Silencing and Counter-Silencing of Salmonella Virulence Genes

描述(由申请人提供)：致病细菌，如肠沙门氏菌，含有水平获得的DNA，位于在宿主-病原体相互作用中起关键作用的质粒或基因组岛上。大多数毒力基因都在转录水平上进行调控，以便在特定的环境条件下协调表达。转录调控的经典模式包括通过与RNA聚合酶相互作用的蛋白质来调节特定DNA序列的结合，以激活或抑制基因表达。我们最近对一种名为H-NS的核样相关蛋白(NAP)的研究表明，许多水平获得的毒力基因受另一种范式控制，在这种范式中，以相对较低的特异性结合DNA的NAP固有的转录沉默被其他DNA结合蛋白的作用所抵消。后者由经典的转录激活因子、抑制因子和替代的西格玛因子组成。这种被称为“异种沉默”的模型提供了一种机制，通过这种机制，水平获得的序列的潜在有害影响可以通过沉默来最小化，而新获得的基因随后通过反沉默被整合到预先存在的调控网络中。已知的DNA结合蛋白如Phop、SlyA、OmpR、SSRB和S(Rpos)对沙门氏菌的毒力是必不可少的。我们认为，受这些蛋白质调控的许多(如果不是大多数)遗传位点实际上是由反沉默机制控制的。本研究旨在通过对单个基因转录调控的生化分析，以及与NAP和反沉默蛋白相互作用的关联，来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通过缓解NAP诱导的DNA僵硬来促进RNA聚合酶开放复合体的形成，或者通过克服NAP对开放复合体的捕获来发挥作用。具体目的是：1.确定Phop调控基因的转录调控机制。典型的沙门氏菌Phop调节子将接受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.筛选的Phop依赖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调节子的单个反沉默基因，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.对H-NS和S共同调控的Sigma因子基因座的功能、生化和生物物理特性进行了分析，并与PhoP和SlyA的抗沉默作用进行了比较。

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.