Staphyloxanthin Plays a Role in the Fitness of Staphylococcus aureus and Its Ability To Cope with Oxidative Stress

Abstract

Staphyloxanthin is a membrane-bound carotenoid of Staphylococcus aureus. Here we studied the interaction of staphyloxanthin with reactive oxygen substances (ROS) and showed by comparative analysis of the wild type (WT) and an isogenic crtM mutant that the WT is more resistant to hydrogen peroxide, superoxide radical, hydroxyl radical, hypochloride, and neutrophil killing.

Staphyloxanthin is an orange-red triterpenoid carotenoid whose biosynthesis and structure have been recently elucidated (14, 18). It is well known that carotenoids function as antioxidants, and it has been suggested that staphyloxanthin can protect Staphylococcus aureus against oxidative stress (12, 13). To further study the protective function of staphyloxanthin, we compared the viability of staphyloxanthin-producing S. aureus Newman (3) with that of its isogenic crtM mutant (19) (Fig. 1), which does not produce staphyloxanthin (but can be complemented by the expression plasmid pTXcrtM) (14), to various radical and nonradical substances generated in vitro. We also tested the ability of staphyloxanthin to act as a radical scavenger.

FIG. 1.

Illustration of the construction of knockout plasmid pRBSXΔcrtM and xylose-inducible crtM expression plasmid pTXcrtM, which is able to complement the crtM mutant in the presence of xylose as an inducer. TT, transcription terminator.

First we investigated the oxidation of staphyloxanthin by hydroxyl radicals (OH^·) and peroxynitrite (ONOO⁻). We generated hydroxyl radicals by the Fenton reaction in an assay containing iron(II) chloride and hydrogen peroxide in equimolar concentrations ranging from 0.01 to 0.5 mM. Although the system is nonspecific, OH^· is likely to be among the oxidants produced, including OOH^· and CH3^· (20). Purified staphyloxanthin (14) in the reaction mixture was rapidly oxidized, as indicated by the decrease in absorbance at 478 nm (Fig. 2A). The absorbance of controls lacking iron(II) chloride, H₂O₂, or both did not decrease. The time course of staphyloxanthin oxidation in the test samples shows that after 2 min of incubation with 0.05, 0.1, and 0.5 mM equimolar amounts of iron chloride and hydrogen peroxide, 40, 50, and 60% of the staphyloxanthin was oxidized, respectively (Fig. 2C). The oxidation of staphyloxanthin by peroxynitrite was monitored for 3 h by monitoring the decrease in absorbance at the absorption maximum of staphyloxanthin in ethanol (470 nm). In this experiment, prolonged incubations were necessary because the concentration of peroxynitrite increased only gradually after addition of SIN-1. Staphyloxanthin oxidation by peroxynitrite anions was indicated by the decrease in absorption and at the same time the shift of the absorption maximum to shorter wavelengths, which indicates the reduction of double bonds (Fig. 2B). The absorbance of the controls lacking SIN-1 did not decrease.

FIG. 2.

Time course of staphyloxanthin oxidation by free radicals. (A) Oxidation by free radicals generated in a nonspecific Fenton reaction. The reaction mixture consisted of 12 μM staphyloxanthin in dimethyl sulfoxide-H₂O (4:1, vol/vol), 0.5 mM iron(II) chloride, and 0.5 mM H₂O₂ and was incubated under air at 25°C. Absorption spectra were recorded before the reaction with iron(II) chloride and H₂O₂ (time zero) and after 2, 15, 60, and 90 min. The absorption maxima are indicated. (B) Oxidation by peroxynitrite generated with SIN-1. The reaction mixture consisted of 12 μM staphyloxanthin and 3 mM SIN-1 in ethanol-H₂O (4:1, vol/vol) and was incubated under air at 25°C. Absorption spectra were recorded before reaction with SIN-1 (time zero) and after 1, 2, and 3 h. The absorption maxima are indicated. (C) Time course of oxidation of purified staphyloxanthin by hydroxyl radicals generated in a Fenton reaction. The reaction mixture consisted of 12.5 μM staphyloxanthin in dimethyl sulfoxide-H₂O (4:1, vol/vol) and equimolar concentrations of FeCl₂ and H₂O₂, i.e., 0.05 mM (•), 0.1 mM (▪), 0.2 mM (▴), and 0.5 mM (⧫). The mixture was incubated under air at 25°C. Oxidation of staphyloxanthin was determined by measuring the decrease in absorption at 478 nm. Data points represent the means of five independent experiments. Error bars indicate the deviation of five independent experiments.

