Genetic Analysis of Salmonella Typhimurium Virulence

描述（由申请人提供）：使RPG能够利用NCRR中心和类似中心的计划（NOT-OD-09-058竞争性修订申请的恢复法案基金）。这项研究的目标是开始了解巨噬细胞如何对沙门氏菌感染作出反应，以及沙门氏菌毒力因子如何改变这种反应。我们将用亲本14028和缺失特异性毒力调节因子或完全缺失III型分泌的同基因衍生物感染细胞，并比较感染细胞的转录组、蛋白质组和磷酸化蛋白质组的差异。一般而言，调节突变体将表达与亲本相同的病原体相关分子模式，但分泌的蛋白质以及其他毒力因子不同，其中一些尚未定义。三重突变体SPI-1/SPI-2/flgB将不表达III型分泌的效应物。该实验将允许在对病原体相关分子模式的应答与修饰或破坏巨噬细胞信号转导途径的表达的毒力因子的表达之间进行明确区分。我们将使用一种新的交联和亲和纯化方法来鉴定给定分泌效应物的靶蛋白。我们将使用生物信息学进行解释，包括网络推理算法来寻找基因表达数据中的因果影响模式，以及为重建哺乳动物细胞中的基因调控网络而开发的算法（Margolin et al.，2006年）。将包括来自若干来源的途径信息（例如NCI-Nature Pathway Interaction Database（Schaefer et al.，2009年）。这一分析将有助于确定沙门氏菌是如何通过破坏特定的宿主功能而在宿主中生存的。 公共卫生相关性：该应用程序的目标是开始了解宿主细胞如何对感染作出反应。身体对感染的反应是由身体先天免疫系统所感知的所有细菌的分子模式决定的。反过来，细菌通过产生毒力因子来阻断宿主的防御机制来保护自己。缺失特定毒力因子的沙门氏菌突变将用于通过比较宿主细胞在多个水平上的变化来评估这些相同毒力因子如何改变宿主应答。

DESCRIPTION (provided by applicant): Enabling RPGs to Leverage NCRR Center and Center-like Programs (NOT-OD-09-058 Recovery Act Funds for Competitive Revision Applications). The goal of this proposed research is to begin to understand how a macrophage responds to Salmonella infection and how Salmonella virulence factors alter that response. We will infect cells with the parent 14028 and isogenic derivatives either missing specific virulence regulators or missing type III secretion altogether and compare differences in the transcriptome, proteome and phosphoproteome of the infected cell. In general the regulator mutants will express the same pathogen associated molecular patterns as the parent but differ in the proteins secreted as well as other virulence factors some of which are not yet defined. The triple mutant SPI-1/SPI-2/flgB will express no type III secreted effectors. This experiment will allow a clear distinction to be made between response to pathogen associated molecular patterns and expression of virulence factors that modify or disrupt expression of macrophage signal transduction pathways. We will use a new cross-linking and affinity purification approach to identify the target protein of a given secreted effector. We will use bioinformatics for interpretation including network inference algorithms to look for patterns of causal influence in gene expression data and an algorithm developed for the reconstruction of gene regulatory networks in mammalian cells (Margolin et al., 2006). Pathway information from several sources will be included (e.g. NCI-Nature Pathway Interaction Database (Schaefer et al., 2009). This analysis will help define how Salmonella is able to survive within the host by subverting particular host functions. PUBLIC HEALTH RELEVANCE: The goal of this application is to begin to understand how a host cell responds to infection. Response of the body to infection is determined by molecular patterns on all bacteria that are sensed by the body's innate immune system. In turn, the bacteria defends itself by producing virulence factors to block host defense mechanisms. Salmonella mutations that are missing specific virulence factors will be used to assess how these same virulence factors alter host response by comparing changes in the host cell at multiple levels.