Function of Vibrio cholerae ToxT

描述（由申请人提供）：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。我们研究的目的是了解控制霍乱弧菌毒力相关基因表达的机制。霍乱弧菌的毒力基因包括那些编码霍乱毒素（CT）的基因，它产生霍乱的特征症状，毒素共调节菌毛（TCP），它参与宿主小肠的定植，以及其他一些基因，它们在毒力中的作用尚不清楚。毒力基因的表达受转录调控因子网络控制；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，了解这种蛋白质的功能是我们工作的重点。本应用程序中描述的实验将检查以下内容：(1)将使用遗传选择/筛选确定ToxT中参与转录激活和dna结合的氨基酸残基，然后对体内和体外突变体进行进一步表征。(2)越来越多的证据表明，弓形虫的活性受转录后调控。我们将确定弓形虫的转录后控制是如何表现的，检查环境中的弓形虫变异在霍乱弧菌中的功能，并在体外和动物模型中评估组成型弓形虫表达的影响。(3)我们将通过比较携带野生型弓形虫和携带缺失弓形虫的霍乱弧菌的蛋白质组学分析，确定所有依赖弓形虫表达的蛋白的身份。这些实验将显著增加我们对弓形虫蛋白功能和被弓形虫激活表达的基因功能的认识，并可能为霍乱疾病提供新的治疗靶点。霍乱仍然是一个重大的世界性问题，特别是在遭受战争、自然灾害或经济困难影响的地区，改善治疗应大大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.