Structure and Function of Vibrio cholerae ToxT

霍乱弧菌ToxT的结构和功能

• 批准号: 6552706
• 负责人: JEFFREY H WITHEY
• 金额: $ 4.42万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6552706
• 关键词: DNA; DNA footprinting; Vibrio cholerae; bacteria infection mechanism; bacterial cytopathogenic effect; bacterial disease; bacterial genetics; bacterial proteins; binding sites; cholera; cholera toxin; disease /disorder model; gel mobility shift assay; gene expression; genetic manipulation; genetic regulation; genetic screening; genetic transcription; infant animal; laboratory mouse; maltose; nucleic acid sequence; operon; postdoctoral investigator; protein structure function; transcription factor; virulence

The bacterium Vibrio cholerae causes the severe diarrheal disease cholera, which remains a serious cause of morbidity in developing areas. For V. cholerae to cause disease, a collection of virulence genes, including the genes encoding cholera toxin and a toxin co-regulated pilus must be expressed appropriately. Virulence gene expression is regulated coordinately by a cascade of transcription factors. The ToxRS and TepsilonPH proteins activate transcription of another transcription activator, ToxT, which then directly activates transcription of over 20 virulence genes. The goals of this project are to understand the structure and function of ToxT. Studies using the infant mouse model will directly test the importance of appropriate temporal expression of virulence genes to colonization by utilizing a strain that expressed ToxT constitutively. The 4 ToxT-activated operons that have not been well characterized will also be studied both in vivo and in vitro. A consensus DNA binding site for ToxT will be determined by identifying and comparing sequences in genes ToxT is known to regulate, and by in vitro selection of DNA sequences to which ToxT binds with high affinity. Residues in ToxT specifically involved in transcription activation will be identified by a genetic screen/selection that isolates ToxT mutants defective in activation but able to bind DNA. Finally, since evidence exists that ToxT activity may be regulated, the mechanism for regulation will be investigated by measuring ToxT levels under non-inducing conditions, and searching for effectors of ToxT activity.

霍乱弧菌引起严重的腹泻病霍乱，这仍然是发展中地区发病的一个严重原因。为了使霍乱弧菌引起疾病，必须适当表达一系列毒力基因，包括编码霍乱毒素和毒素共同调节菌毛的基因。毒力基因表达由一系列转录因子协调调节。 ToxRS 和 TepsilonPH 蛋白激活另一种转录激活剂 ToxT 的转录，然后 ToxT 直接激活 20 多个毒力基因的转录。该项目的目标是了解 ToxT 的结构和功能。使用婴儿小鼠模型的研究将通过利用组成型表达 ToxT 的菌株来直接测试毒力基因的适当时间表达对于定植的重要性。尚未充分表征的 4 个 ToxT 激活操纵子也将在体内和体外进行研究。 ToxT 的共有 DNA 结合位点将通过识别和比较已知 ToxT 调节的基因中的序列以及通过体外选择 ToxT 以高亲和力结合的 DNA 序列来确定。 ToxT 中专门参与转录激活的残基将通过遗传筛选/选择来鉴定，该遗传筛选/选择分离出激活缺陷但能够结合 DNA 的 ToxT 突变体。最后，由于存在证据表明ToxT活性可能受到调节，因此将通过在非诱导条件下测量ToxT水平并寻找ToxT活性的效应物来研究调节机制。