Function of Vibrio cholerae ToxT

描述(申请人提供)：霍乱弧菌是严重腹泻疾病霍乱的病原体，每年影响全球数百万人。本研究的目的是了解霍乱弧菌毒力相关基因表达的调控机制。霍乱弧菌的毒力基因包括编码霍乱毒素(CT)的基因(产生霍乱的特征症状)、参与宿主小肠定植的毒素共调节菌毛(TCP)以及一系列其他基因，这些基因在毒力中的作用尚不清楚。毒力基因的表达受一系列转录调控因子的控制；然而，一种名为ToxT的蛋白质最终激活了几乎所有毒力基因的转录，了解该蛋白的功能是我们工作的重点。本申请中描述的实验将检查以下内容：(1)ToxT中参与转录激活和DNA结合的氨基酸残基将通过遗传选择/筛选确定，然后在体内和体外进一步鉴定突变体。(2)越来越多的证据表明，ToxT活性受转录后调控。我们将确定ToxT转录后调控是如何表现的，检查环境中ToxT变异在霍乱弧菌中的功能，并在体外和动物模型中评估结构性ToxT表达的影响。(3)我们将通过比较野生型毒素T霍乱弧菌和毒素T缺失霍乱弧菌的蛋白质组学分析来确定所有表达ToxT依赖的蛋白质的同源性。这些实验应该会显著增加我们对ToxT蛋白功能和ToxT激活表达的基因功能的了解，并可能导致霍乱疾病的新治疗靶点。霍乱仍然是一个严重的世界性问题，特别是在遭受战争、自然灾害或经济困难影响的地区，改善治疗应该会极大地提高这些地区人民的生活质量。

DESCRIPTION (provided by applicant): Vibrio cholerae is the causative agent of the severe diarrheal disease cholera, which affects millions of people worldwide each year. The goal of our research is to understand the mechanisms for controlling expression of the genes involved in V. cholerae virulence. The V. cholerae virulence genes include those encoding cholera toxin (CT), which produces the characteristic symptoms of cholera, the toxin co-regulated pilus (TCP), which is involved in the colonization of the small intestine of the host, together with a collection of other genes whose roles in virulence are poorly understood. Expression of the virulence genes is controlled by a network of transcriptional regulators; however, one protein, ToxT, ultimately activates transcription of nearly all the virulence genes, and understanding how this protein functions is the focus of our work. Experiments described in this application will examine the following: (1) the amino acid residues in ToxT that are involved in transcriptional activation and DNAbinding will be determined using a genetic selection/screening followed by further characterization of mutants in vivo and in vitro. (2) There is mounting evidence that ToxT activity is regulated post-transcriptionally. We will determine how the post-transcriptional control of ToxT is manifested, examine environmental ToxT variants for functionality in epidemic V. cholerae, and assess the effects of constitutive ToxT expression in vitro and in an animal model. (3) We will determine the identities of all proteins whose expression is ToxT dependent by comparing proteomic analysis of V. cholerae having wildtype toxT and V. cholerae having a toxT deletion. These experiments should significantly increase our knowledge of ToxT protein function and of the functions of genes whose expression is activated by ToxT, and could lead to new therapeutic targets for cholera disease. Cholera remains a significant worldwide problem, particularly in areas suffering from the effect of war, natural disaster, or economic hardship, and improved treatments should greatly increase the quality of life for people in these regions.