The methylglyoxal pathway is a sink for glutathione in Salmonella experiencing oxidative stress

Abstract

Salmonella suffer the cytotoxicity of reactive oxygen species generated by the phagocyte NADPH oxidase in the innate host response. Periplasmic superoxide dismutases, catalases and hydroperoxidases detoxify superoxide and hydrogen peroxide (H2O2) synthesized in the respiratory burst of phagocytic cells. Glutathione also helps Salmonella combat the phagocyte NADPH oxidase; however, the molecular mechanisms by which this low-molecular-weight thiol promotes resistance of Salmonella to oxidative stress are currently unknown. We report herein that Salmonella undergoing oxidative stress transcriptionally and functionally activate the methylglyoxal pathway that branches off from glycolysis. Activation of the methylglyoxal pathway consumes a substantial proportion of the glutathione reducing power in Salmonella following exposure to H2O2. The methylglyoxal pathway enables Salmonella to balance glucose utilization with aerobic respiratory outputs. Salmonella take advantage of the metabolic flexibility associated with the glutathione-consuming methylglyoxal pathway to resist reactive oxygen species generated by the enzymatic activity of the phagocyte NADPH oxidase in macrophages and mice. Taken together, glutathione fosters oxidative stress resistance in Salmonella against the antimicrobial actions of the phagocyte NADPH oxidase by promoting the methylglyoxal pathway, an offshoot metabolic adaptation of glycolysis.

Fig 1. Redox potential in Salmonella experiencing peroxide stress.

(A) Cytoplasmic redox potential was estimated by recording the fluorescent spectra of roGFP2 in bacteria grown in MOPS-GLC medium to OD₆₀₀ of 0.25. Some of the bacterial cultures were treated for 1 min with 400 μM H₂O₂. The relative redox potential denotes the ratio of roGFP2_ox /roGFP2_red emission signals at 510 nm after excitation at 405 and 480 nm. Data are the mean ± S.D. (N = 6). ****, p< 0.0001, as determined by unpaired t-test. (B) Estimations of reduced and oxidized glutathione (GSH and GSSG, respectively) in Salmonella strains grown to an OD₆₀₀ of 0.25 in MOPS-GLC minimal medium, pH 7.2. Where indicated, samples were treated with 400 μM H₂O₂ for 30 min prior to glutathione measurements. Data are the mean ± S.D. (N = 5) *, ***, p < 0.05 and p < 0.001, respectively as determined by unpaired t-test.

Fig 2. Schematic representation of methylglyoxal pathway in Salmonella.

GLC, Glucose; FBP, Fructose 1,6 bis phosphate; DHAP, Dihydroxyacetone phosphate; G-3P, Glyceraldehyde 3-phosphate; PEP, Phosphoenolpyruvate; PYR, Pyruvate.

Fig 3. Oxidative stress stimulates utilization of the methylglyoxal pathway.

Intracellular (A,B) aldehyde and (C) D-lactate concentrations in Salmonella grown to an OD₆₀₀ of 0.25 in MOPS-GLC minimal medium, pH 7.2. Selected samples were treated with 400 μM H₂O₂ for 30 min. Data are the mean ± S.D. (N = 3–8). **, ***, p< 0.01 and p< 0.001, respectively as determined by unpaired t-test. (D) Gene expression analysis quantified by qRT-PCR of specimens isolated from Salmonella grown to an OD₆₀₀ of 0.25 in MOPS-GLC minimal medium. Where indicated, bacterial cultures were treated with 400 μM H₂O₂ for 30 min before the RNA was isolated. The data, normalized to the rpoD housekeeping gene, represent the average fold-change ± S.D (N = 3). Dashed lines depict the 2-fold upregulated marks.

Fig 4. Glyoxalase-II is important for growth of Salmonella strains in glucose.

(A) Aerobic growth of Salmonella in MOPS-GLC minimum medium, pH 7.2, at 37°C in a shaking incubator as assessed by OD₆₀₀. Data are the mean ± S.D (N = 3). (B) The content of reduced and oxidized glutathione (GSH and GSSG, respectively) was measured in wildtype and ΔgloB Salmonella strains grown to an OD₆₀₀ of 0.25 in MOPS-GLC minimum medium, pH 7.2. Data are the mean ± S.D. (N = 5) **, ****, p < 0.01 and p < 0.0001, respectively, as determined by unpaired t-test.

Fig 5. Importance of the methylglyoxal pathway in resistance of Salmonella to peroxide killing.

(A) Bacterial cultures were grown overnight in LB broth, diluted to 2×10⁵ CFU/ml in PBS and treated for 2h with 400 μM H₂O₂. Killing, expressed as percent survival compared to the bacterial burden at time zero, is the mean ± S.D. (N = 16); p<0.0001 as determined by unpaired t-test. (B). The GSH/GSSG content was estimated as described in Fig 1. Where indicated, bacterial cells grown in MOPS-GLC minimum medium to OD₆₀₀ of 0.25 were treated with 400 μM H₂O₂ for 30 min. Data are the mean ± S.D. (N = 5) *, **, ****, p < 0.05, p < 0.01 and p < 0.0001, respectively as determined by two-way ANOVA.

Fig 6. The methylglyoxal pathway facilitates glucose and energy metabolism in Salmonella experiencing peroxide stress.

(A) Intracellular concentrations of glucose and (B) reduced and oxidized nicotinamide adenine nucleotide and (D) L-lactate in Salmonella grown aerobically in MOPS-GLC minimum medium, pH 7.2 to an OD₆₀₀ of 0.25 at 37°C. Where indicated, bacterial cultures were treated with 400 μM H₂O₂ for 30 min. Data are the mean ± S.D. (N = 5–6). ***, ****, p < 0.001 and p < 0.0001, respectively, as determined by two-way ANOVA. (C) Consumption of O₂ by log phase Salmonella grown in MOPS-GLC minimal medium, pH 7.2, was measured polarographically in an APOLLO-4000 free radical analyzer equipped with an ISO-OXY O2 probe. Data are the mean ± S.D (N = 7–10). **** p<0.0001 as determined by unpaired t-test.

Fig 7. Role of the methylglyoxal pathway in resistance of Salmonella to phagocyte NADPH oxidase.

(A) Bacterial burden in bone-marrow-derived macrophages isolated from C57BL/6 and Cybb^-/- mice. Bacterial burden and competitive index of Salmonella strains in (B) livers and (C) spleens of C57BL/6 and Cybb^-/- mice after i.p. inoculation with 100 CFU of equal numbers of wildtype and ΔgloB Salmonella (N = 10). Statistical differences (***, ****, p < 0.001 and p < 0.0001, respectively) were calculated by unpaired t-test.