Characterization of the ETEC Virulence Regulator Rns

描述(由申请人提供)：全球产肠毒素大肠杆菌(ETEC)每年估计导致2.1亿起腹泻疾病和38万人死亡。这种病原体对5岁以下的婴儿和儿童的影响最大。病原体附着在宿主的肠粘膜上是建立感染所必需的。附着通常是通过结合特定宿主受体的菌毛来实现的。作为AraC/XylS家族的一员，RNS在转录水平上正向调控CS1和CS2菌毛的表达。在CS1菌毛蛋白启动子的上游有两个RNS结合位点，在典型的原核生物激活剂所预期的区域内。RNS也积极地自动调节自己的表达，但它是通过一种非常不寻常的DNA结合位点安排来做到这一点的。RNS需要两个DNA结合位点才能激活其自身的启动子PRNS。其中一个是以转录起始点上游224.5个碱基为中心的1号位点，比预期的要远得多。更不同寻常的是3号站点的位置，它位于TSS下游83.5BP的中心。只有少数原核生物激活子在-10六聚体下游有结合位点。然而，我们已经证明，来自志贺氏菌的毒力调节因子VirF、来自ETEC的CFAR和来自肠聚集性大肠杆菌的AggR也能够激活PRN，并且像RNS一样，每个都需要下游结合位点来实现这一功能。到目前为止，RNS是这一组中唯一已开发出体外系统的毒力调节因子。因此，我们的体外系统，加上免费的遗传分析，为我们提供了一个绝佳的机会，将RNS用作一组相关调节器的模型系统。这些调节剂对几种致病细菌的毒力至关重要，这些细菌每年总共导致200多万人死亡，其中主要是儿童和婴儿。对这些同源调控因子的详细了解可能最终导致在疾病治疗和预防方面的新应用，例如通过靶向保守的毒性调节因子的功能来减弱细菌的毒力。由于RNS自身调节的独特特性，我们提出的研究也将提供有关转录及其调控的新的和基本的重要信息。这些包括下游结合位点的激活和非生产性RNAP开放(RPO)复合体对PRN的潜在抑制。这项研究将确定RNS正向自动调节的分子机制，以及PRNS附近非RNS非生产性RPO复合体的生物学意义。

DESCRIPTION (provided by the applicant): Worldwide enterotoxigenic E. coli (ETEC) cause an estimated 210 million episodes of diarrheal disease and 380,000 deaths annually. The impact of this pathogen is greatest among infants and children less than 5 years of age. Adherence of the pathogen to the intestinal mucosa of the host is essential for the establishment of an infection. Attachment is usually achieved by pili which bind specific host receptors. The expression of CS1 and CS2 pili is positively regulated at the level of transcription by Rns a member of the AraC/XylS family. There are two Rns binding sites immediately upstream of the CS1 pilin promoter, within the region expected for a typical prokaryotic activator. Rns also positively autoregulates its own expression, however it does so through a highly unusual arrangement of DNA binding sites. Rns requires two DNA binding sites for the activation of its own promoter Prns. One of these is site 1 which is centered 224.5 bp upstream of the transcription start site (TSS), considerably further upstream than expected. Even more unusual is the location of site 3, which is centered 83.5 bp downstream of the TSS. Only a few prokaryotic activators are known to have binding sites downstream of the -10 hexamer. However we have shown that the virulence regulators VirF from Shigella flexneri, CfaR from ETEC, and AggR from enteroaggregative E. coli are also capable of activating Prns and like Rns, each requires the downstream binding site to do so. To date Rns is the only virulence regulator within this group for which an in vitro system has been developed. Thus our in vitro system, coupled with complimentary genetic analysis, affords us an outstanding opportunity to use Rns as a model system for a group of related regulators. These regulators are essential for the virulence of several pathogenic species of bacteria that collectively kill over 2 million people, predominantly children and infants, every year. A detailed understanding of these homologous regulators may eventually lead to new applications for disease treatment and prevention, such as attenuation of bacterial virulence by targeting the function of conserved virulence regulators. Our proposed studies will also provide new and fundamentally important information about transcription and its regulation because of the unique features of Rns autoregulation. These include activation from a downstream binding site and the potential repression of Prns by nonproductive RNAP open (RPo) complexes. Studies in this proposal will determine the molecular mechanism of Rns positive autoregulation and the biological significance of Rns-independent, nonproductive RPo complexes near Prns.