Function of Vibrio cholerae ToxT

描述（由申请人提供）：霍乱弧菌是严重霍乱的病原体，每年影响全世界数百万人。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。霍乱弧菌毒力基因包括编码霍乱毒素（CT）的那些基因，其产生霍乱的特征性症状，毒素共调节菌毛（TCP），其参与宿主小肠的定殖，以及一系列其他基因，其在毒力中的作用知之甚少。毒力基因的表达是由一个网络的转录调控因子控制，然而，一种蛋白质，ToxT，最终激活几乎所有的毒力基因的转录，并了解这种蛋白质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）将使用遗传选择/筛选，随后在体内和体外进一步表征突变体，来确定ToxT中涉及转录激活和DNA结合的氨基酸残基。(2)越来越多的证据表明，ToxT活性是转录后调节的。我们将确定ToxT的转录后控制是如何表现的，检查环境ToxT变体在流行性霍乱弧菌中的功能，并评估组成型ToxT表达在体外和动物模型中的影响。(3)我们将通过比较具有野生型toxT的霍乱弧菌和具有toxT缺失的霍乱弧菌的蛋白质组学分析来确定其表达是ToxT依赖性的所有蛋白质的身份。这些实验应该显着增加我们的知识ToxT蛋白质的功能和功能的基因，其表达被激活的ToxT，并可能导致霍乱疾病的新的治疗目标。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，改善治疗方法应能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.