Silencing and Counter-Silencing of Salmonella Virulence Genes

沙门氏菌毒力基因的沉默和反沉默

• 批准号: 8534704
• 负责人: Ferric C Fang
• 金额: $ 36.31万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-21 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8534704
• 关键词: Antibiotics; Bacteria sigma factor KatF protein; Bacterial Infections; Biochemical; Bioinformatics; Complex; Control Locus; DNA; DNA Binding; DNA-Binding Proteins; DNA-Directed RNA Polymerase; Data; Evolution; Gastroenteritis; Gene Expression; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Transcription; Genomic Islands; Human; Individual; Mechanics; Methods; Modeling; Molecular; Plasmids; Play; Proteins; Regulon; Research; Role; Salmonella; Salmonella enterica; Sequence-Specific DNA Binding Protein; Sigma Factor; Specificity; Systemic infection; Transcription Coactivator; Transcriptional Regulation; Virulence; base; effective therapy; foodborne outbreak; pathogen; pathogenic bacteria

DESCRIPTION (provided by applicant): Pathogenic bacteria such as Salmonella enterica contain horizontally acquired DNA on plasmids or genomic islands that play a critically important role in host-pathogen interactions. Most virulence genes are regulated at the level of transcription in order to be coordinately expressed under specific environmental conditions. Classical models of transcriptional regulation involve regulated binding of specific DNA sequences by proteins that interact with RNA polymerase to activate or repress gene expression. Our studies of a Nucleoid-Associated Protein (NAP) called H-NS have recently shown that many horizontally acquired virulence genes are controlled by an alternative paradigm in which intrinsic transcriptional silencing by NAPs that bind DNA with relatively low specificity is countered by the actions of other DNA binding proteins. The latter are comprised of classical transcriptional activators, repressors, and alternative sigma factors. This model, designated "xenogeneic silencing," provides a mechanism by which the potentially deleterious impact of horizontally acquired sequences can be minimized by silencing, and newly acquired genes are subsequently integrated into pre-existing regulatory networks through counter-silencing. DNA binding proteins such as PhoP, SlyA, OmpR, SsrB and ¿S (RpoS) are known to be essential for Salmonella virulence. We propose that many, if not most, genetic loci regulated by these proteins are in fact controlled by counter-silencing mechanisms. This application aims to elucidate the molecular mechanisms of silencing and counter-silencing by biochemically analyzing the transcriptional regulation of individual genes and relating expression to interactions between NAPs and counter-silencing proteins. We hypothesize that counter-silencing proteins act by relieving NAP-induced DNA stiffening to facilitate RNA polymerase open complex formation or by overcoming open complex trapping by NAPs. The specific aims are: 1. Determination of Transcriptional Regulatory Mechanisms in the PhoP Regulon. The prototypical Salmonella PhoP regulon will be subjected to bioinformatic and functional analysis to distinguish genetic loci controlled by direct activation and those controlled by counter-silencing mechanisms. 2.Analysis of Silencing and Counter-Silencing Mechanisms for Selected PhoP-dependent Genes. Individual counter-silenced genes from the PhoP regulon will be analyzed using biochemical and biophysical methods to determine the functional and mechanical consequences of DNA binding by NAPs (H-NS, StpA) and counter-silencing by the PhoP and SlyA proteins. 3. Characterization of Counter-Silencing by the Alternative Sigma Factor ¿S. Genetic loci co-regulated by H-NS and ¿S will be subjected to functional, biochemical and biophysical analysis and compared with counter-silencing by PhoP and SlyA.

描述（由申请人提供）：诸如肠沙门氏菌之类的病原菌在质粒或基因组岛上含有水平获得的DNA，这些DNA在宿主与病原体的相互作用中发挥着至关重要的作用。大多数毒力基因在转录水平上受到调节，以便在特定环境条件下协调表达。转录调控的经典模型涉及通过与 RNA 聚合酶相互作用的蛋白质来调节特定 DNA 序列的结合，从而激活或抑制基因表达。我们对称为 H-NS 的核相关蛋白 (NAP) 的研究最近表明，许多水平获得的毒力基因是由另一种范式控制的，其中以相对较低的特异性结合 DNA 的 NAP 的内在转录沉默被其他 DNA 结合蛋白的作用所抵消。后者由经典转录激活因子、阻遏因子和替代西格玛因子组成。这种被称为“异种沉默”的模型提供了一种机制，通过该机制可以通过沉默将水平获得的序列的潜在有害影响最小化，并且新获得的基因随后通过反沉默整合到预先存在的调控网络中。 DNA 结合蛋白如 PhoP、SlyA、OmpR、SsrB 和 ¿S (RpoS) 已知对沙门氏菌毒力至关重要。我们认为，许多（如果不是大多数）受这些蛋白质调节的基因位点实际上是由反沉默机制控制的。本申请旨在通过生化分析单个基因的转录调控并将表达与 NAP 和反沉默蛋白之间的相互作用联系起来，阐明沉默和反沉默的分子机制。我们假设反沉默蛋白通过减轻 NAP 诱导的 DNA 硬化以促进 RNA 聚合酶开放复合物形成或通过克服 NAP 的开放复合物捕获来发挥作用。具体目标是： 1. 确定 PhoP Regulon 的转录调控机制。原型沙门氏菌 PhoP 调节子将接受生物信息学和功能分析，以区分直接激活控制的遗传位点和反沉默机制控制的遗传位点。 2.所选PhoP依赖性基因的沉默和反沉默机制分析。将使用生化和生物物理方法分析来自 PhoP 调节子的各个反沉默基因，以确定 NAP（H-NS、StpA）与 DNA 结合以及 PhoP 和 SlyA 蛋白反沉默的功能和机械后果。 3. 通过替代 Sigma 因子 ¿S 表征反沉默。由 H-NS 和 ¿S 共同调控的基因位点将接受功能、生化和生物物理分析，并与 PhoP 和 SlyA 的反沉默进行比较。