Arginine Metabolism Powers Salmonella Resistance to Oxidative Stress

Abstract

Salmonella invades host cells and replicates inside acidified, remodeled vacuoles that are exposed to reactive oxygen species (ROS) generated by the innate immune response. Oxidative products of the phagocyte NADPH oxidase mediate antimicrobial activity, in part, by collapsing the ΔpH of intracellular Salmonella. Given the role of arginine in bacterial resistance to acidic pH, we screened a library of 54 single-gene mutants in Salmonella that are each involved in, but do not entirely block, arginine metabolism. We identified several mutants that affected Salmonella virulence in mice. The triple mutant ΔargCBH, which is deficient in arginine biosynthesis, was attenuated in immunocompetent mice, but recovered virulence in phagocyte NADPH oxidase deficient Cybb^-/- mice. Furthermore, ΔargCBH Salmonella was profoundly susceptible to the bacteriostatic and bactericidal effects of hydrogen peroxide. Peroxide stress led to a larger collapse of the ΔpH in ΔargCBH mutants than occurred in wild-type Salmonella. The addition of exogenous arginine rescued ΔargCBH Salmonella from peroxide-induced ΔpH collapse and killing. Combined, these observations suggest that arginine metabolism is a hitherto unknown determinant of virulence that contributes to the antioxidant defenses of Salmonella by preserving pH homeostasis. In the absence of phagocyte NADPH oxidase-produced ROS, host cell-derived l-arginine appears to satisfy the needs of intracellular Salmonella. However, under oxidative stress, Salmonella must additionally rely on de novo biosynthesis to maintain full virulence.

Keywords: Salmonella; arginine; innate immunity; metabolism; nox2; oxidative stress; pH; phagocyte NADPH oxidase.

FIG 1

Significant hits from an arginine metabolism-specific single-gene KO library screen in mice. Five C57BL/6 mice were inoculated intraperitoneally with 3,000 CFU of a library containing barcoded deletion mutants of 54 arginine metabolism genes, and then livers and spleens were harvested 3 days after infection. Log₂ fold changes in barcode abundance were determined between the input and output libraries. Fitnesses with an adjusted P-value of less than 0.05 in both organs are marked. Color key: fitness defect (red), no difference (blue), not tested (no color). Figure created with BioRender.com.

FIG 2

l-arginine biosynthesis is important during oxidative stress. (a) Aerobic growth of wild-type (WT) and ΔargCBH mutant Salmonella in EG minimal media in the presence or absence of 80 μg/mL supplemental l-arginine as measured by OD₆₀₀ with a Biotek Synergy H1 plate reader. Data are shown as mean ± SEM (n = 2 to 6). (b) Aerobic growth of the indicated Salmonella mutants in the presence of 150 μM H₂O₂ in EG minimal media supplemented with 80 μg/mL l-arginine as measured by OD₆₀₀ with a Biotek Synergy H1 plate reader. Data are shown as means ± SEM (n = 3 to 9). (c) Competitive index (CI) of the ΔargCBH mutant and WT Salmonella inoculated in competition into C57BL/6 and Cybb^−/− mice. 250 CFU of each strain was inoculated intraperitoneally and then harvested 4.5 days after infection for C57BL/6 mice or 2.5 days after infection for Cybb^−/− mice. Data were analyzed by unpaired, two-tailed t test with Welch’s correction where ****, P < 0.0001. Box and whisker plots represent minimums to maximums, 25th and 75th percentiles, and medians. n = 5 for C57BL/6 and n = 8 for Cybb^−/− mice.

FIG 3

l-arginine biosynthesis-dependent peroxide killing is exacerbated in the presence of oxygen. Survival of wild-type (WT) and ΔargCBH mutant Salmonella grown overnight in EG minimal media supplemented with 20 μg/mL l-arginine after 30 min of treatment with 400 μM H₂O₂ in EG supplemented with 80 μg/mL l-arginine. (a) Salmonella in aerobic experiments were grown overnight and treated with H₂O₂ in the presence of oxygen. (b) Salmonella in aerobic to anaerobic shift experiments were grown overnight in the presence of oxygen and then treated with H₂O₂ under anaerobic conditions. (c) Salmonella in anaerobic experiments were grown overnight and treated with H₂O₂ under anaerobic conditions. Data are shown as means ± SEM (n = 6) and was analyzed by two-way ANOVA with Tukey’s correction where *, P < 0.05; ****, P < 0.0001.

FIG 4

l-arginine levels help to maintain resistance to oxidative stress. Salmonella grown overnight in EG minimal media supplemented with either 20 μg/mL l-arginine (Low Arg.) or 80 μg/mL l-arginine (High Arg.) was resuspended in either EG media (No Arg.) or EG media with 80 μg/mL l-arginine (High Arg.) for 30 min prior to treatment. Susceptibility of wild-type (WT) and ΔargCBH mutant Salmonella to 30 min treatment of 400 μM H₂O₂ in EG media +/− l-arginine. Data are shown as means ± SEM (n = 6) and was analyzed by two-way ANOVA with Tukey’s correction where **, P < 0.01; ***, P < 0.001.

FIG 5

l-arginine levels help to maintain pH homeostasis during oxidative stress. All assays were performed using Salmonella grown overnight in EG minimal media supplemented with either 20 μg/mL l-arginine (Low Arg.) or 80 μg/mL l-arginine (High Arg.) and then resuspended in either EG media (No Arg.) or EG media with 80 μg/mL l-arginine (High Arg.) for 30 min prior to measurements. (a) Effect of supplemental l-arginine on respiration of wild-type (WT) and ΔargCBH mutant Salmonella in EG media +/− l-arginine. Aerobic respiration was measured polarographically. Data are shown as means ± SEM (n = 5). (b) Intracellular pH of wild-type (WT) and ΔargCBH mutant Salmonella measured ratiometrically with pHluorin before and after treatment with 400 μM H₂O₂ in EG media +/− l-arginine. Data are shown as means ± SEM (n = 6) and was analyzed by unpaired, two-tailed t test where *, P < 0.05; ****, P < 0.0001.