Role of sodium pyruvate in maintaining the survival and cytotoxicity of Staphylococcus aureus under high glucose conditions

Abstract

Glucose is a crucial carbon source for the growth of Staphylococcus aureus, but an excess of glucose is detrimental and even leads to cell death. Pyruvate, the central metabolite of glycolysis, has been shown to have anti-inflammatory and antioxidant properties. This study aimed to investigate the protective effect of pyruvate on S. aureus under high glucose conditions. Sodium pyruvate greatly increased the cytotoxicity of S. aureus strain BAA-1717 to human erythrocytes and neutrophils in vitro. However, the cytotoxicity and survival of S. aureus were significantly reduced by high glucose, which was restored to normal levels by the addition of sodium pyruvate. The expression of hlg and lukS in S. aureus was higher in the LB-GP cultures than that in LB-G cultures, but there was no significant difference in cytotoxicity between LB-GP and LB-G cultures. Furthermore, the hemolytic activity of S. aureus supernatants could be inhibited by the cell-free culture medium (CFCM) of LB-G cultures, suggesting that high levels of extracellular proteases were presence in the CFCM of LB-G cultures, resulting in degradation of the hemolytic factors. The expression of sarA, which negatively regulates extracellular protease secretion, was higher in LB-GP cultures than that in LB-G cultures. Additionally, sodium pyruvate increased acetate production in S. aureus, which helps maintain cell viability under acidic environment. In conclusion, pyruvate plays an important role in the survival and cytotoxicity of S. aureus under high glucose conditions. This finding may aid in the development of effective treatments for diabetic foot infections.

Keywords: Staphylococcus aureus; high glucose; pyruvate; sarA; survival; virulence.

Figure 1

Effect of sodium pyruvate on the growth and hemolytic activity of S. aureus. S. aureus grown in CDM medium with different concentration of sodium pyruvate. After cultured for 24 h, the turbidity was measured (A), the CFUs were determined by serial dilutions (B), and the cytotoxicity of culture supernatants to human erythrocytes and neutrophils was analyzed (C,D). S. aureus was grown in LB medium with different concentration of sodium pyruvate, and the turbidity was measured (E), the CFUs were determined by serial dilutions (F), and the cytotoxicity of culture supernatants to human erythrocytes and neutrophils was analyzed (G,H) after being cultured for 24 h. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Figure 2

Effect of sodium pyruvate on the cytotoxicity of S. aureus under high glucose conditions. S. aureus strain BAA-1717 was grown in CDM, CDM-G, CDM-GP, and CDM-P medium. After cultured for 24 h or 48 h, the pigment formation was determined (A,B), and the hemolytic activity of CFCM was analyzed (C,D). S. aureus strain BAA-1717 was grown in LB, LB-G, LB-GP and LB-P medium. After cultured for 24 h or 48 h, the pigment formation was determined (E,F), and the hemolytic activity of CFCM was analyzed (G,H). ^*** p < 0.001.

Figure 3

Sodium pyruvate affects the production of extracellular protease under high glucose conditions. Overnight cultures of S. aureus strain BAA-1717 was diluted 1:100 into LB, LB-G, LB-GP, and LB-P medium. (A) the total protein in the culture supernatants was detected after being cultured for 24 h. The expression of hlg (B) and lukS (C) was examined after being cultured for 6 h. (D) The cytotoxicity of culture supernatants to human neutrophils was examined. (E) The CFCM of LB cultures were pre-incubated with the CFCM of LB-G or LB-GP cultures, and the cytotoxicity to human erythrocytes was examined. (F) Lysis of human erythrocytes by the CFCM of S. aureus when grown in medium with glucose or/and PMSF. (G) The CFCM of LB cultures were pre-incubated with the CFCM of LB-G or LB-GP cultures, and the cytotoxicity to human neutrophils was examined. The expression of sarA (H) and agrA (I) was examined after being cultured for 6 h or 24 h. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Figure 4

Effect of sodium pyruvate on the survival of S. aureus under high glucose conditions. S. aureus was grown in LB, LB-G, LB-GP and LB-P medium, and the CFUs were determined after being cultured for 24 h (A) or 48 h (B). After cultured for 48 h, bacterial cells were analyzed by Gram-staining (C) and TEM assays (D). (E) After cultured for 24 h, bacterial cells were collected and lysed by 10 μg/mL lysostaphin, and the value of OD₅₇₀ was taken at timed intervals. ^* p < 0.05, ^** p < 0.01.

Figure 5

Sodium pyruvate altered glucose metabolism of S. aureus under high glucose conditions. Overnight cultures of S. aureus were added with 20 mM glucose, 20 mM sodium pyruvate or both 20 mM glucose and 20 mM sodium pyruvate. After incubated for 7 h, the turbidity was determined (A), and the levels of glucose (B), lactate (C), acetate(D), and pyruvate (E) in the culture supernatants were detected. (F) Overnight cultures of S. aureus were incubated with lactate or acetate, and the CFUs was determined after incubation for 48 h. (G) Overnight cultures of S. aureus were pre-incubated with 30 mM or 60 mM sodium pyruvate for 1 h, and then the aggregation was induced by 15 mM lactate. ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.