Environmental Modulation of ToxT-dependent Transcription

描述（由申请人提供）：霍乱是一种由霍乱弧菌引起的致命性肠道疾病。这种疾病仍然是世界大多数人的健康威胁，每年造成数千人死亡。我们最近已经证明，毒素T，在霍乱弧菌毒力基因的主要转录激活因子，是负调控某些环境信号，特别是胆汁的存在。我们的研究将集中在解剖环境调节ToxT转录活性的分子机制，利用胆汁作为环境调节因子。我们希望了解并利用这种负调节来开发预防霍乱的新方法。基本上对ToxT的结构/功能一无所知，因此这些研究还包括阐明ToxT蛋白的功能。我们的方法首先涉及表征ToxT的结构域结构。这将通过（i）来实现。嵌合ToxT蛋白的构建和表征，和（ii）.鉴定对DNA结合和转录激活重要的ToxT氨基酸。ToxT的进一步表征将包括通过微阵列分析鉴定霍乱弧菌的所有ToxT调节基因，以及ToxT DNA结合位点的表征。一旦我们对ToxT有了更深入的了解，我们将确定环境信号对ToxT转录活性的调节机制，利用胆汁作为调节因子。这些研究包括：（一）。确定孔蛋白OmpU和OmpT（已知其对胆汁具有差异渗透性）对ToxT活性的胆汁调节的影响，（ii）.鉴定参与ToxT活性的胆汁调节的另外的霍乱弧菌基因，（iii）.鉴定胆汁调节所必需的ToxT氨基酸，和（iv）。测定胆汁对ToxT DNA结合活性的影响。最后，将通过测试含有影响ToxT转录各个方面的突变的霍乱弧菌菌株的毒力特性来评估ToxT活性的环境调节（通过胆汁或其他刺激）的相关性。我们的最终目标是了解如何通过外部因素操纵ToxT，以抑制毒力基因表达并预防霍乱，即，迫使霍乱弧菌防止自身致病这种从根本上不同的霍乱治疗方法可能会导致模仿胆汁作用的新型抗菌策略。

DESCRIPTION (provided by applicant): 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 V. 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 V. cholerae strains containing mutations that 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 could lead to novel antimicrobial strategies mimicking the effects of bile.