The facultative intracellular pathogen Salmonella enterica Typhimurium (STM) resides in a specific membrane-bound compartment termed the Salmonella-containing vacuole (SCV). STM is able to obtain all nutrients required for rapid proliferation, although being separated from direct access to host cell metabolites. The formation of specific tubular membrane compartments, called Salmonella-induced filaments (SIFs) are known to provides bacterial nutrition by giving STM access to endocytosed material and enabling proliferation. Additionally, STM expresses a range of nutrient uptake system for growth in nutrient limited environments to overcome the nutrition depletion inside the host. By utilizing dual fluorescence reporters, we shed light on the nutritional environment of intracellular STM in various host cells and distinct intracellular niches. We showed that STM uses nutrients of the host cell and adapts uniquely to the different nutrient conditions. In addition, we provide further evidence for improved nutrient supply by SIF formation or presence in the cytosol of epithelial cells, and the correlation of nutrient supply to bacterial proliferation.

Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen and acquisition of nutrients from host cells is essential for survival and proliferation of intracellular STM. The nutritional environment of intracellular STM is only partially understood. We deploy bacteria harbouring reporter plasmids to interrogate the environmental cues acting on intracellular STM, and flow cytometry allows analyses on level of single STM. Phosphorus is a macro‐element for cellular life, and in STM inorganic phosphate (Pi), homeostasis is mediated by the two‐component regulatory system PhoBR, resulting in expression of the high affinity phosphate transporter pstSCAB‐phoU. Using fluorescent protein reporters, we investigated Pi availability for intracellular STM at single‐cell level over time. We observed that Pi concentration in the Salmonella‐containing vacuole (SCV) is limiting and activates the promoter of pstSCAB‐phoU encoding a high affinity phosphate uptake system. Correlation between reporter activation by STM in defined media and in host cells indicates Pi concentration less 10 μM within the SCV. STM proliferating within the SCV experience increasing Pi limitations. Activity of the Salmonella pathogenicity island 2 (SPI2)‐encoded type III secretion system (T3SS) is crucial for efficient intracellular proliferation, and SPI2‐T3SS‐mediated endosomal remodelling also reliefs Pi limitation. STM that are released from SCV to enter the cytosol of epithelial cells did not indicate Pi limitations. Addition of Pi to culture media of infected cells partially relieved Pi limitations in the SCV, as did inhibition of intracellular proliferation. We conclude that availability of Pi is critical for intracellular lifestyle of STM, and Pi acquisition is maintained by multiple mechanisms. Our work demonstrates the use of bacterial pathogens as sensitive single‐cell reporters for their environment in host cell or host organisms. Take Away Salmonella strains were engineered to report their intracellular niche and the availability of inorganic phosphate (Pi) on level of single intracellular bacteria Within the Salmonella‐containing vacuole (SCV), Pi is limited and limitation increases with bacterial proliferation Salmonella located in host cell cytosol are not limited in Pi availability Remodelling of the host cell endosomal system mediated by T3SS‐2 reliefs Pi limitation in the SCV

The facultative intracellular pathogen Salmonella enterica Typhimurium (STM) resides in a specific membrane-bound compartment termed the Salmonella-containing vacuole (SCV). STM is able to obtain all nutrients required for rapid proliferation, although being separated from direct access to host cell metabolites. The formation of specific tubular membrane compartment, called Salmonella-induced filaments (SIFs) are known to provides bacterial nutrition by giving STM access to endocytosed material and enabling proliferation. Additionally, STM expresses a range of nutrient uptake system for growth in nutrient limited environments to overcome the nutrition depletion inside the host. By utilizing dual fluorescence reporters, we shed light on the nutritional environment of intracellular STM in various host cells and distinct intracellular niches. We showed that STM uses nutrients of the host cell and adapts uniquely to the different nutrient conditions. In addition, we provide further evidence for improved nutrient supply by SIF formation or presence in the cytosol of epithelial cells, and the correlation of nutrient supply to bacterial proliferation.