Environmental Modulation of ToxT-dependent Transcription

描述(申请人提供)：霍乱是一种通常致命的腹泻疾病，由霍乱弧菌引起。这种疾病仍然威胁着世界大多数地区的健康，每年造成数千人死亡。我们最近已经证明，霍乱弧菌毒力基因的主要转录激活因子ToxT受到某些环境信号的负调控，特别是胆汁的存在。我们的研究将集中在利用胆汁作为环境调节因子来剖析环境调节ToxT转录活性的分子机制(S)。我们希望了解和利用这一负面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，因此这些研究也包括对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(I)完成。嵌合ToxT蛋白的构建和鉴定，以及(Ii)。鉴定对DNA结合和转录激活重要的ToxT氨基酸。对ToxT的进一步鉴定将包括通过微阵列分析鉴定所有ToxT调节的霍乱弧菌基因，以及鉴定ToxT DNA结合位点(S)。一旦我们对ToxT有了更深入的了解，我们将利用胆汁作为调节因子，确定环境信号对ToxT转录活性的调控机制。这些研究包括：(I)。孔蛋白OmpU和OmpT(已知对胆汁具有不同的通透性)对胆汁调节ToxT活性的影响的测定，(Ii)。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)。胆汁调节所必需的ToxT氨基酸的鉴定，以及(Iv)。胆汁对ToxT DNA结合活性的影响最后，将通过测试含有影响ToxT转录的各个方面的突变的霍乱弧菌菌株的毒力特性，来评估环境对ToxT活性的调节(通过胆汁或其他刺激)的相关性。我们的最终目标是学习如何利用外界因素操纵ToxT，以抑制毒力基因的表达，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可能会导致模仿胆汁效果的新的抗菌策略。

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.