Single cell analyses reveal distinct adaptation of typhoidal and non-typhoidal Salmonella enterica serovars to intracellular lifestyle

Author summary Typhoidal Salmonella enterica serovars Typhi (STY) and Paratyphi A (SPA) cause a major disease burden to the human population. The restriction of these pathogens to human hosts limits experimental analyses of molecular mechanisms of diseases. S. enterica serovar Typhimurium is commonly used as surrogate model for typhoidal Salmonella (TS), and allowed the identification of virulence factors for intracellular lifestyle of S. enterica in mammalian host cells. If virulence factors, such as the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) have similar roles for intracellular lifestyle of TS is largely unknown. We analyzed, on single cell level, the intracellular activities of STY and SPA in comparison to STM. STY and SPA deploy SPI2-T3SS to actively manipulate their host cells, but with far lower frequency than STM. Our work supports a model of TS as stealth pathogens that persist in host cells. Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Human-restricted typhoidal S. enterica serovars Typhi (STY) or Paratyphi A (SPA) cause severe typhoid or paratyphoid fever, while many S. enterica serovar Typhimurium (STM) strains have a broad host range and in human hosts usually lead to a self-limiting gastroenteritis. Due to restriction of STY and SPA to primate hosts, experimental systems for studying the pathogenesis of typhoid and paratyphoid fever are limited. Therefore, STM infection of susceptible mice is commonly considered as model system for studying these diseases. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2-T3SS) is a key factor for intracellular survival of Salmonella. Inside host cells, the pathogen resides within the Salmonella-containing vacuole (SCV) and induces tubular structures extending from the SCV, termed Salmonella-induced filaments (SIF). This study applies single cell analyses approaches, which are flow cytometry of Salmonella harboring dual fluorescent protein reporters, effector translocation, and correlative light and electron microscopy to investigate the fate and activities of intracellular STY and SPA. The SPI2-T3SS of STY and SPA is functional in translocation of effector proteins, SCV and SIF formation. However, only a low proportion of intracellular STY and SPA are actively deploying SPI2-T3SS and STY and SPA exhibited a rapid decline of protein biosynthesis upon experimental induction. A role of SPI2-T3SS for proliferation of STY and SPA in epithelial cells was observed, but not for survival or proliferation in phagocytic host cells. Our results indicate that reduced intracellular activities are factors of the stealth strategy of STY and SPA and facilitate systemic spread and persistence of the typhoidal Salmonella.