Staphylococcus aureus strain ATCC BAA-1717 (USA300) were kindly provided by Abace Biotechnology (Beijing, China), and which had remarkable hemolytic activity and pigment formation. The bacteria were routinely cultured at 37°C on 5% sheep blood agar plates (BA, Bio-caring, China) and then grown in lysogeny broth (LB, Solarbio Life Sciences, Beijing, China) at 37°C with shaking at 180 rpm. Overnight cultures of S. aureus strain BAA-1717 were diluted 1:100 into 3 mL fresh LB medium with 20 mM glucose (Sigma-Aldrich) or/and 20 mM sodium pyruvate (Sigma-Aldrich) in a 12 mL tube. All cultures were incubated at 37°C with shaking at 180 rpm, and the culture supernatants were collected at 24 h or 48 h post-inoculation. The cell-free culture medium (CFCM) was obtained by filtered through a PES filter (0.22 μm pore size; Millipore), used immediately or stored at −70°C.

Overnight cultures of S. aureus strain BAA-1717 were diluted 1:100 into a chemical defined medium (CDM, D6540, Solarbio Life Sciences, Beijing, China) or fresh LB medium with or without 5-, 10-, 20- or 40 mM sodium pyruvate. All cultures were incubated at 37°C with shaking at 180 rpm, either in or out of an anaerobic bag. After cultured for 24 h, the OD values were measured at 450 nm and the colony-forming units (CFUs) were determined by plating 5 μL serial dilutions on BA, then incubating them overnight at 37°C and counting the number of colonies. For the survival of S. aureus under acidic environment, overnight cultures of S. aureus strain BAA-1717 was added with 15 mM lactate (Sigma-Aldrich) or 15 mM acetate (Sigma-Aldrich), incubated at 37°C with shaking at 180 rpm for 48 h, and the CFUs was determined.

Quantitative hemolysis assays were performed according to the methods described by Ridder et al. (2021). Briefly, discarded whole blood from healthy human subjects was washed twice with normal saline and then resuspended to a final concentration of 4% (v/v). Equal volumes of 4% human erythrocyte suspension and CFCM of S. aureus were added to 96-well flat-bottom plates and placed in 37°C incubator (static). The plate was centrifuged after incubated for 90 min. The supernatants were transferred to a new 96-well plate and measured at OD₄₅₀ using a microplate reader. To inhibit extracellular protease, S. aureus strain BAA-1717 was grown in LB medium with 10 mM glucose or/and phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich).

Peripheral blood was resuspended in RPMI 1640, layered with Ficoll Hypaque Plus (Sigma-Aldrich), and then centrifuged at 1000 g for 12 min. The red blood cell (RBC) pellet was incubated with red blood cell lysis buffer (CWBiotech, China) at a 9-fold volume for 15 min at 37°C to remove erythrocytes. After centrifugation at 1000 g for 10 min, the cell pellet was washed and resuspended in RPMI 1640 to the desired concentration. Neutrophil lysis was measured by lactate dehydrogenase (LDH) release assay. Briefly, the CFCM of S. aureus was added to 4.0 × 10⁶ neutrophils/mL to a total volume of 400 μL in 24-well plates and incubated at 37°C with 5% CO₂. At the desired times, the samples were centrifuged at 3000 rpm for 5 min, and the supernatants were collected. The LDH activity in the culture supernatants was measured by using an automatic biochemical analyzer 7600 (Hitachi, Japan) according to the manufacturer’s instructions.

Measurement of pigmentation by methanol extraction was conducted as described by Sullivan and Rice (2021). Briefly, overnight cultures of S. aureus strain BAA-1717 was inoculated into 3 mL of LB medium with 20 mM glucose, 20 mM sodium pyruvate or both 20 mM glucose and 20 mM sodium pyruvate. After cultured for 24 h or 48 h, 1 mL of the cultures were centrifuged at 12000 rpm for 10 min. The resulting pellets were washed twice with normal saline, resuspended in 200 μL of methanol, and incubated for 30 min in an incubator with shaking at 180 rpm. The samples were then centrifuged again at 12000 rpm for 10 min, and the OD value of the supernatants was measured at 450 nm by using a microplate reader.

