Structure and Function of Vibrio cholerae ToxT

霍乱弧菌引起严重的腹泻疾病霍乱，这仍然是发展中地区发病率的一个严重原因。要使霍乱弧菌致病，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一种毒素共调节菌毛。毒力基因的表达受一系列转录因子的协调调控。ToxRS和TepsilonPH蛋白激活另一种转录激活因子ToxT的转录，然后直接激活20多种毒力基因的转录。这个项目的目标是了解ToxT的结构和功能。使用幼鼠模型的研究将通过使用组成性表达弓形虫的菌株直接测试适当的毒力基因的时间表达对定植的重要性。4个尚未被很好地表征的弓形虫激活的操纵子也将在体内和体外进行研究。通过鉴定和比较已知弓形虫调控的基因序列，以及在体外选择与弓形虫高亲和力结合的DNA序列，可以确定弓形虫的DNA结合位点。弓形虫中特异性参与转录激活的残基将通过基因筛选/选择来鉴定，分离出激活缺陷但能够结合DNA的弓形虫突变体。最后，由于有证据表明ToxT活性可能受到调节，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.