Single cell analyses reveal phosphate availability as critical factor for nutrition ofSalmonella entericawithin mammalian host cells

Salmonella enterica serovar Typhimurium (STM) is an invasive, facultative intracellular pathogen that resides in a specialized membrane-bound compartment termed Salmonella -containing vacuole (SCV). Essential for survival and proliferation in the SCV is Salmonella pathogenicity island II (SPI2) that encodes a type III secretion system (T3SS). The SPI2-T3SS and the effector translocation maintain SCV integrity and formation of specific tubular membrane compartments, called Salmonella -induced filaments (SIFs). The SCV/SIF continuum allows STM to bypass nutritional restriction in the intracellular environment by acquiring nutrients from the host cell. Phosphate is one of the most abundant elements in living organisms 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 investigate P i availability for STM at single cell level over time within the intracellular habitats of different host cells. We observed that the pstSCAB-phoU encoded phosphate uptake system is essential for intracellular replication of STM because there is a P i ion concentration less 10 μM within the SCV. Additionally, the demand and consumption of P i correlates with intracellular proliferation of STM and we identify a dependency of SPI2 activity and P i starvation.