Recombinant Staphylococcus aureus Peptide methionine sulfoxide reductase MsrB (msrB)

What is MsrB and what is its role in S. aureus?

MsrB (Methionine Sulfoxide Reductase B) is an essential enzyme in S. aureus that specifically reduces R-epimers of methionine sulfoxide back to methionine. This enzymatic activity is crucial for repairing oxidative damage to proteins when bacteria encounter reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI). In S. aureus, there is one msrB gene and three msrA genes (msrA1, msrA2, and msrA3) located in different parts of the chromosome . This repair mechanism is particularly important during host-pathogen interactions, where S. aureus faces oxidative bursts from host immune cells such as polymorphonuclear neutrophils (PMN) . MsrB contributes to the bacterium's defense against oxidative stress and influences various phenotypic characteristics including pigmentation and virulence.

How does MsrB differ functionally from MsrA in S. aureus?

The primary functional difference between MsrB and MsrA in S. aureus is their substrate stereospecificity. MsrB specifically reduces the R-epimers of methionine sulfoxide, while MsrA reduces the S-epimers . This stereoselectivity is highly conserved across species and represents a defining characteristic of these enzymes. In terms of genomic organization, S. aureus possesses three msrA genes (msrA1, msrA2, and msrA3) but only one msrB gene . Interestingly, phenotypic studies have revealed that MsrA1-deficient strains are sensitive to oxidative stress conditions, less pigmented, and less adherent to human lung epithelial cells, with reduced survival in mouse tissues . In contrast, MsrB-deficient strains demonstrate unexpected resistance to oxidants and increased pigmentation . These findings suggest that despite their complementary biochemical functions, MsrA and MsrB play distinct roles in bacterial physiology and virulence.

What structural characteristics define S. aureus MsrB?

S. aureus MsrB is a non-selenoprotein that maintains the same substrate stereospecificity as selenoprotein MsrB variants found in other organisms such as mice . While detailed structural information specific to S. aureus MsrB is limited in the available literature, comparative analysis with other bacterial MsrB proteins suggests it likely contains conserved catalytic cysteine residues crucial for the reduction of R-methionine sulfoxide. Unlike the selenoprotein versions that incorporate selenocysteine in the active site, S. aureus utilizes cysteine residues for catalysis. The enzyme's structure is fundamentally different from MsrA despite catalyzing similar reactions, reflecting their independent evolutionary origins. This structural distinction underlies the stereoselectivity of the two enzyme classes, allowing them to specifically target different epimers of methionine sulfoxide.

How is MsrB regulated in S. aureus during infection?

The regulation of MsrB in S. aureus involves multiple mechanisms responsive to environmental stressors. Cell wall-active antibiotics have been shown to cause elevated synthesis of methionine sulfoxide reductases, including MsrB, in S. aureus . Research with S. aureus has demonstrated that upregulation of msrA1 occurs within the phagosomes of polymorphonuclear neutrophils (PMN), and this modulation is partly dependent on the VraSR two-component regulatory system (TCS) involved in cell wall homeostasis . Similar regulatory mechanisms likely apply to msrB expression. Additionally, PMN granule-rich extract stimulates the upregulation of msrA1, suggesting that components of host immune cells directly influence the expression of methionine sulfoxide reductases . This regulatory network highlights the importance of these enzymes in bacterial adaptation during host-pathogen interactions.

How do msrA and msrB mutations differentially affect S. aureus phenotypes?

Mutations in msrA and msrB genes result in strikingly different phenotypic outcomes in S. aureus. MsrA1-deficient strains exhibit increased sensitivity to oxidative stress conditions, decreased pigmentation, reduced adherence to human lung epithelial cells, and diminished survival in mouse tissues . In contrast, MsrB-deficient strains demonstrate resistance to oxidants and increased pigmentation . When comparing the triple msrA mutant (lacking msrA1, msrA2, and msrA3) with the quadruple mutant (lacking all msrA genes plus msrB), complementation experiments revealed that it was MsrA1, not MsrB, that proved critical for adherence and phagocytic resistance . These differential effects suggest distinct roles in bacterial physiology despite their complementary biochemical functions in reducing different epimers of methionine sulfoxide. The unexpected phenotype of msrB mutants may reflect complex regulatory interrelationships between oxidative stress responses, pigment production, and virulence factors in S. aureus.

