Recombinant Enterococcus faecalis Adapter protein mecA (mecA)

What is Recombinant Enterococcus faecalis Adapter protein mecA?

Recombinant Enterococcus faecalis Adapter protein mecA is a full-length protein (220 amino acids) derived from E. faecalis strain ATCC 700802/V583, with UniProt accession number Q830U4. This protein functions as an adapter molecule involved in cellular signaling pathways within E. faecalis. It is typically produced through recombinant DNA technology using E. coli expression systems, resulting in a protein with >85% purity as determined by SDS-PAGE analysis . Unlike the mecA gene in Staphylococcus aureus (which encodes penicillin-binding protein 2a and confers methicillin resistance), E. faecalis mecA serves as an adapter protein with distinct functional properties in cellular processes.

What are the optimal storage and handling conditions for recombinant E. faecalis mecA protein?

For optimal stability and activity preservation, recombinant E. faecalis mecA protein should be stored at -20°C/-80°C, with different shelf life expectations based on formulation:

• Liquid form: 6 months shelf life at -20°C/-80°C

• Lyophilized form: 12 months shelf life at -20°C/-80°C

Working aliquots can be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be strictly avoided as they lead to protein degradation and activity loss . When handling the protein for experimental purposes, maintain cold chain practices and use sterile techniques to prevent contamination.

What are the recommended reconstitution protocols for experimental applications?

For reconstitution of lyophilized mecA protein:

• Briefly centrifuge the vial prior to opening to consolidate contents

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being optimal) for long-term stability

• Aliquot in appropriate volumes to minimize freeze-thaw cycles

• Store reconstituted protein at -20°C/-80°C for extended shelf life

For experimental assays, further dilution in appropriate buffers may be necessary depending on specific application requirements and detection methods employed.

How can researchers investigate potential interactions between mecA and bacteriophage resistance mechanisms?

Recent research on E. faecalis bacteriophage vB_EfaS_efap05-1 provides insights into potential experimental approaches:

• Phage adsorption assays: To determine if mecA influences phage attachment, researchers can perform adsorption assays comparing wild-type strains with mecA-overexpressing or deletion mutants. This approach has been effective in studying how proteins like ComEA affect bacteriophage binding .

• Resistance development monitoring: Culture E. faecalis with bacteriophages in the presence of varying mecA concentrations to observe if mecA modulates the development of phage resistance. Studies have shown that mutations in membrane proteins and polysaccharide biosynthesis genes can lead to phage resistance in E. faecalis .

• Receptor competition assays: Pre-incubate bacteria with purified recombinant mecA before phage exposure to determine if it competitively inhibits phage binding, suggesting a direct receptor-like function .

What methodologies are effective for studying mecA protein interactions with cell wall components?

To investigate potential interactions between mecA and cell wall components, consider these approaches:

• Pull-down assays: Immobilize purified recombinant mecA on an affinity matrix and expose it to cell wall fractions to identify binding partners through mass spectrometry.

• Surface plasmon resonance (SPR): Measure real-time binding kinetics between mecA and purified cell wall components, particularly focusing on peptidoglycan fragments.

• Bacterial two-hybrid systems: Construct fusion proteins to detect potential protein-protein interactions between mecA and cell wall biosynthesis enzymes.

• Peptidoglycan binding assays: Similar to studies with SagA peptidoglycan hydrolase in E. faecium, researchers can assess whether mecA binds to specific peptidoglycan structures through co-precipitation experiments .

How can researchers investigate the role of mecA in environmental stress responses?

The following experimental approaches could elucidate mecA's role in stress response:

• Transcriptomic profiling: Compare gene expression profiles in wild-type and mecA knockout/overexpression strains under various stressors (antibiotics, pH shifts, nutrient limitation). This approach mirrors the methodology used to study E. faecalis responses to collagen exposure .

• Stress survival assays: Subject wild-type and mecA-modified strains to various environmental stressors and measure survival rates to determine if mecA confers protection.

• Protein localization studies: Use fluorescently tagged mecA to track its subcellular localization during stress responses to identify potential translocation or aggregation patterns.

What approaches can determine if mecA contributes to virulence or colonization capabilities?