Overnight cultures of S. aureus strain BAA-1717 were diluted 1:100 into fresh LB medium with 20 mM glucose, 20 mM sodium pyruvate or both 20 mM glucose and 20 mM sodium pyruvate. After cultured for 48 h, a smear of bacterial culture was air dried, heat fixed, and then stained with a commercial Gram-staining solution (BASO Diagnostics, China). Briefly, the staining process included 10 s in crystal violet solution, 10 s in iodine solution, a 20 s wash in decolorizer, then a final counter stain with safranin solution for 10 s. The stained slides were examined under a microscope (1000×).

S. aureus strain BAA-1717 cultures were prepared by diluting overnight cultures 1:100 into fresh LB medium with 20 mM glucose, or both 20 mM glucose and 20 mM sodium pyruvate, and further cultured for 48 h. These cultures were collected and centrifugated at 12000 rpm for 10 min, washed twice with normal saline and then re-suspended in 2.5% glutaraldehyde solution with Millonig’s phosphate buffer. These samples were sent to the Transmission Electron Microscopy (TEM) Laboratory at the Department of Pathology, Xiangya Hospital for examination and photography using a Hitachi HT7700 electron microscope.

Lysostaphin lysis assays were performed as previously reported with minor modifications (Grundling et al., 2006). Briefly, overnight cultures of S. aureus BAA-1717 were diluted 1:100 into fresh LB medium with 20 mM glucose, 20 mM sodium pyruvate or both 20 mM glucose and 20 mM sodium pyruvate, and cultured at 37°C, 180 rpm for 24 h. These cultures were collected and washed twice with PBS and resuspended to an OD₅₇₀ of 1.8–2.1. This value was set as 100% at 0 min. Lysostaphin (Sigma-Aldrich) was added at final concentrations of 10 μg/mL, and the OD₅₇₀ values were recorded at timed intervals, and data were plotted as percent OD₅₇₀ values of the initial reading.

S. aureus strain BAA-1717 was grown in LB medium at 37°C with shaking at 180 rpm for overnight. For glucose-induced aggregation, overnight cultures were added with 20 mM glucose, 20 mM sodium pyruvate or both 20 mM glucose and 20 mM sodium pyruvate, and further cultured at 37°C for 7 h under static conditions. For lactate-induced aggregation, overnight cultures were pre-incubated with 30 mM or 60 mM sodium pyruvate for 1 h, then 15 mM lactate was added, and further cultured at 37°C for 7 h under static conditions. 150 μL of the supernatants was transferred to the wells of 96-well tissue culture plates, and the absorption was measured at 450 nm using a microplate reader. After cultured for 7 h, the medium was centrifuged at 12000 rpm for 10 min, and the content of glucose, lactate, pyruvate and total protein in the supernatants were analyzed by an automatic biochemical analyzer 7600 according to the manufacturer’s instructions. The content of acetate in the supernatants was analyzed by Acetate Colorimetric Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions.

S. aureus strain BAA-1717 were grown in LB medium supplemented with 20 mM glucose, 20 mM sodium pyruvate or both 20 mM glucose and 20 mM sodium pyruvate, and incubated at 37°C with shaking at 180 rpm. After cultured for 6 h or 24 h, bacterial cells were collected and centrifuged at 12000 rpm for 10 min. The cell pellets were re-suspended in 200 μL PBS with 10 μg/mL lysostaphin and incubated at room temperature for 30 min. Total RNA was extracted and purified by using nucleic acid extraction kit (paramagnetic particle method) (Shanghai BioGerm Medical Technology Co., Ltd.) according to the manufacturer’s instructions. The RNA quality and concentration were evaluated by using a NanoDrop 1000 (Thermo Fisher Scientific). Then, DNA was removed by DNase I, and then total RNA was reverse transcribed to cDNA using a reverse transcription kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. The gyrB gene was used as an internal reference to normalize the expressions of genes of interest and PCRs were performed in 50 μL reaction mixtures. The relative quantification method (2^-△△Ct) was used to analyze the transcription level of target genes. The primers used in this study were reported in previous study (Abdelhady et al., 2014; Shi et al., 2019; Shang et al., 2022). All analyses were conducted in triplicate.

