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（由适用提供）：在质粒或基因组岛上水平获得的DNA等致病细菌，例如在宿主 - 病原体相互作用中起着至关重要的作用。大多数病毒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聚合酶相互作用以激活或抑制基因表达的蛋白质对特定DNA序列的结合。我们对称为H-NS的核苷相关蛋白（NAP）的研究最近表明，许多水平获得的病毒基因受到替代范式的控制，在这种范式中，通过与其他DNA结合蛋白的作用相反，通过与相对低特异性结合DNA的固有转录沉默。后者包括经典的转录激活剂，复制品和替代Sigma因子。该模型设计的“异种沉默”提供了一种机制，可以通过沉默来最大程度地减少水平获得序列的潜在删除影响，随后将新获取的基因通过违反定性整合到预先存在的调节网络中。 DNA结合蛋白，例如PHOP，SLYA，OMPR，SSRB和S（RPOS）对于沙门氏菌病毒至关重要。我们假设许多蛋白质调节的遗传局部遗传局部实际上是通过反沉降机制控制的。该应用旨在通过生物化学分析单个基因的转录调节，并将表达与小睡和反沉淀蛋白之间的相互作用有关，以阐明沉默和反沉降的分子机制。我们假设反沉淀蛋白可以缓解NAP诱导的DNA僵硬来促进RNA聚合酶开放式复合物形成或克服小NAP的开放式复合物捕获。具体目的是：1。确定PHOP调节中的转录调节机制。原型沙门氏菌PHOP调节子将进行生物信息学和功能分析，以区分通过直接激活控制的遗传基因座和由反沉降机制控制的基因座。 2。分析所选PHOP依赖性基因的沉默和反沉降机制。将使用生物化学和生物物理方法分析来自PHOP调节的单个反沉淀基因，以确定通过NAPS（H-NS，STPA）和PHOP和SLYA蛋白通过NAPS（H-NS，STPA）与DNA结合的功能和机械后果。 3。通过替代sigma因子对反沉降的表征。S。由H-NS和s共同调节的遗传基因座将经过功能，生化和生物物理分析，并与PHOP和SLYA的反沉降进行比较。