Contribution of Asparagine Catabolism to Salmonella Virulence

Abstract

Salmonellae are pathogenic bacteria that cause significant morbidity and mortality in humans worldwide. Salmonellae establish infection and avoid clearance by the immune system by mechanisms that are not well understood. We previously showed that l-asparaginase II produced by Salmonella enterica serovar Typhimurium (S Typhimurium) inhibits T cell responses and mediates virulence. In addition, we previously showed that asparagine deprivation such as that mediated by l-asparaginase II of S Typhimurium causes suppression of activation-induced T cell metabolic reprogramming. Here, we report that STM3997, which encodes a homolog of disulfide bond protein A (dsbA) of Escherichia coli, is required for l-asparaginase II stability and function. Furthermore, we report that l-asparaginase II localizes primarily to the periplasm and acts together with l-asparaginase I to provide S Typhimurium the ability to catabolize asparagine and assimilate nitrogen. Importantly, we determined that, in a murine model of infection, S Typhimurium lacking both l-asparaginase I and II genes competes poorly with wild-type S Typhimurium for colonization of target tissues. Collectively, these results indicate that asparagine catabolism contributes to S Typhimurium virulence, providing new insights into the competition for nutrients at the host-pathogen interface.

Keywords: Salmonella; T cells; asparaginase; asparagine; catabolism; host response; metabolism; nitrogen metabolism; pathogenesis; virulence.

FIG 1

STM3997 is required for S. Typhimurium to cause inhibition of T cells. (A) Expression of surface TCR-β by anti-CD3ε/CD28-stimulated T cells left uninfected (UI) or cultured with wild-type S. Typhimurium (WT) or ΔSTM3993–8 S. Typhimurium (ΔSTM3993–8). (B) Expression of surface TCR-β by anti-CD3ε/CD28-stimulated T cells left uninfected (UI) or cultured with wild-type S. Typhimurium (WT), ΔSTM3993–8 S. Typhimurium (ΔSTM3993–8), ΔSTM3993–8 S. Typhimurium carrying plasmid pBAD18-Cm (ΔSTM3993–8/Empty Vector) or ΔSTM3993–8 S. Typhimurium carrying derivatives of pBAD18-Cm expressing STM3993 (ΔSTM3993–8/pSTM3993), STM3994 (ΔSTM3993–8/pSTM3994), STM3995 (ΔSTM3993–8/pSTM3995), STM3996 (ΔSTM3993–8/pSTM3996), STM3997 (ΔSTM3993–8/pSTM3997), or STM3998 (ΔSTM3993–8/pSTM3998). (C) Genetic organization of the chromosomal region deleted in ΔSTM3993–8 S. Typhimurium. (D) Expression of surface TCR-β by anti-CD3ε/CD28-stimulated T cells left uninfected (UI) or cultured with wild-type S. Typhimurium (WT), ΔSTM3997 S. Typhimurium (ΔSTM3997), ΔSTM3997 S. Typhimurium carrying pBAD18-Cm (ΔSTM3997/Empty Vector) or ΔSTM3997 S. Typhimurium carrying a derivative of pBAD18-Cm bearing STM3997 (ΔSTM3997/pSTM3997). (E) Blastogenesis of anti-CD3ε/CD28-stimulated T cells treated as described for panel D. (F) IL-2 secretion by anti-CD3ε/CD28-stimulated T cells treated as described for panel D. Data show means with standard errors of the means obtained from at least five independent experiments. Data were analyzed by repeated measures one-way ANOVA with Tukey's multiple comparisons posttest; P values of <0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences for designated posttest comparisons (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s. not significant). GMFI, geometric mean fluorescent intensity.

