Salmonella enterica serovar Typhi impairs CD4 T cell responses by reducing antigen availability

Abstract

Salmonella enterica serovar Typhi is associated with a disseminated febrile illness in humans, termed typhoid fever, while Salmonella enterica serovar Typhimurium causes localized gastroenteritis in immunocompetent individuals. One of the genetic differences between both pathogens is the presence in S. Typhi of TviA, a regulatory protein that shuts down flagellin (FliC) expression when bacteria transit from the intestinal lumen into the intestinal mucosa. Here we investigated the consequences of TviA-mediated flagellum gene regulation on flagellin-specific CD4 T cell responses in a mouse model of S. Typhimurium infection. Introduction of the S. Typhi tviA gene into S. Typhimurium suppressed antigen presentation of dendritic cells to flagellin-specific CD4 T cells in vitro. Furthermore, TviA-mediated repression of flagellin expression impaired the activation and proliferation of naive flagellin-specific CD4 T cells in Peyer's patches and mesenteric lymph nodes, which was accompanied by increased bacterial dissemination to the spleen. We conclude that TviA-mediated repression of flagellin expression reduces antigen availability, thereby weakening flagellin-specific CD4 T cell responses.

FIG 1

TviA represses flagellin expression in vitro and during infection of cultured mononuclear cells. (A) Schematic of the viaB locus of S. Typhi. Sizes and locations of deletions present in the S. Typhi ΔtviB-vexE mutant (SW74) and the S. Typhi ΔviaB mutant (SW347) are indicated. (B and C) S. Typhi wild-type (Ty2), ΔtviB-vexE mutant (SW74), ΔviaB mutant (SW347), and ΔviaB fliC mutant (SW483) strains as well as S. Typhimurium wild-type (IR715), phoN mutant (SW478), phoN::tviA mutant (SW474), and phoN ΔfliC fljB mutant (SW681) strains were cultured in TYE medium for 3 h. Expression of FliC was determined by Western blotting using Hd-specific antiserum (αHd) (B) or Hi-specific antiserum (αHi) (C). As a loading control, the expression of GroEL was examined (αGroEL). The approximate location of marker proteins is indicated on the left. (D and E) Transcription of fliC (D) and fljB (E) in S. Typhimurium wild-type (IR715), phoN mutant (SW478), and tviA-expressing phoN::tviA mutant (SW474) strains during growth in TYE broth. Levels of mRNA were determined by quantitative RT-PCR and normalized to levels of 16S rRNA. (F) Murine mononuclear cells (JAWSII cell line) were infected with the indicated S. Typhimurium strains for 4 h. Transcription of fliC, normalized to 16S rRNA levels, was determined by quantitative RT-PCR. Bars represent the geometric means ± standard errors. *, P < 0.05; ***, P < 0.001; ND, none detected; ns, not statistically significant.

FIG 2

Impact of TviA-mediated gene regulation on antigen presentation in cell culture. CD11c⁺ dendritic cells isolated from C57BL/6 Nramp^+/+ mice were treated with medium alone (DPBS) or infected with various amounts of the indicated S. Typhimurium strains suspended in DPBS. The MOI is indicated below each panel. Thirty minutes after infection, cells were washed and subsequently cocultured for 16 h with flagellin-specific SM1 T cells. (A) Representative histograms of CD69 expression on gated SM1 T cells (CD4⁺ CD90.1⁺). (B) Bars represent the geometric means ± standard errors of activated SM1 T cells as a percentage of all SM1 T cells. The experiment was performed in triplicate. *, P < 0.05; ***, P < 0.001.

FIG 3

TviA modulates flagellin-specific, naive CD4 T cell responses in vivo. The expansion of adoptively transferred SM1 T cells (CD4⁺ CD90.1⁺) in Peyer's patches in response to infection with various S. Typhimurium strains was investigated. Mice were orally infected for 5 days with a phoN mutant (SW478), a phoN::tviA mutant (SW474), or a phoN ΔfliC fljB mutant (SW681) or were mock treated (transfer only). (A) Representative plots show flagellin-specific SM1 T cells (CD4⁺ CD90.1⁺). (B) Bars represent the geometric means ± standard errors of gated SM1 T cells (CD4⁺ CD90.1⁺) for groups of 5 mice. *, P < 0.05.

FIG 4

Effect of TviA on activation and proliferation of flagellin-specific, naive SM1 T cells in Peyer's patches and mesenteric lymph nodes. A total of 1 × 10⁶ CFSE-labeled, flagellin-specific SM1 T cells were adoptively transferred into C57BL/6 Nramp^+/+ mice. Groups of these mice were mock treated (transfer only) or orally infected with the indicated S. Typhimurium strains. T cells were isolated from Peyer's patches (A and C) and mesenteric lymph nodes (B and D) 5 days after infection. CFSE staining as well as CD11a expression on SM1 T cells were examined by flow cytometry (gated on CD4⁺ CD90.1⁺ SM1 T cells). (A and B) Representative plots for CD11a and CFSE staining (top) and representative histogram plots for CFSE staining in SM1 T cells (bottom). (C and D) Bars represent the geometric means ± standard errors of CD11a-expressing, CFSE-low SM1 T cells in groups of 3 mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 5

TviA augments dissemination to the spleen. Flagellin-specific SM1 T cells were adoptively transferred into Nramp^+/+ mice. Animals were mock treated (n = 3) or orally inoculated with the S. Typhimurium phoN mutant (SW478) (n = 5) or the phoN::tviA mutant (SW474) (n = 5). The bacterial load in Peyer's patches (A), mesenteric lymph node (B), or spleen (C) was determined 3 days after infection. Bars represent the geometric means ± standard errors. *, P < 0.05.