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. 2014;6(3):353-64.
doi: 10.1159/000355915. Epub 2013 Nov 15.

Methionine sulfoxide reductases protect against oxidative stress in Staphylococcus aureus encountering exogenous oxidants and human neutrophils

Yun Yun Pang  1 , Jamie Schwartz, Sarah Bloomberg, Jeffrey M Boyd, Alexander R Horswill, William M Nauseef
Affiliations

Methionine sulfoxide reductases protect against oxidative stress in Staphylococcus aureus encountering exogenous oxidants and human neutrophils

Yun Yun Pang et al. J Innate Immun. 2014.

Abstract

To establish infection successfully, Staphylococcus aureus must evade clearance by polymorphonuclear neutrophils (PMN). We studied the expression and regulation of the methionine sulfoxide reductases (Msr) that are involved in the repair of oxidized staphylococcal proteins and investigated their influence on the fate of S. aureus exposed to oxidants or PMN. We evaluated a mutant deficient in msrA1 and msrB for susceptibility to hydrogen peroxide, hypochlorous acid and PMN. The expression of msrA1 in wild-type bacteria ingested by human PMN was assessed by real-time PCR. The regulation of msr was studied by screening a library of two-component regulatory system (TCS) mutants for altered msr responses. Relative to the wild-type bacteria, bacteria deficient in Msr were more susceptible to oxidants and PMN. Upregulation of staphylococcal msrA1 occurred within the phagosomes of normal PMN and PMN deficient in NADPH oxidase activity. Furthermore, PMN granule-rich extract stimulated the upregulation of msrA1. Modulation of msrA1 within PMN was shown to be partly dependent on the VraSR TCS. Msr contributes to staphylococcal responses to oxidative attack and PMN. Our study highlights a novel interaction between the oxidative protein repair pathway and the VraSR TCS that is involved in cell wall homeostasis.