Next we tested the viability of S. aureus after incubation with H₂O₂. The numbers of viable S. aureus Newman wild-type (WT) and crtM mutant cells decreased with increasing concentrations of H₂O₂ (50, 75, 100, and 150 mM) but to different extents (Fig. 3A). The number of CFU of the controls lacking H₂O₂ did not change. The survival of the bacteria in the test samples correlated with the H₂O₂ concentration. Both strains were susceptible to high concentrations of H₂O₂, but the crtM mutant was more susceptible than the WT at all concentrations. The viability of S. aureus was also investigated after incubation with superoxide radicals generated by both hypoxanthine and xanthine oxidase (XO) and phenazine methosulfate (PMS) and NADH. The generation of the radicals was monitored by measuring the reduction of cytochrome c. WT and the crtM mutant S. aureus cells were both sensitive to killing by hypoxanthine-XO (Fig. 3B), but the WT always revealed a better survival frequency independent of the presence of catalase, which increases XO activity, which is inhibited by H₂O₂ (Fig. 3B). The number of CFU of the control did not change. Both S. aureus WT and the crtM mutant were susceptible to 1.0 mM PMS and succinate (a source of NADH), which generate the superoxide radical O₂⁻. Killing correlated with time, but the mutant was more susceptible than the WT (Fig. 4A), which showed twofold higher survival than the mutant. The number of CFU of the control did not change after incubation. Phenazine oxidizes NADH, resulting in production of O₂⁻, which can react nonenzymatically with H₂O₂ to form hydroxyl radical (6) and singlet oxygen (10), leading to the peroxidation of polyunsaturated fatty acids of membrane lipids (4, 17).

FIG. 3.

Effects of H₂O₂ and superoxide radical on the survival of WT and ΔcrtM mutant S. aureus Newman. (A) After 24 h of growth in basic medium, 5 × 10⁶ CFU ml⁻¹ were incubated in phosphate-buffered saline containing the indicated concentrations of H₂O₂ in the dark at 0°C. After 45 min, the reaction was stopped by destroying the remaining H₂O₂ with 2 U ml⁻¹ catalase and incubation for 20 min. Diluted cells (0.1 ml) were spread on BM agar plates. Colonies were counted after 24 h of incubation at 37°C. Values are expressed as a percentage of the CFU in the control culture lacking H₂O₂. Values are the averages of five independent experiments. Error bars indicate the deviation of five independent experiments. (B) After 24 h of growth in basic medium, 5 × 10⁶ CFU ml⁻¹ were incubated in HEPES buffer containing 10 mM hypoxanthine and 0.1 U of xanthine oxidase (XO) with or without 2 U of catalase at 25°C. After incubation for 30 and 60 min, the reaction was stopped by addition of 10 μM allopurinol. Diluted cells (0.1 ml) were spread on BM agar plates. Colonies were counted after 24 h of incubation at 37°C. Values are expressed as a percentage of the number of CFU in the control culture containing only hypoxanthine (10 mM) and lacking XO. Values represent the average of five independent experiments. Error bars indicate the deviation of five independent experiments.

FIG. 4.

Effects of PMS and MPO on the survival of WT and ΔcrtM mutant S. aureus Newman. (A) After 24 h of growth in basic medium, cells were harvested and washed twice in HEPES buffer and 5 × 10⁶ CFU ml⁻¹ were incubated in 20 mM HEPES buffer containing 1 mM PMS and 2 mM succinate at 25°C. After the indicated time, 0.1 ml of diluted cells was spread on BM agar plates. Colonies were counted after 24 h of incubation at 37°C. Values are expressed as a percentage of the number of CFU in the control culture containing only succinate (2 mM) and lacking PMS. Values are the average of five independent experiments. Error bars indicate the deviation of five independent experiments. (B) After 24 h of growth in basic medium, cells were harvested and washed twice and 5 × 10⁶ CFU ml⁻¹ in phosphate-buffered saline at pH 7.4 were mixed with 0.05 U of MPO and 10 μM H₂O₂ and incubated at 25°C for 90 min. Diluted cells (0.1 ml) were spread on BM agar plates. Colonies were counted after 24 h of incubation at 37°C. The number of CFU is expressed as a percentage of the control containing only H₂O₂ (10 μM). Values are the average of five independent experiments. Error bars indicate the deviation of five independent experiments.

