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: 8704382
• 负责人: Ferric C Fang
• 金额: $ 38.63万
• 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/8704382
• 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; 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; biophysical techniques; 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（由申请方提供）：致病菌（如肠道沙门氏菌）在质粒或基因组岛上含有水平获得的DNA，这些DNA在宿主-病原体相互作用中起着至关重要的作用。大多数毒力基因在转录水平上受到调控，以便在特定的环境条件下协调表达。转录调控的经典模型涉及通过与RNA聚合酶相互作用以激活或抑制基因表达的蛋白质来调节特定DNA序列的结合。我们对一种称为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受到另一种范式的控制，在这种范式中，以相对低的特异性结合DNA的NAP的内在转录沉默被其他DNA结合蛋白的作用抵消。后者由经典的转录激活因子、抑制因子和替代性σ因子组成。这种称为“异种沉默”的模型提供了一种机制，通过这种机制，水平获得的序列的潜在有害影响可以通过沉默最小化，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。已知DNA结合蛋白如PhoP、SlyA、OmpR、SsrB和<$S（RpoS）对于沙门氏菌毒力是必需的。我们认为，许多，如果不是大多数，由这些蛋白质调控的遗传位点实际上是由反沉默机制控制的。本申请旨在通过生物化学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相关联来阐明沉默和反沉默的分子机制。我们推测，反沉默蛋白的作用，通过缓解NAP诱导的DNA硬化，以促进RNA聚合酶开放复合物的形成或克服开放复合物捕捉NAP。具体目标是：1. PhoP调节子中转录调控机制的确定。将对原型沙门氏菌PhoP调节子进行生物信息学和功能分析，以区分由直接激活控制的遗传位点和由反沉默机制控制的遗传位点。2.选择的PhoP依赖基因的沉默和反沉默机制的分析。将使用生物化学和生物物理方法分析来自PhoP调节子的单个反沉默基因，以确定NAP（H-NS，StpA）的DNA结合以及PhoP和SlyA蛋白的反沉默的功能和机械后果。3.通过替代西格玛因子表征反沉默。由H-NS和S共调节的遗传基因座将进行功能、生物化学和生物物理分析，并与PhoP和SlyA的反沉默进行比较。