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Figures

Fig. 1
Fig. 1
Msr activity in wild-type and msr mutants. Conversion of dabsyl-methionine sulfoxide to dabsyl-methionine by lysates from wild-type, ΔmsrA1B and complemented ΔmsrA1B mutants were measured. Bacteria were treated with buffer (non-treated) or with vancomycin for 1 h. Data represent the mean ± SEM (n = 3 experiments).
Fig. 2
Fig. 2
Susceptibility of wild-type and msr mutants to oxidants and PMN. Wild-type, ΔmsrA1B and complemented ΔmsrA1B strains were grown to the mid-log phase, resuspended in PBS and treated with H2O2 (a), HOCl (b) or HOCl generated by the MPO-H2O2 system (c) at the stated concentrations for 1 h. After 1 h at 37°C, the bacteria were plated and CFU enumerated after overnight growth. Data represent CFU/ml as a percentage of non-treated control for each strain at time 0. Data are the mean ± SEM (n = 6 experiments for a and b, and n = 3 experiments for c). At each concentration of the oxidant, a paired t test was used to compare the wild-type and ΔmsrA1B strains (* p < 0.05, ** p < 0.01). d Each bacterial strain was opsonized in pooled human serum and then fed to normal or DPI-treated PMN (MOI of 5:1). Ingested bacteria were recovered at the indicated time points and CFU were enumerated. At each time point, the viability of a strain is expressed as bacteria recovered from normal PMN as a percent of the same strain recovered from DPI-treated PMN at time 0. A paired t test was used to compare the wild-type and ΔmsrA1B strains at each time point (* p < 0.05). Data represent the mean ± SEM (n = 6 experiments).
Fig. 3
Fig. 3
msrA1 expression in wild-type mutant ingested by normal and oxidase-deficient PMN. Serum-opsonized wild-type S. aureus in the mid-log (a) and stationary phase (b) were incubated alone or fed to PMN at an MOI of 1:1. At 0, 15, 30 and 60 min after phagocytosis, both ingested bacteria had recovered from PMN and bacteria incubated alone were prepared for real-time PCR. The expression of msrA1 was normalized to the expression of gapdh, and fold change in gene expression was relative to that in opsonized S. aureus prior to exposure to PMN. Data represent the mean ± SEM (n = 3 experiments). At each time point, a paired t test was used to analyze the difference between the ingested and non-ingested bacteria (* p < 0.05, ** p < 0.01). c Opsonized mid-log phase wild-type S. aureus was incubated alone or fed at an MOI of 1:1 to suspended normal PMN, DPI-treated PMN or PMN derived from 1 of 4 unrelated individuals with X-linked CGD. Data represent the mean ± SEM (n = 4 experiments). Data after 60 min were analyzed by 1-way ANOVA followed by a Tukey post-test (* p < 0.05).
Fig. 4
Fig. 4
msrA1 expression in S. aureus treated with oxidants and vancomycin (vanc). Wild-type S. aureus cultured in minimal media was treated with H2O2 or HOCl in the presence or absence of vancomycin (7 µg/ml) or with vancomycin alone for 1 h at 37°C. Bacteria were then assessed for msrA1 expression by real-time PCR and the data were normalized to gapdh expression. The data represent the fold change in msrA1 expression relative to the non-treated control (mean ± SEM, n = 6). A paired t test was used to compare sample conditions (* p < 0.05, ** p < 0.01).
Fig. 5
Fig. 5
Regulation of msrA1 by TCS. a Wild-type and mutant strains of S. aureus that were each deficient in one TCS were grown to the mid-log phase and treated with vancomycin for 1 h (table 1). TCS mutants are labeled in the graph with the gene locus or TCS name where known. The expression of msrA1 measured by real-time PCR was normalized to the expression of gapdh. Fold change in msrA1 is expressed relative to the untreated wild-type strain. The data represent the mean of two independent experiments. b Wild-type and ΔvraSR grown to the stationary phase were diluted to an OD550 of 0.3 and then treated with buffer or with D-cycloserine. Msr activity in bacterial lysates was measured. Data shown represent the mean ± SEM (n = 4). A paired t test was performed between the cycloserine-treated wild-type and mutant samples (* p < 0.05). c Serum-opsonized wild-type, ΔvraSR and complemented ΔvraSR grown to the mid-log phase were fed to PMN (MOI of 1:1), and msrA1 expression in the ingested bacteria was measured and normalized to gapdh expression. Fold change is expressed relative to opsonized wild-type bacteria at time 0. Data represent the mean ± SEM (n = 9) and are analyzed by 1-way ANOVA with a Tukey post-test (* p < 0.05, *** p < 0.001).
Fig. 6
Fig. 6
msrA1 expression in S. aureus treated with PMN granule proteins. Wild-type, ΔvraSR and complemented ΔvraSR strains were treated with PMN granule extracts (Gex), PBS (negative control) or RB (vehicle control). The levels of msrA1 in the samples at 0 and 30 min were assessed by real-time PCR. The expression levels were normalized to the expression of gapdh. Data represent the mean ± SEM (n = 3 experiments). To compare the RB sample with the Gex sample at 30 min, a paired t test was used for each strain (* p < 0.05).
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References

    1. Klebanoff SJ, Kettle AJ, Rosen H, Winterbourn CC, Nauseef WM. Myeloperoxidase: a front-line defender against phagocytosed microorganisms. J Leukoc Biol. 2013;93:185–198. - PMC - PubMed
    1. Vogt W. Oxidation of methionyl residues in proteins: tools, targets, and reversal. Free Radic Biol Med. 1995;18:93–105. - PubMed
    1. Singh K, Singh VK. Expression of four methionine sulfoxide reductases in Staphylococcus aureus. Int J Microbiol. 2012;2012:719594. - PMC - PubMed
    1. Singh VK, Moskovitz J, Wilkinson BJ, Jayaswal RK. Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin. Microbiology. 2001;147:3037–3045. - PubMed
    1. Singh VK, Moskovitz J. Multiple methionine sulfoxide reductase genes in Staphylococcus aureus: expression of activity and roles in tolerance of oxidative stress. Microbiology. 2003;149:2739–2747. - PubMed

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