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

Abstract

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 (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 investigate Pi 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 Pi ion concentration less 10 micromolar within the SCV. Additionally, the demand and consumption of Pi correlates with intracellular proliferation of STM and we identify a dependency of SPI2 activity and Pi starvation.

Comprehensive Single Cell Analyses of the Nutritional Environment of Intracellular Salmonella enterica

“…Together with our results, we conclude that bacteria in the cytosol have the best access to nutrients of the host cell, allowing fast proliferation. In addition to the factors analyzed in this study, the availability of phosphate and C-sources has to be considered ( Röder and Hensel, 2020 ; Röder and Hensel, 2021 ), as well as xenophagic or pyroptotic host cell responses to cytosolic STM. A large SIF-SCV continuum increases the frequency of fusion with vesicles, and allows the bacteria to replicate in the vacuolar compartment.…”

“…As cultural growth leads to continuous reduction of nutrient concentrations, the actual inducing concentration is likely lower for most nutritional factors. For a more detailed calibration, shift experiments with transfer in media with various amounts of nutrients may be considered as performed for analyses of phosphate availability ( Röder and Hensel, 2020 ), or inhibition of bacterial growth. The presence of a second, non-regulated fluorescent protein has been used to identify in the intracellular bacterial population.…”

Fluorescent protein‐based reporters reveal stress response of intracellularSalmonella entericaat level of single bacterial cells

“…However, the long turn‐over of sfGFP did not allow to analyse a potential decrease of stress signals acting on intracellular STM. The addition of tags for degradation, such as the LVA tag (Andersen et al, 1998) is possible and allows generation of dual colour reporters with increased dynamic range (Röder, Felgner, & Hensel, 2021; Röder & Hensel, 2021). We observed that induction of all reporters in intracellular STM was higher compared to in vitro exposure to defined stressors.…”

Comprehensive single cell analyses of the nutritional environment of intracellularSalmonella enterica

“…As cultural growth leads to continuous reduction of nutrient concentrations, the actual inducing concentration is likely lower for most nutritional factors. For a more detailed calibration, shift experiments with transfer in media with various amounts of nutrients be considered as performed for analyses of phosphate availability (Röder and Hensel, 2020b), or inhibition of bacterial growth. The presence of a second, non-regulated fluorescent protein has been used to identify in the intracellular bacterial population.…”