Environmental Modulation of ToxT-dependent Transcription

ToxT 依赖性转录的环境调节

• 批准号: 6574703
• 负责人: Karl E Klose
• 金额: $ 24.81万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6574703
• 关键词: bacterial proteins; bacterial toxins; bile; binding sites; cholera; enzyme linked immunosorbent assay; gene expression; genetic transcription; infant animal; laboratory mouse; microarray technology; polymerase chain reaction; pore forming protein; protein structure function; southern blotting; virulence; western blottings

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.

描述（由申请人提供）：霍乱是一种通常致命的腹泻疾病，由霍乱弧菌引起。这种疾病仍然是世界上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，弓形虫是霍乱弧菌毒力基因的主要转录激活因子，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剖析环境调节ToxT转录活性的分子机制，利用胆汁作为环境调节因子。我们希望了解和利用这种负调控，以开发新的手段来预防霍乱。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)嵌合ToxT蛋白的构建和表征，以及（ii）鉴定对DNA结合和转录激活重要的ToxT氨基酸来完成。弓形虫的进一步鉴定将包括通过微阵列分析鉴定霍乱弧菌所有弓形虫调节基因，以及弓形虫DNA结合位点的鉴定。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转录活性的调节机制。这些研究包括：(i)确定孔蛋白OmpU和OmpT（已知它们对胆汁的渗透性不同）对胆汁调节弓形虫活性的影响，（ii）鉴定参与胆汁调节弓形虫活性的其他霍乱弧菌基因，（iii）。鉴定胆汁调节所需的弓形虫氨基酸，以及（iv）测定胆汁对弓形虫DNA结合活性的影响。最后，环境对弓形虫活性（通过胆汁或其他刺激）调节的相关性将通过检测含有影响弓形虫转录各方面突变的霍乱弧菌菌株的毒力特性来评估。我们的最终目标是学习如何通过外部因素操纵弓形虫，从而抑制毒力基因表达，预防霍乱，即迫使霍乱弧菌阻止自身致病。这种完全不同的霍乱治疗方法可能导致模仿胆汁作用的新型抗菌策略。