PagR mediates the precise regulation of Salmonella pathogenicity island 2 gene expression in response to magnesium and phosphate signals in Salmonella Typhimurium

Abstract

To establish systemic infections, Salmonella enterica serovar Typhimurium (S. Typhimurium) requires Salmonella pathogenicity island 2 (SPI-2) to survive and replicate within macrophages. High expression of many SPI-2 genes during the entire intracellular growth period within macrophages is essential, as it contributes to the formation of Salmonella-containing vacuole and bacterial replication. However, the regulatory mechanisms underlying the sustained induction of SPI-2 within macrophages are not fully understood. Here, we revealed a time-dependent regulation of SPI-2 expression mediated by a novel regulator PagR (STM2345) in response to the low Mg 2+ and low phosphate (P i ) signals, which ensured the high induction of SPI-2 during the entire intramacrophage growth period. Deletion of pagR results in reduced bacterial replication in macrophages and attenuation of systemic virulence in mice. The effects of pagR on virulence are dependent on upregulating the expression of slyA, a regulator of SPI-2.At the early (0-4 hr) and later (after 4 hr) stage post-infection of macrophages, pagR is induced by the low P i via PhoB/R two-component systems and low Mg 2+ via PhoP/Q systems, respectively. Collectively, our findings revealed that the PagR-mediated regulatory mechanism contributes to the precise and sustained activation of SPI-2 genes within macrophages, which is essential for S. Typhimurium systemic virulence.

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

“…Deletion of pstSCAB was recently reported to attenuate systemic virulence of STM in the murine infection model (Zhang et al, 2019), and our work extends these findings to further components of the phosphate metabolism. The reduced replication of a Δ phoB strain in RAW264.7 macrophages and a reduced systemic virulence in mice was recently reported (Jiang et al, 2020). Defect of PhoU leads to constitutive expression of the Pst transporter and unphysiological accumulation of P i in STM cytosol, indicating that the proper phosphate homeostasis is of critical importance.…”

“…Recently, PagR was identified as S. enterica -specific integrator of low P i and low Mg 2+ levels leading to activation of expression of SPI2-T3SS genes (Jiang et al, 2020). The study proposed that PhoB is activated by low P i , and PhoQ in response to low Mg 2+ , both activated PagR.…”

Downregulation of a novel flagellar synthesis regulator AsiR promotes intracellular replication and systemic pathogenicity of Salmonella Typhimurium

“…The mutant strains (ΔasiR, ∆flhDC, and ∆asiR∆flhDC) were constructed with the λ Red recombinase system [31]. The transcriptional fusions asiR-lux, ssrA-lux, and flhDC-lux were generated as described previously [13]. The amplification products of respective promoter regions were digested and cloned into the XhoI-BamHI site upstream of the lux genes in the plasmid pMS402.…”

“…Immunofluorescence was performed as previously described [13]. Briefly, at 2 and 16 h postinfection (hpi), the infected RAW264.7 cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 5 min, and then blocked with 5% BSA for 1 h. The intracellular S. Typhimurium cells were stained using the mouse raised monoclonal anti-S. Typhimurium LPS antibody (1:100 dilution, Abcam) and FITC-conjugated goat antimouse IgG antibody (1:200 dilution, Abcam).…”

“…Several regulatory proteins have been previously identified to be involved in the regulatory network and have been reported to contribute to Salmonella intracellular replication in response to various environmental stimuli. PagR, a LacI family transcriptional regulator, is induced by the low Mg 2+ and low P i stimuli in host macrophages, and it activates the expression of Salmonella pathogenicity island (SPI)-2 genes, which are essential for Salmonella intracellular replication [13]. The two-component regulatory systems, PhoP/Q and EnvZ/ OmpR, also activate the expression of SPI-2 genes in response to low Mg 2+ , acidic pH, and the presence of antibacterial peptides inside host macrophages (PhoP/ Q) [14] or acidic pH (EnvZ/OmpR) [15].…”