Myeloperoxidase (MPO) plays a crucial role in bacterial killing by generating hypochlorous acid within neutrophil phagosomes, where it constitutes about 5% of the total neutrophil protein (1, 16). In vitro, MPO in the presence of H₂O₂ and chloride or iodide ions leads to hypochlorous acid and to the killing of many bacteria and fungi (7, 8). We analyzed the sensitivity of WT and crtM mutant S. aureus to MPO in the presence of H₂O₂ and Cl⁻. Both strains were killed, but the crtM mutant cells were killed much faster and in greater numbers than WT cells (Fig. 4B). The bactericidal effect was dependent upon the incubation time, but the overall difference between the strains remained almost constant during the 90 min of incubation; e.g., there was a fourfold difference after 15 min of incubation. The viability of the WT and the crtM mutant was not affected by incubation of the cells with NO^· donors (not shown). NO^· also has little effect on the viability of other bacterial species, e.g., Escherichia coli, Salmonella sp., and Proteus vulgaris (2, 9).

Finally, we investigated the killing of S. aureus by human neutrophils, which consume more O₂ after ingestion of bacteria (15). Since all of the ROS analyzed in this work are also produced during the oxidative burst, it was of interest to compare the killing of the WT and the crtM mutant by human neutrophils. Killing of both strains by human neutrophils increased with time, but a higher percentage of the mutant cells were killed (Fig. 5). After 15 and 60 min of incubation, the survival of the WT was 1.3- and 1.8-fold, respectively, higher than that of the crtM mutant. Liu et al. (13) described an approximately 10-fold higher survival frequency of the WT compared to the crtM mutant in human neutrophils, and they also showed that this effect is not explained by differences in the rate of phagocytosis, because the uptake of WT S. aureus was comparable to that of the crtM mutant. We saw the same tendency, although the differences between the WT and the crtM mutant were less pronounced. One explanation for this discrepancy could be that we used stationary-phase cells throughout our study, where staphyloxanthin production is greatest but where cells might also become more resistant to peroxides and radical species. Other groups have also described better survival of carotenoid-producing cells within human neutrophils (5, 11).

FIG. 5.

Killing of WT and ΔcrtM mutant S. aureus Newman by human neutrophils. After 24 h of growth in basic medium, cells were harvested and washed twice in potassium-phosphate buffer (pH 7.2) containing 0.05% human serum albumin. Bacteria (5 × 10⁶ CFU ml⁻¹) were mixed with neutrophils (2.5 × 10⁶/ml). Human serum was added to a final concentration of 10%, and 150 μl of prewarmed Hanks balanced salt solution was also added. Samples (500 μl) were shaken at 37°C, and the incubation was stopped after the indicated time by diluting the samples 100-fold in ice-cold distilled water. The diluted samples (0.1 ml) were spread on BM agar plates, and colonies were counted after 24 h of incubation at 37°C. The number of CFU after incubation with neutrophils is expressed as a percentage of the initial count. Values are the average of five independent experiments. Error bars indicate the deviation of five independent experiments. The significance of experimental differences was evaluated by unpaired Student test.

Our data indicate that staphyloxanthin scavenges free radicals with its conjugated double bonds. Since staphyloxanthin is located in the cell membrane, it probably primarily protects lipids but might also be involved in protecting proteins and DNA. Enzymes such as catalase and SOD most likely contribute to a larger extent to the survival of cells during stress and the host response, but staphyloxanthin plays an additional role in the defense against damage by ROS, thereby enhancing the virulence and fitness of the cells. Staphyloxanthin can be regarded as a biological antioxidant against hydrogen peroxide and hydroxyl radicals and might be useful as a therapeutic radical scavenger.