Structure and Function of Vibrio cholerae ToxT

霍乱弧菌ToxT的结构和功能

• 批准号: 6752377
• 负责人: JEFFREY H WITHEY
• 金额: $ 5.05万
• 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 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6752377
• 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.

细菌弧菌霍乱引起严重的腹泻病霍乱，这仍然是发展区域发病率的严重原因。为了使霍乱谷引起疾病，必须适当地表达毒力基因的集合，包括编码霍乱毒素和毒素共同调节的基因。毒力基因表达由转录因子的级联反应协调。 ToxR和Tepsilonph蛋白激活了另一个转录激活剂TOXT的转录，然后直接激活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直接检验毒力基因适当的时间表达对定植的重要性，该菌株构成了构成的菌株。在体内和体外，还将研究尚未表征尚未表征的4个TOXT激活的操纵子。 TOXT的共识DNA结合位点将通过鉴定和比较基因TOXT中的序列来确定可以调节的序列，并在体外选择TOXT与高亲和力结合的DNA序列。特有参与转录激活的TOXT中的残基将通过遗传筛选/选择，该遗传筛选/选择分离出TOXT突变体在激活中有缺陷但能够结合DNA。最后，由于存在可能调节TOXT活动的证据，因此将通过在非诱导条件下测量TOXT水平以及寻找TOXT活动的效应子来研究调节机制。