Characterization of the ETEC Virulence Regulator Rns

ETEC 毒力调节剂 Rns 的表征

• 批准号: 7344773
• 负责人: GEORGE Patrick MUNSON
• 金额: $ 31.48万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7344773
• 关键词: Adherence; Age-Years; Bacteria; Binding; Binding Sites; Biological; Biological Models; Cessation of life; Child; Chromatin Loop; Complex; Coupled; DNA; DNA Binding; Decompression Sickness; Diarrhea; Disease; Distal; Family; Fimbriae Proteins; Genetic Transcription; Homeostasis; In Vitro; Infant; Infection; Intestinal Mucosa; Lead; Location; Molecular; Phase; Pilum; Prevention; Recruitment Activity; Regulation; Repression; Shigella flexneri; Site; Solutions; System; Transcription Initiation Site; Virulence; attenuation; enteroaggregative Escherichia coli; enterotoxigenic Escherichia coli; genetic analysis; killings; member; monomer; novel; pathogen; prevent; promoter; receptor

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.

描述（由申请人提供）：全世界范围内产肠毒素大肠杆菌 (ETEC) 每年导致估计 2.1 亿次腹泻疾病和 380,000 人死亡。这种病原体对婴儿和 5 岁以下儿童的影响最大。病原体粘附在宿主肠粘膜上对于感染的建立至关重要。附着通常通过结合特定宿主受体的菌毛来实现。 CS1 和 CS2 菌毛的表达在转录水平上受到 AraC/XylS 家族成员 Rns 的正向调节。 CS1 菌毛蛋白启动子的上游有两个 Rns 结合位点，位于典型原核激活子的预期区域内。 Rns 还积极地自动调节其自身的表达，但它是通过极其不寻常的 DNA 结合位点排列来实现的。 Rns 需要两个 DNA 结合位点来激活其自身的启动子 Prns。其中之一是位点 1，它位于转录起始位点 (TSS) 上游 224.5 bp 处，比预期的上游要远得多。更不寻常的是位点 3 的位置，它位于 TSS 下游 83.5 bp 处。已知只有少数原核激活剂在 -10 六聚体下游具有结合位点。然而，我们已经证明，福氏志贺氏菌的毒力调节因子 VirF、ETEC 的 CfaR 和肠聚集性大肠杆菌的 AggR 也能够激活 Prns 和 Rns，每个都需要下游结合位点才能激活。迄今为止，Rns 是该组中唯一已开发出体外系统的毒力调节剂。因此，我们的体外系统与免费的遗传分析相结合，为我们提供了一个绝佳的机会，可以使用 Rns 作为一组相关调节因子的模型系统。这些调节因子对于几种致病细菌的毒力至关重要，这些细菌每年总共导致超过 200 万人死亡，其中主要是儿童和婴儿。对这些同源调节因子的详细了解最终可能会带来疾病治疗和预防的新应用，例如通过针对保守毒力调节因子的功能来减弱细菌毒力。由于 Rns 自动调节的独特特征，我们提出的研究还将提供有关转录及其调节的新的、根本上重要的信息。其中包括下游结合位点的激活以及非生产性 RNAP 开放 (RPo) 复合物对 Prns 的潜在抑制。本提案中的研究将确定 Rns 正向自动调节的分子机制以及 Prns 附近不依赖 Rns 的非生产性 RPo 复合物的生物学意义。