Silencing and Counter-Silencing of Salmonella Virulence Genes

描述（由申请人提供）：致病性细菌，如肠沙门氏菌，在质粒或基因组岛上含有水平获得的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聚合酶相互作用来激活或抑制基因表达的蛋白质对特定DNA序列的调节结合。我们对一种名为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，在这种范式中，特异性相对较低的NAP结合DNA的内在转录沉默被其他DNA结合蛋白的作用所抵消。后者由经典的转录激活因子、抑制因子和可选的sigma因子组成。这种被称为“异种沉默”的模型提供了一种机制，通过沉默可以将水平获得序列的潜在有害影响降到最低，并且新获得的基因随后通过反沉默整合到先前存在的调节网络中。已知PhoP、SlyA、OmpR、SsrB和¿S （RpoS）等DNA结合蛋白对沙门氏菌的毒力至关重要。我们提出，许多，如果不是大多数，由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。本应用程序旨在通过生化分析单个基因的转录调控以及nap与反沉默蛋白之间相互作用的表达，阐明沉默和反沉默的分子机制。我们假设反沉默蛋白的作用是缓解nap诱导的DNA硬化，促进RNA聚合酶开放复合体的形成，或克服nap捕获开放复合体。具体目标是：1。PhoP调控子转录调控机制的确定。我们将对沙门氏菌PhoP调控子原型进行生物信息学和功能分析，以区分由直接激活控制的基因位点和由反沉默机制控制的基因位点。2.磷依赖基因的沉默和反沉默机制分析。我们将使用生化和生物物理方法分析PhoP调控子中的单个反沉默基因，以确定nap （H-NS, StpA）结合DNA和PhoP和SlyA蛋白的反沉默的功能和机械后果。3. 用备选西格玛因子表征反沉默。将对H-NS和¿S共同调控的基因位点进行功能、生化和生物物理分析，并与PhoP和SlyA的反沉默进行比较。

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.