Genetic Analysis of Salmonella Typhimurium Virulence

描述（由申请人提供）：使rpg能够利用NCRR中心和类似中心的项目（NOT-OD-09-058 Recovery Act Funds for Competitive Revision Applications）。这项研究的目的是开始了解巨噬细胞对沙门氏菌感染的反应以及沙门氏菌毒力因子如何改变这种反应。我们将用缺失特定毒力调节因子或完全缺失III型分泌的亲本14028和等基因衍生物感染细胞，并比较受感染细胞的转录组、蛋白质组和磷蛋白质组的差异。一般来说，调节突变体将表达与亲本相同的病原体相关分子模式，但在分泌的蛋白质以及其他一些尚未确定的毒力因子方面有所不同。三突变体SPI-1/SPI-2/flgB不表达III型分泌效应物。该实验将明确区分对病原体相关分子模式的反应和修饰或破坏巨噬细胞信号转导途径表达的毒力因子的表达。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，以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等人，2006年）。将包括来自多个来源的途径信息（例如NCI-Nature Pathway Interaction Database (Schaefer et al., 2009)）。这一分析将有助于确定沙门氏菌是如何通过破坏宿主的特定功能而在宿主体内存活的。

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.