Structure and Function of Vibrio cholerae ToxT

霍乱弧菌引起严重的腹泻疾病霍乱，霍乱在发展中地区仍是严重的致病原因。霍乱弧菌要致病，必须适当表达一系列毒力基因，包括编码霍乱毒素的基因和毒素共同调节的菌毛。毒力基因的表达受一系列转录因子的协调调节。ToxRS和TepsilonPH蛋白激活另一个转录激活因子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。利用幼鼠模型进行的研究将通过利用结构性表达ToxT的菌株来直接测试毒力基因的适当时间表达对定植的重要性。还将在体内和体外研究尚未很好表征的4个ToxT激活的操纵子。通过鉴定和比较已知ToxT调控基因中的序列，以及通过体外选择ToxT与之高亲和力结合的DNA序列，将确定ToxT的共识DNA结合部位。ToxT中特定参与转录激活的残基将通过基因筛选/选择来鉴定，该选择分离出激活缺陷但能够结合DNA的ToxT突变体。最后，由于有证据表明ToxT活性可能被调节，因此将通过测量非诱导条件下的ToxT水平并寻找ToxT活性的效应因子来研究调节机制。

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.