Single-cell analyses reveal phosphate availability as critical factor for nutrition of Salmonella enterica within mammalian host cells

Abstract

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 (P_i), 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 P_i availability for intracellular STM at single-cell level over time. We observed that P_i 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 P_i concentration less 10 μM within the SCV. STM proliferating within the SCV experience increasing P_i 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 P_i limitation. STM that are released from SCV to enter the cytosol of epithelial cells did not indicate P_i limitations. Addition of P_i to culture media of infected cells partially relieved P_i limitations in the SCV, as did inhibition of intracellular proliferation. We conclude that availability of P_i is critical for intracellular lifestyle of STM, and P_i 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 (P_i) on level of single intracellular bacteria
• Within the Salmonella-containing vacuole (SCV), P_i is limited and limitation increases with bacterial proliferation
• Salmonella located in host cell cytosol are not limited in P_i availability
• Remodelling of the host cell endosomal system mediated by T3SS-2 reliefs P_i limitation in the SCV