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: 8338998
• 负责人: Ferric C Fang
• 金额: $ 38.19万
• 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/8338998
• 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. PUBLIC HEALTH RELEVANCE: Salmonella is a pathogen of global importance, causing costly food-borne outbreaks of gastroenteritis as well as potentially lethal systemic infections. This project expands the concept of xenogeneic silencing in pathogenic bacteria, a conceptual breakthrough our lab has discovered in the understanding of regulatory network evolution and control of virulence gene expression. Characterizing the molecular mechanisms of virulence gene expression will have broad implications for Salmonella and many other human pathogens, many uncover new antibiotic targets, and provide new avenues for more effective treatment of bacterial infections.

描述（由申请人提供）：肠沙门氏菌等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，以便协调表达。在特定环境条件下，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调节特定 DNA 序列的结合，以激活或抑制基因表达。称为 H-NS 的 NAP 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。该模型由经典转录激活因子、阻遏因子和替代 sigma 因子组成，称为“异种沉默”，提供了一种机制，通过该机制可以最大限度地减少水平获得序列的潜在有害影响。沉默，新获得的基因随后通过反沉默 DNA 结合蛋白（例如 PhoP、SlyA、OmpR、SsrB 和 ¿）整合到预先存在的调控网络中。已知 S (RpoS) 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。本申请旨在阐明沉默和反沉默的分子机制。 -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，我们发现反沉默蛋白通过减轻 NAP 诱导的 DNA 硬化来发挥作用。促进 RNA 聚合酶开放复合物形成或克服 NAP 的开放复合物捕获。 具体目标是： 1. 确定 PhoP 调节子中的转录调节机制 对原型沙门氏菌 PhoP 调节子进行生物信息学和功能分析，以区分受控基因位点。 2.选定对象的沉默与反沉默机制分析PhoP 依赖性基因。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 反沉默的功能和机械后果。 3. 通过替代 Sigma 因子表征反沉默 ¿ S. 由 H-NS 和 ¿ 共同调控的遗传位点S 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。 公共卫生相关性：沙门氏菌是一种具有全球重要性的病原体，可导致代价高昂的食源性胃肠炎爆发以及潜在致命的全身感染。该项目扩展了病原菌异种沉默的概念，这是我们实验室在理解上发现的概念性突破。毒力基因表达的调控网络进化和控制的表征将对沙门氏菌和许多其他人类病原体产生广泛的影响，许多发现新的抗生素靶点，并为更有效地治疗细菌感染提供新途径。