Staphylococcus aureus Peptide Methionine Sulfoxide Reductases Protect from Human Whole-Blood Killing

Abstract

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H₂O₂ stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.

Keywords: MRSA; Staphylococcus aureus; methionine sulfoxide; methionine sulfoxide reductases; oxidative stress; pathogenesis.

FIG 1

Methionine sulfoxide reductases repair oxidized methionine residues. (A) USA300 wild-type and Δmsr strains were lysed, and Msr activity was quantified by HPLC. The Δmsr strain has no measurable Msr activity. Data are means ± standard deviations from biological triplicate measurements. (B) USA300 wild-type and Δmsr strains were treated with HOCl; then, using a methionine reactive probe, Ox4, the extent of methionine oxidation was assessed. A darker signal indicates less methionine oxidation, and as expected, the Δmsr strain, which is unable to repair methionine sulfoxide, has decreased levels of free methionine.

FIG 2

Methionine sulfoxide reductases protect S. aureus from HOCl killing. (A) USA300 wild-type and Δmsr strains were treated with 0 or 100 μM HOCl. Growth was monitored by optical density at 600 nm for 24 h. Data are means ± standard deviations for measurements acquired in biological triplicates. (B) USA300 wild-type and Δmsr strains were treated with 0 or 3.2 mM paraquat (PQ). Growth was monitored by optical density at 600 nm for 24 h. Data are means ± standard deviations for measurements acquired in six biological replicates. (C) USA300 wild-type and Δmsr strains were treated with 0 or 16 μg/ml pyocyanin (PCN). Growth was monitored by optical density at 600 nm for 24 h. Data are means ± standard deviations for measurements acquired in biological quadruplicates. (D) USA300 wild-type and Δmsr strains were treated with 0 or 500 μM H₂O₂. Growth was monitored by optical density at 600 nm for 24 h. Data are means ± standard deviations for measurements acquired in biological triplicates.

FIG 3

S. aureus lacking all four Msr enzymes is fully virulent in a murine model of bacteremia. Female 8-week-old BALB/cJ mice were infected retro-orbitally with USA300 wild-type or Δmsr strains. After 4 or 10 days of monitoring, the heart (A), kidneys (B), liver (C), lungs (D), and spleen (E) were harvested, homogenized, and dilution plated on solid medium to enumerate viable bacteria. Gray symbols indicate bacterial burdens below the limit of detection, and so the limit of detection was used for statistical analyses. Data are means ± standard deviations, and P values were calculated by Mann-Whitney test.

FIG 4

S. aureus lacking all four Msr enzymes is attenuated for dissemination to the spleen in a murine model of intraperitoneal infection. Female 8-week-old BALB/cJ mice were infected intraperitoneally with USA300 wild-type or Δmsr strains. After 4 days of monitoring, the heart, kidneys, liver (A), lungs (B), and spleen (C) were harvested, homogenized, and dilution plated on solid medium to enumerate viable bacteria. No viable bacteria were detected in the heart or kidneys. Gray symbols indicate bacterial burdens below the limit of detection, and so the limit of detection was used for statistical analyses. Data are means ± standard deviations, and P values were calculated by Mann-Whitney test.

FIG 5

S. aureus lacking all four Msr enzymes is attenuated for pathogenesis in a murine skin infection model. Skin wounds were created on the backs of female 8-week-old BALB/cJ mice and then infected with USA300 wild-type or Δmsr strains. After 2 days of monitoring, the skin lesions were harvested, homogenized, and dilution plated on solid medium to enumerate viable bacteria. Data are means ± standard deviations, and the P value was calculated by Mann-Whitney test.

FIG 6

Msr enzymes protect S. aureus against neutrophil killing and human whole-blood killing. (A) USA300 wild-type and Δmsr strains were incubated with PMNs (MOI = 1) isolated from human blood, and killing was assessed by plating for CFU. (B) USA300 wild-type and Δmsr strains were incubated with PMNs (MOI = 1) isolated from murine bone marrow, and killing was assessed by plating for CFU. (C) USA300 wild-type and Δmsr strains were incubated with human whole blood (MOI = 1), and killing was assessed by plating for CFU. (D) USA300 wild-type and Δmsr strains were incubated with murine whole blood (MOI = 1), and killing was assessed by plating for CFU. All data are means ± standard deviations, and P values were calculated by t test. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.