA结合。我们最近发现了一个 脂肪酸（FAs）与当前霍乱弧菌流行株毒力基因表达之间的新联系 随着发现FA代谢的主要调节因子FadR，通过一种新的途径影响ToxT的翻译， 未知机制该提案将建立在ToxT结构和功能数据，以及我们的 最近的研究涉及FadR，以阐明参与调控的几个关键机制， 毒力级联的表达。在目的1中，我们提出阐明控制 ToxT的二聚化，并研究UFA和碳酸氢盐对该过程的相反影响。在 目的2：阐明FadR影响ToxT在V. 胆汁。这些研究将有助于我们了解毒力基因是如何 表达在霍乱弧菌中受到调控，可能为发现抗病毒药物提供新的途径。

Vibrio cholerae O1 causes the fatal epidemic disease cholera. The ability of V. cholerae to cause disease in humans is dependent upon two primary virulence factors, the toxin-coregulated pilus (TCP), a critical colonization factor, and cholera toxin (CT). The expression of these factors is controlled by a highly regulated transcriptional cascade that serves as a paradigm for the regulation of bacterial virulence. Expression of the cascade is initiated at the tcpPH promoter by a cooperative interaction between the regulators AphA and AphB. TcpPH and ToxRS are homologous pairs of transmembrane regulators that then cooperate to activate expression from the toxT promoter. ToxT, an AraC-type regulator, directly activates the expression of TCP and CT. Transcriptional activation of the virulence cascade is strongly dependent upon a variety of stimuli from the external environment. The long-term goals of this proposal are to understand the molecular basis of virulence gene regulation in V. cholerae so as to facilitate the development of new strategies to control its infectivity. Through a collaborative effort involving laboratories with expertise in structural biology, virulence gene regulation and pathogenesis, we have found that exogenous unsaturated fatty acids (UFAs), which are components of human bile, are capable of binding to ToxT and impairing its ability to activate virulence gene expression. UFAs bind into a ligand pocket in the N-terminal domain of ToxT and inhibit its dimerization as well as its ability to bind to DNA. Bicarbonate, which neutralizes the acid that comes from the stomach, has been shown to function as a second in vivo signal that, in contrast to UFAs, stimulates ToxT and enhances its DNA binding through an unknown mechanism. We have recently identified a new link between fatty acids (FAs) and virulence gene expression in the current pandemic strain of V. cholerae with the discovery that the master regulator of FA metabolism, FadR, influences the translation of ToxT by an unknown mechanism. This proposal will build upon the ToxT structural and functional data, as well as our recent studies involving FadR, in order to elucidate several key mechanisms involved in regulating the expression of the virulence cascade. In Aim 1, we propose to elucidate the allosteric mechanisms controlling the dimerization of ToxT and to investigate the opposing effects of UFAs and bicarbonate on this process. In Aim 2, we propose to elucidate the mechanism by which FadR influences the translation of ToxT in V. cholerae. These studies will contribute significantly toward our understanding of how virulence gene expression is regulated in V. cholerae and will likely provide new avenues for antivirulence drug discovery.