What is the relationship between MsrB and antibiotic resistance in S. aureus?

The relationship between MsrB and antibiotic resistance in S. aureus involves several interconnected mechanisms. Cell wall-active antibiotics have been shown to induce elevated synthesis of methionine sulfoxide reductases, including MsrB, in S. aureus . This upregulation suggests that Msr enzymes may contribute to bacterial responses to antibiotic stress. The VraSR two-component system, which is involved in cell wall homeostasis and regulates msrA1 expression within neutrophil phagosomes, provides a mechanistic link between cell wall integrity and oxidative protein repair pathways . Since many antibiotics induce oxidative stress as part of their bactericidal action, alterations in oxidative stress management via msrB could potentially modify responses to antibiotics. The increased pigmentation observed in msrB mutants might provide additional protection against oxidative damage from antibiotics. Understanding this relationship has potential implications for developing new therapeutic strategies that might target Msr enzymes in combination with existing antibiotics to overcome resistance mechanisms.

What are the most effective methods for recombinant expression and purification of S. aureus MsrB?

For recombinant expression and purification of S. aureus MsrB, several strategic approaches have proven effective based on work with similar redox-sensitive bacterial enzymes. Expression in E. coli BL21(DE3) or Rosetta strains with pET-based vectors containing an N-terminal 6xHis tag offers a good starting point. Induction conditions should be optimized using lower temperatures (16-25°C) and moderate IPTG concentrations (0.1-0.5 mM) to enhance proper folding. Including reducing agents such as DTT (1-2 mM) in growth media and all purification buffers is critical to maintain enzyme stability and prevent oxidation of catalytic cysteine residues. Purification typically employs immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for initial capture, followed by size exclusion chromatography for higher purity. The purified enzyme should be stored in buffer containing reducing agents and glycerol, with aliquots flash-frozen to preserve activity. This general approach has been successfully applied to other bacterial MsrB proteins and can be adapted specifically for S. aureus MsrB, with adjustments based on protein-specific characteristics.

What assays can reliably measure MsrB activity in S. aureus?

Several complementary assays can reliably measure MsrB activity in S. aureus, each with specific advantages for different research questions. For direct measurement of stereospecific activity, the dabsyl-methionine-R-sulfoxide reduction assay using HPLC separation is highly specific but requires specialized equipment. A more accessible approach employs a coupled enzyme assay with the thioredoxin system, where MsrB activity is measured through NADPH consumption monitored at 340 nm. This method more closely resembles physiological conditions. For studying protein substrate specificity, mass spectrometry-based approaches can detect the reduction of specific methionine sulfoxide residues in oxidized proteins. Studies with H. influenzae MsrAB demonstrated this approach, showing the enzyme could repair oxidative damage to specific proteins with up to 100% efficiency . When choosing an assay, researchers should consider the specific research question, available equipment, and whether they need to distinguish between MsrA and MsrB activities, as both enzymes reduce methionine sulfoxide but with different stereospecificity.

How can researchers create and validate msrB knockout mutants in S. aureus?

Creating and validating msrB knockout mutants in S. aureus requires careful genetic manipulation and thorough confirmation. Based on strategies described in the literature, researchers have successfully created msrB mutants in the wild-type background . The allelic replacement strategy involves designing primers to amplify upstream and downstream flanking regions of msrB, cloning these fragments into a temperature-sensitive shuttle vector with an antibiotic resistance marker between flanking regions, and selecting for double crossover events. Alternatively, CRISPR-Cas9 approaches offer more efficient and cleaner deletions. Validation should include multiple methods: genetic verification through PCR amplification across the deletion junction, sequencing to confirm precise deletion, RT-PCR to confirm absence of msrB transcript, and protein-level confirmation through Western blotting or enzyme activity assays. Functional characterization should assess oxidative stress response, pigmentation changes, and virulence-associated phenotypes. Complementation studies, reintroducing msrB on a plasmid, are essential to confirm that observed phenotypes result specifically from msrB deletion rather than polar effects or secondary mutations. This comprehensive validation ensures reliable interpretation of subsequent experiments.

What approaches identify physiological protein substrates of S. aureus MsrB?

