Regulation of phenotypic heterogeneity permits Salmonella evasion of the host caspase-1 inflammatory response

Abstract

Sensing and adapting to the environment is one strategy by which bacteria attempt to maximize fitness in an unpredictable world; another is the stochastic generation of phenotypically distinct subgroups within a genetically clonal population. In culture, Salmonella Typhimurium populations are bistable for the expression of flagellin. We report that YdiV controls this expression pattern by preventing transcription of the sigma factor that recruits RNA polymerase to the flagellin promoter. Bistability ensues when the sigma factor is repressed in a subpopulation of cells, resulting in two phenotypes: flagellin expressors and flagellin nonexpressors. Although the ability to swim is presumably a critical survival trait, flagellin activates eukaryotic defense pathways, and Salmonella restrict the production of flagellin during systemic infection. Salmonella mutants lacking YdiV are unable to fully repress flagellin at systemic sites, rendering them vulnerable to caspase-1 mediated colonization restriction. Thus, a regulatory mechanism producing bistability also impacts Salmonella virulence.

Fig. 1.

An EAL domain protein modulates cytotoxicity by repressing fliC. (A) A panel of 22 GGDEF/EAL domain mutants was screened for the ability to trigger pyroptosis of bone marrow-derived macrophages. Macrophages were infected for 90 min at a multiplicity of infection (MOI) of 10. Only ΔydiV (STM1344) was significantly more cytotoxic than WT Salmonella (P < 0.05). Error bars represent the SD of two independent experiments. (B) Macrophages infected with ΔydiV (MOI = 12) release mature IL-1β into the supernatant more rapidly than macrophages infected with WT (MOI = 14). Values were determined using densitometry as described in Materials and Methods. Uninfected and ΔsipB (noncytotoxic control; MOI = 11) samples were collected at 40 min. (C) Hypercytotoxicity of ΔydiV is dependent on fliC. Dashed lines represent strains in which the ΔfliC mutation was complemented. pV, vector control. (D) ΔydiV overexpresses FliC. WT and ΔydiV cultures were grown to exponential phase in LB and evaluated by Western blot analysis. Four independent samples of each strain are shown. FliC expression was normalized to the DnaK loading control.

Fig. 2.

Bistability of fliC depends on ydiV. (A) Transcriptional fusion of the fliC promoter to gfp reveals distinct populations of fliC-OFF and fliC-ON cells in WT when cultures are interrogated by flow cytometry. (B) In contrast, ΔydiV demonstrates a unimodal distribution of predominantly fliC-ON cells. In A and B, 10 independent trials (colored traces) per strain are shown; the black trace represents GFP-negative control. MFIs of the fliC-ON populations are not significantly different between the two strains. fliC-ON is defined as all cells with fluorescence greater than that of the reporter-minus strain (average MFI, 1,084.7 for WT and 1,235.3 for ΔydiV:; P = 0.08). (C) When expression of ydiV is driven from the araBAD promoter, increasing concentrations of inducer correspondingly reduce the fliC-ON population (red trace, 0.0002% l-arabinose; blue trace, 0.002%; green trace, 0.2%). All experiments were performed using exponential-phase cultures.

Fig. 3.

YdiV controls the heterogeneity of fliA (σ²⁸) transcription. (A) The Salmonella flagellar gene regulatory cascade. (B) YdiV does not repress flhDC transcription. The average MFI of 10 independent trials per strain is reported. The black line represents GFP-negative control. (C) A lacZ translational reporter fusion to flhD reveals that YdiV does not repress the translation of class I proteins. csrA is required for efficient translation of the flhDC mRNA (32); thus, a ΔcsrA mutant serves as a negative control. (D) Western blot analysis demonstrates that WT and ΔydiV Salmonella contain similar amounts of FlhC protein. Four independent samples of each strain are shown. FlhC and FliC expression were normalized to the DnaK loading control for quantification. The ΔfliC mutant does not harbor the FlhC::3× FLAG construct. (E) YdiV represses fliA transcription. The black line represents GFP-negative control. All experiments were performed using exponential-phase cultures.

Fig. 4.

ΔydiV Salmonella overexpress fliC in systemic tissues. C57BL/6 mice were orally infected with 10⁶ ΔydiV or WT Salmonella harboring the PfliC::gfp reporter construct. Splenocytes were harvested from infected animals, and fliC expression by intracellular bacteria was quantified by flow cytometry as described in Materials and Methods. (Left) On average, 10 times more ΔydiV than WT transcribed fliC in the spleen. (Right) To facilitate direct comparison of gene expression from ΔydiV and WT bacteria, the two groups of mice were colonized to similar levels (Materials and Methods). Data from two independent experiments are combined.

Fig. 5.

YdiV is required to evade pyroptosis during infection. (A–C) Serum levels of TNFα and IL-1β were measured as described in Materials and Methods after oral infection with WT or ΔydiV. Concentrations of TNFα (A) and IL-1β (B) were higher in the sera after infection with ΔydiV. (C) To facilitate direct comparisons, the mice shown in A and B were colonized to similar levels with either WT or ΔydiV Salmonella (Materials and Methods). (D) Mice were orally gavaged with 10⁶ cfu of ΔydiV (strain 2) and ΔydiV ΔfliC (strain 1), for a total of 2 × 10⁶ cfu/mouse. Tissue colonization was measured at the indicated times, and the CIs were calculated as described in Materials and Methods. ΔydiV outcompetes ΔydiV ΔfliC in colonizing the Peyer's patches after 1 d of infection (n = 9 mice; data combined from two independent experiments). However, on day 6, ΔydiV ΔfliC outcompetes ΔydiV in the systemic tissues. This advantage disappears when the same experiment is performed in caspase-1 KO animals. *P < 0.01; **P < 0.001;***P < 0.0001.