Statistical analysis was performed with GraphPad Prism software version 8.3. Significance levels were calculated by using one way ANOVA or two-way ANOVA analysis. Statistical significance was defined as p < 0.05. All error bars depict the standard deviation. Each experiment was repeated at least three times.

S. aureus strain BAA-1717 was firstly grown in a CDM medium without carbon sources under either aerobic or anaerobic conditions, and sodium pyruvate was added as the only carbon source available to the bacteria. The growth of S. aureus was significantly increased by sodium pyruvate under both anaerobic and aerobic conditions, with a higher tendency observed under aerobic condition (Figures 1A,B). Subsequently, the cytotoxicity of S. aureus was analyzed in vitro, and found that the cytotoxicity of S. aureus CFCM to human erythrocytes and neutrophils was significantly increased by sodium pyruvate under both anaerobic and aerobic conditions (Figures 1C,D). To further investigate the effect of pyruvate on the cytotoxicity of S. aureus, then the bacteria was grown in LB medium supplemented with different concentrations of sodium pyruvate. Unlike the ascending trend observed in CDM, the growth of S. aureus in LB medium was unaffected by sodium pyruvate under both anaerobic and aerobic conditions (Figures 1E,F). However, the cytotoxicity of S. aureus CFCM to human erythrocytes and neutrophils was significantly increased by sodium pyruvate under aerobic conditions, which remained extremely low under anaerobic conditions (Figures 1G,H). These data indicate that sodium pyruvate increases the cytotoxicity of S. aureus under aerobic conditions in vitro.

S. aureus is the most described Gram-positive pathogen in DFUs (Macdonald et al., 2021), whether pyruvate affects the cytotoxicity of S. aureus under high glucose conditions is unclear. S. aureus strain BAA-1717 was firstly grown in CDM medium with high glucose and sodium pyruvate, and found that the pigment formation was significantly increased in the CDM-P culture after cultured for 48 h, but no significant difference was observed between CDM-G and CDM-GP cultures (Figures 2A,B). The hemolytic activity of S. aureus remained extremely low in CDM, CDM-G and CDM-GP cultures, but was higher in CDM-P cultures (Figures 2C,D). Next, S. aureus was grown in LB medium with glucose and sodium pyruvate, and found that both pigment formation and hemolytic activity were significantly decreased by high glucose, which was not affected by the addition of sodium pyruvate after being cultured for 24 h (Figures 2E,G). As the culture time increased to 48 h, the pigment formation and hemolytic activity of S. aureus grown in LB-GP medium was greatly increased and restored to the levels of LB and LB-P cultures (Figures 2F,H). These data indicate that the reduced hemolytic activity and pigment formation of S. aureus under high glucose conditions is restored by sodium pyruvate.