Identifying physiological protein substrates of S. aureus MsrB requires a multi-faceted approach combining proteomics, biochemical analysis, and validation studies. Comparative redox proteomics between wild-type and msrB-deficient S. aureus under oxidative stress can reveal proteins with increased methionine-R-sulfoxide levels in the mutant, indicating potential substrates. Affinity-based substrate trapping using catalytically inactive MsrB mutants as "bait" in pull-down experiments can capture interacting proteins for mass spectrometric identification. In vitro screening of candidate proteins involves oxidizing purified S. aureus proteins and testing MsrB's ability to repair specific methionine residues. Based on studies with H. influenzae MsrAB, researchers identified 29 outer membrane and periplasmic proteins as likely substrates, including proteins involved in metabolism and adhesion . Similar approaches could identify the substrate repertoire of S. aureus MsrB, focusing on proteins related to the phenotypes observed in msrB mutants, such as those involved in pigmentation, oxidative stress response, and cell adhesion. Validation of key substrates should include examining the functional consequences of oxidation and repair.

What is the catalytic mechanism of S. aureus MsrB?

The catalytic mechanism of S. aureus MsrB, while not explicitly detailed in the available literature, likely follows the general mechanism established for bacterial MsrB enzymes. As a non-selenoprotein MsrB (unlike the selenoprotein version found in mice) , S. aureus MsrB utilizes catalytic cysteine residues. The reaction begins with nucleophilic attack by the catalytic cysteine on the sulfur atom of methionine sulfoxide, forming a sulfenic acid intermediate on the catalytic cysteine. This intermediate is then resolved through interaction with other cysteine residues or reducing partners like thioredoxin, which regenerates the active enzyme. The stereoselectivity for R-epimers of methionine sulfoxide is determined by the precise architecture of the active site, which differs fundamentally from MsrA despite catalyzing similar reactions. Research with purified recombinant mouse MsrB has demonstrated this stereospecificity, with the enzyme exhibiting high specificity for reduction of the R forms of free and protein-bound methionine sulfoxide . Detailed characterization of the S. aureus enzyme would require enzyme kinetic studies, site-directed mutagenesis of candidate catalytic residues, and identification of physiological reducing partners.

How do environmental conditions affect MsrB expression and activity in S. aureus?

Environmental conditions significantly modulate MsrB expression and activity in S. aureus, reflecting its role in stress adaptation. Oxidative stress conditions, including exposure to hydrogen peroxide (H₂O₂) and hypochlorous acid (HOCl), likely influence msrB expression as part of the oxidative stress response . Cell wall-active antibiotics induce elevated synthesis of methionine sulfoxide reductases, including MsrB, in S. aureus, suggesting a connection between cell wall stress and oxidative protein damage repair systems . The interaction with host immune cells presents particularly important environmental cues; studies with S. aureus showed that upregulation of msrA1 occurs within neutrophil phagosomes and is partly dependent on the VraSR two-component system involved in cell wall homeostasis . Similar regulatory mechanisms may apply to msrB. Studies with H. influenzae MsrAB demonstrated that optimal enzymatic activity occurs around 30°C, close to the temperature of upper respiratory tract environments . This temperature sensitivity could be relevant for S. aureus MsrB as well, particularly during colonization of different body sites with varying temperatures.

How can MsrB be targeted for potential therapeutic applications against S. aureus infections?

Developing therapeutic strategies targeting MsrB in S. aureus requires careful consideration of its unique characteristics and the complex phenotype of msrB mutants. The unexpected finding that MsrB-deficient strains show resistance to oxidants and increased pigmentation suggests that simple inhibition of MsrB alone might not produce the desired antimicrobial effect. A more promising approach might involve targeting both MsrA and MsrB simultaneously to comprehensively disable the methionine sulfoxide reduction system, as MsrA1 appears critical for adherence and phagocytic resistance . Combination therapies pairing Msr inhibitors with oxidative stress-inducing antibiotics could exploit the relationship between oxidative damage and antibiotic efficacy. Development of compounds that selectively inhibit bacterial Msr enzymes without affecting human homologs would be essential to minimize side effects. The complex interplay between methionine sulfoxide reduction, oxidative stress response, and virulence factor production suggests that MsrB inhibition might be most effective as part of a multi-target approach. Future therapeutic development should focus on understanding the compensatory mechanisms that activate in msrB mutants and designing strategies to circumvent these adaptive responses.