Decoding host-pathogen crosstalk that drives intracellular niche formation during Salmonella infection

PROJECT SUMMARY Of the foodborne pathogens, non-typhoidal Salmonella enterica (NTS) cause the largest burden of illness and death worldwide. Upon entering the gut, Salmonella can traverse the mucosal barrier by inducing its uptake into intestinal epithelial cells, followed by growth in lamina propria phagocytes. In recent years, it has been discovered that intracellular Salmonella could colonize two niches in host cells - within a membrane-bound vacuole and free in the cytosol - with profound consequences on host cell death and bacterial survival strategies. But our understanding of these parallel intracellular colonization strategies is currently limited by approaches that are not suited to untangle bacterial and host factors in subpopulations of infected cells. In the proposed work, we will study intracellular niches of Salmonella enterica serovar Typhimurium within human intestinal epithelial cells and macrophages. Combining a sensitive bacterial fluorescent reporter, fluorescence-assisted cell sorting (FACS) and dual RNA-seq methods, we will analyze cell type-specific bacterial and host transcripts from subpopulations of infected cells that contain cytosolic vs. vacuolar bacteria. We will characterize bacterial and host transcripts during Salmonella infection of intestinal epithelial cells, distinguishing between cytosolic vs. vacuolar phenotypes (Aim 1.1). In parallel, we will characterize Salmonella infection of macrophages (Aim 1.2). Combining dual RNA-seq of sorted populations and bioinformatics, we will use genetic perturbations to test the contribution of bacterial and host gene candidates to the intracellular localization phenotypes (Aim 1.3). By defining niche-specific and host cell type-specific interaction networks, we can pinpoint the most crucial vulnerabilities of S.Tm during infection of human hosts.