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, 74 (8), 4950-3

Staphyloxanthin Plays a Role in the Fitness of Staphylococcus Aureus and Its Ability to Cope With Oxidative Stress

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Staphyloxanthin Plays a Role in the Fitness of Staphylococcus Aureus and Its Ability to Cope With Oxidative Stress

Alexandra Clauditz et al. Infect Immun.

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.

Figures

FIG. 1.
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.
FIG. 2.
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.
FIG. 3.
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.
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.
FIG. 5.
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.

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