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

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.

The Neisseria gonorrhoeae Methionine Sulfoxide Reductase (MsrA/B) Is a Surface Exposed, Immunogenic, Vaccine Candidate.

Control of the sexually transmitted infection gonorrhea is a major public health challenge, due to the recent emergence of multidrug resistant strains of Neisseria gonorrhoeae, and there is an urgent need for novel therapies or a vaccine to prevent gonococcal disease. In this study, we evaluated the methionine sulfoxide reductase (MsrA/B) of N. gonorrhoeae as a potential vaccine candidate, in terms of its expression, sequence conservation, localization, immunogenicity, and the functional activity of antibodies raised to it. Gonococcal MsrA/B has previously been shown to reduce methionine sulfoxide [Met(O)] to methionine (Met) in oxidized proteins and protect against oxidative stress. Here we have shown that the gene encoding MsrA/B is present, highly conserved, and expressed in all N. gonorrhoeae strains investigated, and we determined that MsrA/B is surface is exposed on N. gonorrhoeae. Recombinant MsrA/B is immunogenic, and mice immunized with MsrA/B and either aluminum hydroxide gel adjuvant or Freund's adjuvant generated a humoral immune response, with predominantly IgG1 antibodies. Higher titers of IgG2a, IgG2b, and IgG3 were detected in mice immunized with MsrA/B-Freund's adjuvant compared to MsrA/B-aluminum hydroxide adjuvant, while IgM titers were similar for both adjuvants. Antibodies generated by MsrA/B-Freund's in mice mediated bacterial killing via both serum bactericidal activity and opsonophagocytic activity. Anti-MsrA/B was also able to functionally block the activity of MsrA/B by inhibiting binding to its substrate, Met(O). We propose that recombinant MsrA/B is a promising vaccine antigen for N. gonorrhoeae.

Methionine sulfoxide reductase A protects against lipopolysaccharide-induced septic shock via negative regulation of the proinflammatory responses.

Methionine sulfoxide reductase A (MsrA) is a major antioxidant enzyme that specifically catalyzes the reduction of methionine S-sulfoxide. In this study, we used MsrA gene-knockout (MsrA-/-) mice and bone marrow-derived macrophages (BMDMs) to investigate the role of MsrA in the regulation of inflammatory responses induced by lipopolysaccharide (LPS). MsrA-/- mice were more susceptible to LPS-induced lethal shock than wild-type (MsrA+/+) mice. Serum levels of the proinflammatory cytokines IL-6 and TNF-α induced by LPS were higher in MsrA-/- than in MsrA+/+ mice. MsrA deficiency in the BMDMs also increased the LPS-induced cytotoxicity as well as TNF-α level. Basal and LPS-induced reactive oxygen species (ROS) levels were higher in MsrA-/- than in MsrA+/+ BMDMs. Phosphorylation levels of p38, JNK, and ERK were higher in MsrA-/- than in MsrA+/+ BMDMs in response to LPS, suggesting that MsrA deficiency increases MAPK activation. Furthermore, MsrA deficiency increased the expression and nuclear translocation of NF-κB and the expression of inducible nitric oxide synthase, a target gene of NF-κB, in response to LPS. Taken together, our results suggest that MsrA protects against LPS-induced septic shock, and negatively regulates proinflammatory responses via inhibition of the ROS-MAPK-NF-κB signaling pathways.

Autoantibody against one of the antioxidant repair enzymes, methionine sulfoxide reductase A, in systemic sclerosis: association with pulmonary fibrosis and vascular damage.

Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis and vascular changes in the skin and internal organs with autoimmune background. It has been suggested that oxidative stress plays an important role in the development of SSc. To determine the prevalence and clinical correlation of autoantibody to methionine sulfoxide reductase A (MSRA), one of the antioxidant repair enzymes, in SSc, serum anti-MSRA autoantibody levels were examined in patients with SSc by enzyme-linked immunosorbent assay using recombinant MSRA. The presence of anti-MSRA antibody was evaluated by immunoblotting. To determine the functional relevance of anti-MSRA antibody in vivo, we assessed whether anti-MSRA antibody was able to inhibit MSRA enzymatic activity. Serum anti-MSRA antibody levels in SSc patients were significantly higher compared to controls and this autoantibody was detected in 33% of SSc patients. Serum anti-MSRA levels were significantly elevated in SSc patients with pulmonary fibrosis, cardiac involvement, or decreased total antioxidant power compared with those without them. Anti-MSRA antibodies also correlated positively with renal vascular damage determined as pulsatility index by color-flow Doppler ultrasonography of the renal interlobar arteries and negatively with pulmonary function tests. Furthermore, anti-MSRA antibody levels correlated positively with serum levels of 8-isoprostane and heat shock protein 70 that are markers of oxidative and cellular stresses. Remarkably, MSRA activity was inhibited by IgG isolated from SSc sera containing IgG anti-MSRA antibody. These results suggest that elevated anti-MSRA autoantibody is associated with the disease severity of SSc and may enhance the oxidative stress by inhibiting MSRA enzymatic activity.

Analysis of methionine/selenomethionine oxidation and methionine sulfoxide reductase function using methionine-rich proteins and antibodies against their oxidized forms.

Methionine (Met) residues are present in most proteins. However, this sulfur-containing amino acid is highly susceptible to oxidation. In cells, the resulting Met sulfoxides are reduced back to Met by stereospecific reductases MsrA and MsrB. Reversible Met oxidation occurs even in the absence of stress, is elevated during aging and disease, but is notoriously difficult to monitor. In this work, we computationally identified natural Met-rich proteins (MRPs) and characterized three such proteins containing 21-33% Met residues. Oxidation of multiple Met residues in MRPs with H(2)O(2) and reduction of Met sulfoxides with MsrA/MsrB dramatically influenced the mobility of these proteins on polyacrylamide gels and could be monitored by simple SDS-PAGE. We further prepared antibodies enriched for reduced and Met sulfoxide forms of these proteins and used them to monitor Met oxidation and reduction by immunoblot assays. We describe applications of these reagents for the analysis of MsrA and MsrB functions, as well as the development of the assay for high-throughput analysis of their activities. We also show that all Met sulfoxide residues in an MRP can be reduced by MsrA and MsrB. Furthermore, we prepared a selenomethionine form of an MRP and found that selenomethionine selenoxide residues can be efficiently reduced nonenzymatically by glutathione and other thiol compounds. Selenomethionine selenoxide residues were not recognized by antibodies specific for the Met sulfoxide form of an MRP. These findings, reagents, assays, and approaches should facilitate research and applications in the area of Met sulfoxide reduction, oxidative stress, and aging.