FIG 2

STM3997 encodes a periplasmic thiol-disulfide oxidoreductase required to maintain the steady-state level of l-asparaginase II. (A) Western blot analysis of whole-cell lysates of wild-type S. Typhimurium (WT), S. Typhimurium lacking the l-asparaginase II gene STM3106 (ΔSTM3106), S. Typhimurium lacking the DsbA gene STM3997 (ΔSTM3997), ΔSTM3997 S. Typhimurium carrying plasmid pBAD18-Cm (ΔSTM3997/Empty Vector), or ΔSTM3997 S. Typhimurium carrying a derivative of pBAD18-Cm expressing STM3997 (ΔSTM3997/pSTM3997) using anti-l-asparaginase II antibody (α-l-Asnase-II) or anti-DnaK antibody (α-DnaK). (B) Quantification of Western blot data shown in panel A. DnaK was used as a loading control for Western blotting. Electrophoretic densitometry was used to quantify the relative levels of l-asparaginase II normalized to DnaK. Data are representative of (A) or show means with standard errors of the means obtained from (B) four independent experiments. Data in panel B were analyzed by repeated measures one-way ANOVA with Tukey's multiple comparisons posttest; P values of <0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences for designated posttest comparisons (***, P < 0.001; **, P < 0.01; n.s. not significant).

FIG 3

Cysteine residues at positions 99 and 127 are essential for l-asparaginase II stability and function. (A) Western blot analysis of whole-cell lysates of ΔSTM3106 S. Typhimurium carrying plasmid pSTM3106 (ΔSTM3106/pSTM3106 strain) or derivatives of pSTM3106 containing site-specific mutations in STM3106 that result in alanine substitutions for cysteine residues at positions 99 (ΔSTM3106/pSTM3106_C99A strain), 127 (ΔSTM3106/pSTM3106_C127A strain), or both 99 and 127 (ΔSTM3106/pSTM3106_C99A/C127A strain). Expression of l-asparaginase II and DnaK was detected with anti-l-asparaginase II antibody (α-l-Asnase-II) and anti-DnaK antibody (α-DnaK), respectively. DnaK was used as a loading control for Western blotting. (B) Quantification of Western blot data shown in panel A. Electrophoretic densitometry was used to quantify the relative levels of l-asparaginase II normalized to DnaK. (C) Expression of surface TCR-β by anti-CD3ε/CD28-stimulated T cells left uninfected (UI) or cultured with the ΔSTM3106/pSTM3106, ΔSTM3106/pSTM3106_C99A, or ΔSTM3106/pSTM3106_C127A strain. Data are representative of (A) or show means with standard errors of the means obtained from (B and C) at least four independent experiments. Data were analyzed by repeated measures one-way ANOVA with Tukey's multiple comparisons posttest (B and C); P values of <0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences for designated posttest comparisons (****, P < 0.0001; **, P < 0.01; *, P < 0.05; n.s. not significant). See also Fig. S1 in the supplemental material.

FIG 4

l-Asparaginase II localizes primarily to the periplasm and is used by S. Typhimurium to catabolize asparagine and assimilate nitrogen. (A) Western blot analysis of whole-cell lysates (WCL) of S. Typhimurium, cytoplasmic or periplasmic fractions of S. Typhimurium, or TCA-precipitated proteins from cell-free culture supernatants of S. Typhimurium (Secreted) grown in lysogeny broth. Expression of l-asparaginase II, MBP, and DnaK was detected with anti-l-asparaginase II antibody (α-l-Asnase-II), anti-MBP antibody (α-MBP), and anti-DnaK antibody (α-DnaK), respectively. MBP and DnaK were used as fractionation controls. (B) Growth of wild-type S. Typhimurium (WT) and ΔSTM3106 S. Typhimurium (ΔSTM3106) in M9 minimal medium with ammonium chloride (NH₄Cl) as the sole nitrogen source. (C) Growth of wild-type S. Typhimurium (WT), ΔSTM3106 S. Typhimurium (ΔSTM3106), ΔSTM3106 S. Typhimurium carrying plasmid pBAD18-Cm (ΔSTM3106/Empty Vector), and ΔSTM3106 S. Typhimurium carrying a derivative of pBAD18-Cm bearing STM3106 (ΔSTM3106/pSTM3106) in M9 minimal medium with asparagine (Asn) as the sole nitrogen source. Data are representative of (A) or show means with standard errors of the means obtained from (B and C) at least three independent experiments. Data were analyzed by repeated measures two-way ANOVA with Sidak (B) or Tukey's (C) multiple comparisons posttest; P values of <0.05 were considered to be statistically significant. Number (#) symbols indicate statistically significant differences for designated posttest comparisons between WT and ΔSTM3106 S. Typhimurium at indicated time points. See also Fig. S1 in the supplemental material.

