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 (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

Intracellular niche-specific profiling reveals transcriptional adaptations required for the cytosolic lifestyle of Salmonella enterica

“…, and Zn 2+ and S. Typhimurium are experiencing oxidative stress but not amino acid starvation (Figs 3, 5 and 8) [42,43,67,91,134].…”

Bright individuals: Applications of fluorescent protein‐based reporter systems in single‐cell cellular microbiology

“…FPs with short MT are suitable for timeresolved analyses, as they allow to display even small changes in expression. Therefore, this feature is frequently deployed in gene expression analyses or metabolic activity measurements to pro- direct FPs to proteasomal degradation and by this reduce their halflife (Andersen et al, 1998;Keiler et al, 1996;Röder et al, 2021b).…”

“…Comparison of stability of sfGFP and sfGFP-LVA indicated reduction of half-life reduction of more than 2 h by the addition of LVA tag (Röder et al, 2021b). The influence of destabilization tags on FP half-life can be analyzed using an inducer-controlled reporter system.…”

“…Accordingly, DFRs are generated with promoters activated upon nutritional limitation, or induction occurs by the availability of specific nutrients. DFR with various promoters for nutrient uptake systems have been used to monitor the nutritional landscape of STM with mammalian host cells (Röder et al, 2021a(Röder et al, , 2021b). An overview of DFR used in these studies is given in Table 1.…”

A Dual Regulatory Role of the PhoU Protein in Salmonella Typhimurium

“…Here, we investigated the roles of the PhoU protein from Salmonella enterica serovar Typhimurium. Although maintaining phosphate levels appears to be critical for Salmonella pathogenesis ( 16 , 19 ), the role(s) of the PhoU protein in pho expression and Salmonella pathogenesis has not been investigated yet. Based on protein homology modeling, we identified the PhoU residues that are required for PhoR interaction ( Fig.…”