Pyruvate has been shown to induce the production of extracellular proteins and virulence factors, such as Panton-Valentine Leucocidin (PVL), resulting in increased virulence of S. aureus (Harper et al., 2018). As shown in Figure 3A, the total protein in LB-GP cultures was higher than that in LB-G cultures, which was comparable to the level of LB and LB-P cultures. The expression of hlg and lukS (a PVL encoding gene) in LB-GP cultures was higher than that in LB-G cultures (Figures 3B,C). PVL has been shown to disrupt the body’s defense system by lysing human polymorphonuclear cells (Loffler et al., 2010). However, the reduced cytotoxicity of S. aureus CFCM to human erythrocytes and neutrophils by high glucose was not affected by the addition of sodium pyruvate after being cultured for 24 h (Figures 2G, 3D). As the culture time increased to 48 h, the cytotoxicity of S. aureus CFCM from LB-GP cultures to human neutrophils was significantly increased compared to LB-G cultures (Figure 3D). Additionally, the cytotoxicity of LB culture CFCM to human erythrocytes was significantly decreased when pre-incubated with the CFCM of LB-G cultures, but unaffected by the CFCM of LB-GP cultures (Figure 3E). Additionally, the reduced hemolytic activity of S. aureus by high glucose could be restored by PMSF, a serine protease inhibitor (Figure 3F). However, the cytotoxicity of LB culture CFCM to human neutrophils was unaffected when pre-incubated with the CFCM of LB-G or LB-GP cultures (Figure 3G). These data indicate that high levels of extracellular proteases are present in the CFCM of LB-G cultures, which degrade the hemolytic factors of S. aureus. Previous studies have shown that the production of extracellular proteases is negatively regulated by sarA (Karlsson and Arvidson, 2002; Ramirez et al., 2020). Next, the expression of sarA was examined by real-time RT-PCR, and found that the expression of sarA was lower in both LB-G and LB-GP cultures than that of LB and LB-P cultures after being cultured for 6 h. As the culture time increased to 24 h, the expression of sarA in LB-GP cultures was greatly increased and restored to the levels of LB and LB-P cultures (Figure 3H). The expression of agrA was lower in LB-G cultures, which was greatly increased by addition of sodium pyruvate (Figure 3I). Therefore, sodium pyruvate reduces the production of extracellular protease, leading to restore the reduced cytotoxicity of S. aureus under high glucose conditions.

As shown in Figure 4A, the CFUs of S. aureus grown in LB-G and LB-GP medium were reduced nearly 10-fold compared to LB and LB-P cultures, and no significant difference was observed between LB-G and LB-GP cultures after being cultured for 24 h. As the culture time increased to 48 h, the CFUs of S. aureus from LB-G cultures decreased by more than 100-fold. In contrast, the CFUs of S. aureus from LB-GP cultures were greatly increased and returned to the levels of LB and LB-P cultures (Figure 4B). Next, Gram-staining assay was used to examine the morphologic changes of S. aureus. The bacteria from LB-G cultures were more aggregated and larger in size than that of LB cultures, which was significantly alleviated in the LB-GP cultures (Figure 4C). TEM assays were used to further determine the structural changes of S. aureus affected by sodium pyruvate under high glucose conditions, and found that the cells from LB-G cultures were slightly larger in size and had more compact cytoplasm than those from LB and LB-GP cultures (Figure 4D). Previous studies have shown that S. aureus grown in rich-medium (high glucose) is highly resistant to lysostaphin (Wu et al., 2019; Luo et al., 2020). Herein, S. aureus from LB-G cultures was highly resistance to lysostaphin, which was greatly attenuated when grown in LB-GP medium (Figure 4E). These data indicate that sodium pyruvate restores the survival of S. aureus under high glucose conditions by supporting a second round of growth during stationary phase.

Our previous study has shown that high glucose induces aggregation of S. aureus (Luo et al., 2019). We investigate whether pyruvate affects the aggregation of S. aureus under high glucose conditions. As shown in Figure 5A, sodium pyruvate alone did not affect S. aureus aggregation, but the aggregation of S. aureus induced by high glucose was greatly inhibited by the addition of sodium pyruvate. Subsequently, the glycolytic metabolites in the culture supernatants were analyzed. Glucose was undetected in the LB and LB-P cultures, which was higher in the LB-G cultures compared to LB-GP cultures (Figure 5B). The levels of lactate, acetate and pyruvate were higher in the LB-GP cultures compared to LB-G cultures (Figures 5C-E). The content of acetate in the supernatants of LB-GP cultures was as high as 30 mM, which is much higher than that of lactate (13 mM). Inactivation of the Pta-AckA pathway significantly reduces the production of acetate, resulting in reduced growth rate and viability of S. aureus (Sadykov et al., 2013; Marshall et al., 2016). The CFUs of stationary-phase S. aureus strain BAA-1717 was significantly decreased by the addition of lactate, but was unaffected by acetate (Figure 5F). Additionally, high level of lactate induced the aggregation of S. aureus, which was also significantly inhibited by pre-incubation with sodium pyruvate (Figure 5G). These data indicate that sodium pyruvate enhances the production of acetate, maintaining the survival of S. aureus under high glucose conditions.