FIG 5

l-Asparaginase II and l-asparaginase I act together to provide S. Typhimurium the ability to assimilate nitrogen. (A) Growth of wild-type S. Typhimurium (WT) and ΔSTM1294 ΔSTM3106 S. Typhimurium (ΔSTM1294 ΔSTM3106) in M9 minimal medium with ammonium chloride (NH₄Cl) as the sole nitrogen source. (B) Growth of wild-type S. Typhimurium (WT), ΔSTM1294 ΔSTM3106 S. Typhimurium (ΔSTM1294 ΔSTM3106), ΔSTM1294 ΔSTM3106 S. Typhimurium carrying plasmid pBAD18-Cm (ΔSTM1294 ΔSTM3106/Empty Vector), ΔSTM1294 ΔSTM3106 S. Typhimurium carrying a derivative of pBAD18-Cm expressing STM1294 (ΔSTM1294 ΔSTM3106/pSTM1294), and ΔSTM1294 ΔSTM3106 S. Typhimurium carrying a derivative of pBAD18-Cm expressing STM3106 (ΔSTM1294 ΔSTM3106/pSTM3106) in M9 minimal medium with asparagine (Asn) as the sole nitrogen source. Data show means with standard errors of the means obtained from at least three independent experiments. Data were analyzed by repeated measures two-way ANOVA with Sidak (A) or Tukey's (B) multiple comparisons posttest; P values of <0.05 were considered to be statistically significant. Number (#) symbols indicate statistically significant differences for designated posttest comparisons between WT and ΔSTM1294 ΔSTM3106 S. Typhimurium at indicated time points. See also Fig. S2 in the supplemental material.

FIG 6

Asparagine catabolism contributes to S. Typhimurium virulence. (A) Survival and growth of wild-type S. Typhimurium (WT), ΔSTM1294 S. Typhimurium (ΔSTM1294), ΔSTM3106 S. Typhimurium (ΔSTM3106) or ΔSTM1294 ΔSTM3106 S. Typhimurium (ΔSTM1294 ΔSTM3106) inside bone marrow-derived macrophages cultured from 129X1/SvJ mice. (B to D) Competitive indices of the ΔSTM1294 ΔSTM3106 strain versus wild-type S. Typhimurium recovered from mesenteric lymph nodes (MLN; B), livers (C) and spleens (D) of 129X1/SvJ mice (n = 6 to 8 per group per time point) at various times after intragastric inoculation with 1 × 10⁸ CFU of a 1:1 mixture of differentially marked bacterial strains. Data were analyzed by repeated measures two-way ANOVA with Tukey's multiple comparisons posttest (A), repeated-measures one-way ANOVA with Dunnett's multiple comparisons posttest (B to D; data were compared to the expected 1:1 output ratio of the ΔSTM1294 ΔSTM3106 strain versus WT S. Typhimurium). P values of <0.05 were considered to be statistically significant. Asterisks indicate statistically significant differences for designated posttest comparisons (***, P < 0.001; **, P < 0.01; *, P < 0.01; n.s. not significant). See also Fig. S3 in the supplemental material.

FIG 7

Model showing the competition for nutritional asparagine between S. Typhimurium and T cells. Our published findings (14, 15) and results described in the present study support a model in which S. Typhimurium competes with the host for nutritional asparagine, causing the local depletion of a key resource required for T cell activation. See the text for details.