Salmonella enters a dormant state within human epithelial cells for persistent infection

Salmonella Typhimurium ( S . Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. S . Typhimurium colonizes different intracellular niches and is able to either actively divide at various rates or remain dormant to persist. A comprehensive tool to determine these distinct S . Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter, Salmonella INtracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This identified a S . Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S . Typhimurium. This subpopulation entered a dormant state in a vesicular compartment distinct from the conventional Salmonella -containing vacuoles (SCV) as well as the previously reported niche of dormant S . Typhimurium in macrophages. The dormant S . Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormant S . Typhimurium is not triggered by the loss of SPI-2 effector secretion but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S . Typhimurium represents an important pathogen niche and provides an alternative strategy for S . Typhimurium pathogenicity and its persistence.