Genetic Analysis of Salmonella Typhimurium Virulence

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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.