The flagellar regulator TviA reduces pyroptosis by Salmonella enterica serovar Typhi

Abstract

To discern virulent from innocuous microbes, the innate immune system senses events associated with bacterial access to immunoprivileged sites such as the host cell cytosol. One such pathway is triggered by the cytosolic delivery of flagellin, the major subunit of the flagellum, by bacterial secretion systems. This leads to inflammasome activation and subsequent proinflammatory cell death (pyroptosis) of the infected phagocyte. In this study, we demonstrate that the causative agent of typhoid fever, Salmonella enterica serovar Typhi, can partially subvert this critical innate immune recognition event. The transcriptional regulator TviA, which is absent from Salmonella serovars associated with human gastroenteritis, repressed the expression of flagellin during infection of human macrophage-like (THP-1) cells. This mechanism allowed S. Typhi to dampen inflammasome activation, leading to reduced interleukin-1β (IL-1β) secretion and diminished cell death. Likewise, the introduction of the tviA gene in nontyphoidal Salmonella enterica serovar Typhimurium reduced flagellin-induced pyroptosis. These data suggest that gene regulation of virulence factors enables S. Typhi to evade innate immune recognition by concealing a pathogen-induced process from being sensed by the inflammasome.

FIG 1

Effect of flagellin on the interaction of S. Typhi and S. Typhimurium with human macrophage-like cells. (A) PMA-differentiated THP-1 cells were infected with the S. Typhi wild-type strain (Ty2) or the S. Typhimurium wild-type strain (IR715) or mock treated, and the concentration of IL-1β in the supernatant was determined by an enzyme-linked immunosorbent assay (n = 3). (B) In vitro expression of flagellin by the S. Typhi wild-type strain, an isogenic ΔfliC mutant, the S. Typhimurium wild-type strain, or an isogenic ΔfliC fljB mutant. S. Typhi and S. Typhimurium flagellins were detected by Western blotting using Salmonella H antiserum d (αHd) and antiserum i (αHi), respectively. As a loading control, the amount of the chaperone GroEL (αGroEL) was determined. The approximate locations of standard proteins are indicated. (C and D) PMA-differentiated THP-1 cells were treated with DMSO (vehicle control) or 25 μM caspase inhibitor Z-WEHD-FMK and subsequently infected with the S. Typhi wild-type strain (Ty2), an isogenic ΔfliC mutant (SW359), the S. Typhimurium wild-type strain (IR715), or an isogenic ΔfliC fljB mutant (SW473) or mock treated. After 8 h, the intracellular bacterial load (C) and the amount of IL-1β released into the supernatant (D) were determined (n = 4). Bars represent geometric means ± standard errors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not statistically significant.

FIG 2

Effect of TviA-mediated flagellin repression on cell death and IL-1β secretion induced by S. Typhi. (A and B) PMA-differentiated THP-1 cells were infected for 8 h with the indicated S. Typhi strains precultured in TYE broth. (A) Cell death was determined by using an LDH release assay. (B) IL-1β secretion into the supernatant was measured by an enzyme-linked immunosorbent assay. FliC-dependent cell death and IL-1β release are indicated by white bars, while LDH release and IL-1β secretion independent of FliC (i.e., amount induced by a ΔviaB ΔfliC mutant [dashed lines]) are indicated by gray bars (n = 6). (C and D) Quantification of S. Typhi fliC levels inside macrophage-like cells. PMA-differentiated THP-1 cells were infected with the S. Typhi wild-type strain, a ΔviaB mutant, a ΔtviB-vexE mutant, and a ΔviaB ΔfliC mutant for 3 h. (C) Total RNA was extracted, and relative fliC mRNA levels, normalized to the amount of 16S rRNA, were determined by quantitative RT-PCR (n = 4). (D) Lysates of infected THP-1 cells were analyzed by Western blotting for expression of flagellin (αHd antiserum). As a loading control, the alpha subunit of the bacterial RNA polymerase (RpoA) was quantified. The approximate locations of marker proteins are indicated on the left. Bars represent geometric means ± standard errors. *, P < 0.001; **, P < 0.01; ns, not statistically significant; ND, none detected.

FIG 3

Heterologous expression of TviA in S. Typhimurium reduces IL-1β secretion. The indicated S. Typhimurium strains were cultured in TYE broth prior to infections. (A and B) Murine BMMs were infected with the indicated S. Typhimurium strains, and IL-1β secretion (A) (n = 3) as well as cell death (B) (n = 4) were measured after 16 h. FliC-dependent IL-1β release and cell death are indicated by white bars, while LDH release and IL-1β secretion independent of FliC (i.e., amount induced by a ΔfliC fljB mutant [dashed lines]) are indicated by gray bars. (C) Immortalized BMMs isolated from wild-type animals were infected with the indicated S. Typhimurium strains for 3 h. Total RNA was extracted from infected cells, and relative fliC mRNA levels, normalized to 16S rRNA levels, were determined by quantitative RT-PCR (n = 4). Bars represent geometric means ± standard errors. *, P < 0.001; **, P < 0.01; ns, not statistically significant.

FIG 4

Effect of TviA on inflammasome activation. S. Typhimurium strains were cultured in TYE broth prior to infections. (A and B) Immortalized murine BMMs were infected with the indicated S. Typhimurium strains for 16 h. (A) Cell lysates and supernatants were analyzed by Western blotting using caspase-1-specific antiserum. The approximate locations of marker proteins are indicated. (B) Immortalized BMMs isolated from wild-type animals or Nlrc4-deficient animals were infected with S. Typhimurium strains or mock treated, and IL-1β secretion was quantified (n = 3). (C) Groups of C57BL/6 animals (n = 4) were intraperitoneally infected with an equal mixture of the wild type (IR715) and the phoN::tviA mutant (SW474) or the ΔfliC fljB mutant (SW473) and the ΔfliC fljB phoN::tviA mutant (SW718). Bacterial numbers in the spleen were determined at 48 h postinfection, and the ratio of tviA-expressing strain over the strain lacking the tviA gene (competitive index) was calculated. Bars represent geometric means ± standard errors. *, P < 0.001; **, P < 0.01; ns, not statistically significant.