Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. The ty-phoidal S. enterica serovars Paratyphi A (SPA) and Typhi (STY) are human-restricted, and cause severe systemic diseases, while many S. enterica serovars like Typhimurium (STM) have a broad host range and in human hosts usually lead to self-limiting gastroenteritis. There are key differences between typhoidal (TS) and non-typhoidal (NTS) Salmonella in pathogenesis, but research on TS is challenging due to host restriction. Since STM causes a typhoid-like disease in mice, it was widely used as model organism to mimic human TS infection. Although results gained by research on STM could provide major insights in Salmonella virulence in general, the specific virulence mechanisms of TS are far from being understood. Both TS and NTS are able to invade mammalian cells and to replicate within host cells, including epithelial cells and macrophages. After invasion or phagocytic uptake, Salmonella resides in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). The subsequent in-tracellular lifestyle is dependent on the translocation of effector proteins via a type 3 secretion system (T3SS) which is encoded by genes on Salmonella pathogenicity island 2 (SPI2). During the intracellular lifestyle, vesicular compartments of host cells are manipulated by effector pro-teins of the SPI2-T3SS and Salmonella-induced filaments (SIF) are formed. It is currently un-known if observations regarding the molecular pathogenesis made for STM are applicable to TS serovars SPA and STY. In this work, the intracellular lifestyles of TS were investigated on single cell level. Analyses of intracellular activities of STY and SPA in various host cells showed that STY and SPA deploy SPI2-T3SS to actively manipulate their host cells, but with far lower frequency than STM. A role of SPI2-T3SS for proliferation of STY and SPA in epithelial cells was observed, but not for sur-vival or proliferation in phagocytic host cells. Reduced intracellular activities and pronounced SCV integrity of STY and SPA might contribute to the stealth strategy of STY and SPA, facilitat-ing systemic spread and persistence. Furthermore, by analyses of intracellular transcriptomic architecture during human epithelial cell infection of SPA and STM, different gene expression patterns in key virulence and metabolic pathways were identified. Elevated expression of SPI1 and flagella-chemotaxis genes by intracellular SPA results in cytosolic, flagella-mediated motility and increased invasiveness of SPA. Distinct gene expression patterns of carbon utilization path-ways, flagella-chemotaxis and SPI1 genes might contribute to the invasive and systemic disease developed following SPA infection in humans. Live cell imaging revealed that SPA invades host cells in a cooperative manner with multiple bacteria per invasion site, leading to error-prone macropinocytosis with increased membrane damage of the early SCV. After release into the cytosol, motile bacteria showed reduced autophagosomal capture. The results provide new insights into the virulence profile of STY and SPA by unravelling pre-viously unknown intracellular phenotypes and virulence traits. The established 3D and 2D intes-tinal organoid models offer new tools for analyses of human-restricted pathogens in a more in vivo relevant context.

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