A multi-faceted approach is recommended:

• Animal infection models: Compare colonization and virulence between wild-type and mecA-deficient strains in appropriate animal models.

• Adhesion and invasion assays: Quantify bacterial attachment to and invasion of host cells using tissue culture systems.

• Biofilm formation analysis: Assess the impact of mecA expression levels on biofilm development using crystal violet staining and confocal microscopy.

• Immune response measurement: Determine if mecA affects host immune recognition by quantifying cytokine responses to wild-type versus mecA-deficient strains.

• Comparative proteomics: Profile the bacterial proteome under various conditions to identify co-regulated proteins that might function alongside mecA in virulence pathways .

What are the key considerations for designing structure-function experiments with recombinant mecA protein?

When investigating structure-function relationships, researchers should:

• Generate truncated variants: Create systematic deletions of mecA domains to identify regions essential for specific functions.

• Site-directed mutagenesis: Target conserved residues for substitution to determine their importance in protein function.

• Circular dichroism spectroscopy: Assess secondary structure changes resulting from mutations or environmental conditions.

• Thermal shift assays: Evaluate protein stability under various conditions to optimize experimental parameters.

• Crystallization trials: Determine three-dimensional structure through X-ray crystallography, following approaches similar to those used for the SagA-NlpC/p60 catalytic domain from E. faecium .

How can researchers reliably quantify mecA expression levels in various experimental conditions?

For accurate quantification:

• qRT-PCR: Design primers specific to mecA for transcript quantification, normalizing to validated reference genes.

• Western blotting: Develop specific antibodies against mecA or use epitope tags for immunodetection.

• ELISA: Establish sandwich ELISA protocols for quantitative measurement in complex samples.

• Mass spectrometry: Implement targeted proteomics approaches like selected reaction monitoring (SRM) for absolute quantification.

• Reporter gene fusions: Create transcriptional or translational fusions to fluorescent proteins for live-cell monitoring of expression dynamics.

What are the critical quality control parameters for functional assays involving recombinant mecA?

To ensure experimental reliability:

Each batch of recombinant mecA should undergo these quality assessments before use in critical experiments to ensure reproducible results .

How might mecA research intersect with bacteriophage therapy development against E. faecalis infections?

The growing problem of antibiotic resistance in E. faecalis has intensified interest in bacteriophage therapy as an alternative treatment approach . Understanding the role of mecA in potential phage resistance mechanisms could:

• Inform the selection of therapeutic phages less likely to encounter resistance

• Allow prediction of resistance development patterns

• Guide the design of combination therapies targeting both bacterial and resistance mechanisms

• Enable monitoring of treatment efficacy through mecA expression analysis

Research exploring how E. faecalis bacteriophage vB_EfaS_efap05-1 interacts with bacterial surface components provides a framework for investigating potential mecA involvement in phage attachment or resistance mechanisms .

What methodological approaches can reveal potential interactions between mecA and host immune components?

Building on research that demonstrated E. faecium peptidoglycan fragments can activate NOD2 receptors , researchers could:

• Co-culture experiments: Expose immune cells to wild-type versus mecA-deficient E. faecalis and measure differential immune responses.

• Purified protein stimulation: Determine if recombinant mecA directly activates pattern recognition receptors using reporter cell lines.

• In vivo infection models: Compare immune cell recruitment and cytokine production in animals infected with strains expressing different levels of mecA.

• Transcriptomics of host-pathogen interface: Analyze host gene expression changes in response to mecA-varying strains.

• Immunoprecipitation studies: Identify potential direct interactions between mecA and host immune components.

How can advanced microscopy techniques enhance understanding of mecA localization and dynamics?

Advanced imaging approaches can provide unique insights:

• Super-resolution microscopy: Techniques like STORM or PALM can visualize mecA distribution with nanometer precision.

• Single-molecule tracking: Follow individual mecA molecules in live cells to determine mobility and interaction dynamics.

• FRET microscopy: Detect proximity between mecA and potential binding partners through fluorescence resonance energy transfer.

• Correlative light-electron microscopy: Combine functional imaging with ultrastructural context to precisely localize mecA relative to cellular structures.

• 4D imaging: Track mecA dynamics throughout the bacterial cell cycle to identify temporal regulation patterns.