Environmental Modulation of ToxT-dependent Transcription

霍乱是一种经常致命的腹泻疾病，由霍乱弧菌引起。这种病 仍然是世界上大多数人的健康威胁，每年造成数千人死亡。我们有 最近证明，霍乱弧菌毒力基因的主要转录激活因子ToxT是 受某些环境信号的负调控，特别是胆汁的存在。我们的研究 重点剖析环境调控ToxT转录的分子机制(S) 活动，利用胆汁作为环境调节因子。我们希望了解和利用这一点 负向管制，以发展预防霍乱的新方法。从本质上讲，人们对 ToxT的结构/功能，因此这些研究还包括对ToxT蛋白功能的阐明。 我们的方法首先涉及表征ToxT的域结构。这将通过以下方式实现 (I)。嵌合ToxT蛋白的构建和鉴定，以及(Ii)。ToxT氨基酸的鉴定 对DNA结合和转录激活很重要。ToxT的进一步表征将包括 霍乱弧菌ToxT调控基因的基因芯片鉴定及特性分析 ToxTDNA结合位点的研究(S)。一旦我们对ToxT有了更彻底的了解，我们就会确定 利用胆汁作为环境信号调控ToxT转录活性的机制 调节因子。这些研究包括：(I)。多孔蛋白OmpU和OmpT的药效测定 (Ii)胆汁对毒素T活性的调节作用。 与胆汁调节毒素T活性有关的其他霍乱弧菌基因的鉴定，(Iii)。识别 胆汁调节所必需的ToxT氨基酸，以及(Iv)。胆汁对弓形虫DNA影响的测定 结合活性。最后，环境对ToxT活性的调节(胆汁或其他 刺激)将通过测试含有突变的霍乱弧菌菌株的毒力特性进行评估 影响ToxT转录的各个方面。我们的最终目标是学习如何通过以下方式操纵ToxT 外界因素，以抑制毒力基因表达，预防霍乱，即迫使霍乱弧菌 防止自身致病。这种完全不同的霍乱治疗方法应该会导致 新的抗菌策略模仿胆汁的效果，这将在抗击霍乱方面有用。

Cholera is an often-fatal diarrheal disease caused by the bacterium Vibrio cholerae. This disease remains a health threat for the majority of the world, causing thousands of deaths every year. We have recently demonstrated that ToxT, the primary transcriptional activator of virulence genes in K cholerae, is negatively regulated by certain environmental signals, and specifically by the presence of bile. Our studies will focus on dissecting the molecular mechanism(s) of environmental modulation of ToxT transcriptional activity, utilizing bile as an environmental modulatory factor. We wish to understand and exploit this negative regulation to develop novel means to prevent cholera. Essentially nothing is known about the structure/function of ToxT, so these studies also include the elucidation of the functions of the ToxT protein. Our approach first involves characterizing the domain structure of ToxT. This will be accomplished by (i). construction and characterization of chimeric ToxT proteins, and (ii). identification of ToxT amino acids important for DNA binding and transcriptional activation. Further characterization of ToxT will include the identification of all the ToxT-regulated genes of V. cholerae by microarray analysis, and the characterization of the ToxT DNA binding site(s). Once we have a more thorough understanding of ToxT, we will determine the mechanism of modulation of ToxT transcriptional activity by environmental signals, utilizing bile as the modulatory factor. These studies include: (i). determination of the effect of the porins OmpU and OmpT (which are known to be differentially permeable to bile) on bile modulation of ToxT activity, (ii). identification of additional V. cholerae genes involved in bile regulation of ToxT activity, (iii). identification of ToxT amino acids necessary for bile regulation, and (iv). determination of the effects of bile on ToxT DNA binding activity. Finally, the relevance of environmental modulation of ToxT activity (by bile or other stimuli) will be assessed by testing the virulent properties of K cholerae strains containing mutations which affect various aspects of ToxT transcription. Our ultimate goal is to learn how to manipulate ToxT by external factors in order to repress virulence gene expression and prevent cholera, i.e. to force V. cholerae to prevent itself from causing disease. This fundamentally different approach to cholera therapy should lead to novel antimicrobial strategies mimicking the effects of bile which will be useful in combating cholera.