L-asparaginase II produced by Salmonella typhimurium inhibits T cell responses and mediates virulence

Abstract

Salmonella enterica serovar Typhimurium avoids clearance by the host immune system by suppressing T cell responses; however, the mechanisms that mediate this immunosuppression remain unknown. We show that S. Typhimurium inhibit T cell responses by producing L-Asparaginase II, which catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia. L-Asparaginase II is necessary and sufficient to suppress T cell blastogenesis, cytokine production, and proliferation and to downmodulate expression of the T cell receptor. Furthermore, S. Typhimurium-induced inhibition of T cells in vitro is prevented upon addition of L-asparagine. S. Typhimurium lacking the L-Asparaginase II gene (STM3106) are unable to inhibit T cell responses and exhibit attenuated virulence in vivo. L-Asparaginases are used to treat acute lymphoblastic leukemia through mechanisms that likely involve amino acid starvation of leukemic cells, and these findings indicate that pathogens similarly use L-asparagine deprivation to limit T cell responses.

Figure 1. Only Viable S. Typhimurium and Those Capable of Synthesizing New Proteins Are Able to Downmodulate TCR Expression and Suppress T Cell Blastogenesis

(A and B) Expression of surface TCR-β by CD25⁺ T cells left uninfected (UI, gray in histograms) or cultured with viable S. Typhimurium (STm) or formaldehyde-fixed S. Typhimurium (Fixed STm); p = 0.0002. (C and D) Blastogenesis of CD25⁺ T cells treated as in (A); p = 0.0054. (E and F) Expression of surface TCR-β by CD25⁺ T cells left uninfected (UI, gray in histograms) or cultured with untreated S. Typhimurium (STm) or chloramphenicol-treated S. Typhimurium (Cm-treated STm); p < 0.0001. (G and H) Blastogenesis of CD25⁺ T cells treated as in (E); p = 0.0004. Data are representative of (A), (C), (E), and (G), or show the mean with SEM (B, D, F, and H) from at least three independent experiments.

Figure 2. STM3106 Is Required for S. Typhimurium to Downmodulate TCR Expression, Suppress T Cell Blastogenesis, Block Cytokine Production, and Inhibit T Cell Proliferation

(A) Expression of surface TCR-β by CD25⁺ T cells left uninfected (UI) or cultured with either WT or ΔSTM3104-7 S. Typhimurium; p < 0.0001. (B) Genetic organization of the chromosomal region deleted in ΔSTM3104-7 S. Typhimurium. (C) Expression of surface TCR-β by CD25⁺ T cells left UI or cultured with WT, ΔSTM3104, ΔSTM3105, ΔSTM3106, or ΔSTM3107 S. Typhimurium; p < 0.0001. (D and E) Expression of surface TCR-β by CD25⁺ T cells left UI (gray in histograms) or cultured with WT S. Typhimurium, ΔSTM3106 S. Typhimurium, ΔSTM3106 S. Typhimurium carrying plasmid pBAD18-Cm (ΔSTM3106/pBAD), or ΔSTM3106 S. Typhimurium carrying a derivative of pBAD18-Cm encoding STM3106 (ΔSTM3106/pBAD-STM3106); p < 0.0001. (F and G) Blastogenesis of CD25⁺ T cells treated as in (D); p < 0.0001. (H and I) Expression of intracellular IFN-γ by CD25⁺ T cells treated as in (D); p < 0.0001. (J and K) Expression of intracellular IL-2 by CD25⁺ T cells treated as in (D); p = 0.0001. (L and M) Proliferation of CFSE-labeled T cells left UI or cultured with WT or ΔSTM3106 S. Typhimurium; p = 0.0021. Data are representative of (D), (F), (H), (J), and (L), or show the mean with SEM (A, C, E, G, I, K, and M) from at least three independent experiments. See also Figure S1.

Figure 3. L-Asparaginase II Encoded by STM3106 Is Necessary and Sufficient to Cause Downmodulation of the TCR

(A) STM3106 encodes L-Asparaginase II, an extracytoplasmic enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. (B) Expression of surface TCR-β by CD25⁺ T cells incubated with filtered medium harvested from T cells left uninfected (UI) or infected with WT S. Typhimurium (STm). This medium had been subjected to immunodepletion using L-Asparaginase II-specific antibody (α-L-Asparaginase II Ab), control antibody (Control Ab), or no antibody (No Ab); p < 0.0001. (C) Expression of surface TCR-β by CD25⁺ T cells left UI, cultured with STm, or treated with purified L-Asparaginase II; p = 0.0018. (D) Expression of surface TCR-β by untreated, L-asparagine-treated (L-Asn), L-aspartate-treated (L-Asp), or L-glutamine-treated (L-Gln) CD25⁺ T cells left UI or cultured with STm; p < 0.0001. Data show the mean with SEM from at least three independent experiments. See also Figure S2.

Figure 4. An S. Typhimurium ansB Mutant Unable to Express L-Asparaginase II Is Attenuated for Virulence in Mouse Models of Infection

(A) Survival of 129X1/SvJ mice (n = 15 per group) inoculated intragastrically with 5 ×10⁷ cfu of WT (squares) or ΔSTM3106 (triangles) S. Typhimurium; p = 0.0183. (B) Bacterial loads per gram of tissue harvested from 129X1/SvJ mice (n = 15 per group) 60 days after intragastric inoculation with 5 × 10⁷ cfu of WT (squares) or ΔSTM3106 (triangles) S. Typhimurium; p = 0.0003 for liver. At the time of harvest, eight mice infected with WT S. Typhimurium and four mice infected with ΔSTM3106 S. Typhimurium had succumbed. (C and D) Bacterial loads per gram of liver (C) and spleen (D) tissue harvested from 129X1/SvJ mice (n = 5 per group per time point) at various times after intragastric inoculation with 5 × 10⁷ cfu of WT (squares) or ΔSTM3106 (triangles) S. Typhimurium; p = 0.0001 for liver and p = 0.2905 for spleen. (E) Bacterial loads per gram of liver tissue harvested from F1 (C57BL/6J × 129X1/SvJ) hybrid mice (n = 8 per group) 10 days after intravenous inoculation with 5 × 10³ cfu of WT S. Typhimurium, ΔSTM3106 S. Typhimurium, or ΔSTM3106 S. Typhimurium carrying a derivative of pWSK29 encoding STM3106 (ΔSTM3106/pWSK29-STM3106); p = 0.0111. Data show the mean with SEM. See also Figure S3.