Structure and Function of Vibrio cholerae ToxT

霍乱弧菌引起严重的霍乱，这仍然是发展中地区发病的一个严重原因。对于霍乱弧菌引起疾病，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调节菌毛的基因。毒力基因的表达受一系列转录因子的协同调控。ToxRS和TepsilonPH蛋白激活另一种转录激活因子ToxT的转录，然后ToxT直接激活20多个毒力基因的转录。本项目的目标是了解ToxT的结构和功能。使用幼年小鼠模型的研究将通过利用组成型表达ToxT的菌株直接测试毒力基因的适当时间表达对定殖的重要性。还将在体内和体外研究尚未充分表征的4种ToxT活化的操纵子。ToxT的共有DNA结合位点将通过鉴定和比较已知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。将通过遗传筛选/选择来鉴定ToxT中特异性参与转录激活的残基，所述遗传筛选/选择分离在激活中有缺陷但能够结合DNA的ToxT突变体。最后，由于存在ToxT活性可能受到调节的证据，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。

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.