Recombinant Staphylococcus aureus UPF0754 membrane protein MW1787 (MW1787)

What is UPF0754 membrane protein MW1787 in Staphylococcus aureus?

UPF0754 membrane protein MW1787 is a full-length membrane protein encoded by the MW1787 gene in Staphylococcus aureus. It belongs to the UPF0754 protein family, which consists of uncharacterized protein families with unknown functions. This protein spans 374 amino acids (1-374aa) and is identified by the UniProt ID Q8NVV4 . As an integral membrane protein, it's part of the approximately 25% of proteins encoded by the genome that are embedded in cellular membranes . MW1787 is found in clinically relevant strains such as S. aureus MW2, which has been fully genome-sequenced and is known to be methicillin-resistant (MRSA) .

How is recombinant Staphylococcus aureus UPF0754 membrane protein MW1787 expressed?

Recombinant MW1787 protein can be expressed in various host systems, with E. coli being the most common for research applications. The protein is typically expressed with a fusion tag (commonly His-tag at the N-terminus) to facilitate purification . While E. coli and yeast expression systems offer the highest yields and shorter turnaround times, expression in insect cells with baculovirus or mammalian cells can provide many of the post-translational modifications necessary for correct protein folding or activity maintenance . For functional studies, the expression system choice depends on research objectives - E. coli is suitable for structural studies while eukaryotic systems may better preserve activity for functional assays.

How should recombinant MW1787 protein be stored and reconstituted?

Recombinant MW1787 protein is typically supplied as a lyophilized powder and requires proper storage and reconstitution to maintain activity. The recommended storage protocol includes:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

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

• Add glycerol to 5-50% final concentration (50% is standard) and aliquot for long-term storage

• Store working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as they can damage protein structure and function

The protein is typically reconstituted in Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain stability .

What experimental approaches can characterize the function of UPF0754 membrane protein MW1787?

Characterizing MW1787 function requires multiple complementary approaches:

• Genetic approaches:

  • Gene knockout studies in S. aureus to observe phenotypic changes

  • Complementation assays to verify phenotype restoration

  • Site-directed mutagenesis to identify essential residues

• Structural approaches:

  • X-ray crystallography or cryo-EM to determine 3D structure

  • Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

  • Molecular dynamics simulations to predict conformational changes

• Interaction studies:

  • Co-immunoprecipitation to identify binding partners

  • Bacterial two-hybrid screening to map protein-protein interactions

  • Cross-linking mass spectrometry to capture transient interactions

• Functional assays:

  • Membrane potential measurements to assess ion transport activity

  • Reconstitution in liposomes to study transport capabilities

  • Cell invasion assays to evaluate roles in pathogenesis

When designing these experiments, researchers should consider that multi-spanning membrane proteins often require chaperones for proper assembly, as shown by studies of the PAT complex that protects transmembrane domains containing unshielded hydrophilic side chains during folding .

How does UPF0754 membrane protein MW1787 potentially contribute to S. aureus pathogenesis?

While the specific role of MW1787 in S. aureus pathogenesis remains under investigation, several lines of evidence suggest potential contributions:

• Expression patterns: MW1787 is present in virulent strains including MW2 (a community-acquired MRSA strain), suggesting potential relevance to infection processes .

• Membrane protein function: As an integral membrane protein, MW1787 could participate in:

  • Nutrient acquisition during infection

  • Cell wall maintenance under host-imposed stress

  • Evasion of host immune responses

  • Antibiotic resistance mechanisms

• Adaptation to host environment: Recent studies on S. aureus evolution in macrophages demonstrate that membrane proteins can undergo adaptive changes that enhance survival within immune cells . MW1787 may similarly be involved in adaptation to host environments.

• Potential treatment target: Novel approaches targeting S. aureus, such as engineered proteins called centyrins, have shown efficacy against S. aureus infection in preclinical models . Understanding MW1787 function could reveal whether it represents a viable therapeutic target.

The presence of this protein in the extensively studied MW2 strain provides an opportunity to evaluate its role in virulence through comparative studies with other clinical isolates .

What methodologies are optimal for studying MW1787 interactions with host cells?

Studying MW1787 interactions with host cells requires specialized approaches:

• Infection models:

  • Macrophage infection assays similar to those described for S. aureus evolutionary studies

  • Human blood survival assays to assess contributions to immune evasion

  • Tissue-specific cell culture models relevant to S. aureus infection sites

• Visualization techniques:

  • Fluorescently-tagged MW1787 constructs for live-cell imaging

  • Super-resolution microscopy to track protein localization during infection

  • Correlative light and electron microscopy to visualize at multiple scales

• Functional blocking studies:

  • Anti-MW1787 antibodies to inhibit potential extracellular functions

  • Competitive peptides derived from MW1787 sequence

  • Small molecule inhibitors identified through screening approaches

• Host response assessment:

  • Transcriptomic analysis of host cells exposed to wild-type vs. MW1787-deficient S. aureus

  • Cytokine profiling to evaluate inflammatory response differences

  • Phagocytosis and killing assays to assess immune cell function

The experimental evolution approach described for S. aureus in macrophages provides a valuable methodology to understand how membrane proteins like MW1787 may adapt during host-pathogen interactions .

How can structural information about MW1787 be obtained and utilized?

Obtaining and utilizing structural information about MW1787 involves several specialized techniques:

• Structure determination approaches:

  • X-ray crystallography: Requires detergent-solubilized and purified protein, often challenging for membrane proteins

  • Cryo-electron microscopy: Increasingly preferred for membrane proteins, avoiding crystallization

  • NMR spectroscopy: Suitable for dynamic analyses of specific domains

• Sample preparation considerations:

  • Detergent screening is critical for extraction while maintaining native conformation

  • Nanodiscs or amphipols can provide more native-like membrane environments

  • Lipid composition should mimic S. aureus membrane for functional studies

• Computational approaches:

  • Homology modeling based on related structures

  • Molecular dynamics simulations to predict conformational changes

  • Structure-based virtual screening for potential inhibitors

• Structure-function applications:

  • Identification of key residues for mutagenesis studies

  • Design of domain-specific antibodies for function blocking

  • Rational design of inhibitors targeting critical structural features

The presence of multiple transmembrane domains with hydrophilic residues suggests MW1787 may require specialized chaperones like the PAT complex during assembly, which would need to be considered when designing reconstitution experiments .

What purification strategies yield highest quality recombinant MW1787 protein?

Purifying membrane proteins like MW1787 requires specialized approaches:

• Solubilization optimization:

  • Screen multiple detergents (DDM, LMNG, CHAPS) for efficient extraction

  • Test detergent-to-protein ratios systematically

  • Consider membrane mimetics (nanodiscs, liposomes) for downstream applications

• Chromatography workflow:

  • Immobilized metal affinity chromatography (IMAC) using the His-tag

  • Size exclusion chromatography to remove aggregates

  • Ion exchange chromatography for additional purity

• Quality assessment:

  • SDS-PAGE analysis (>90% purity standard)

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Circular dichroism to confirm secondary structure integrity

  • Functional assays specific to predicted activities

A typical purification protocol would include:

How can functional assays be developed to assess MW1787 activity?

Developing functional assays for MW1787 requires consideration of its potential roles:

• Transport activity assessment:

  • Liposome reconstitution with fluorescent indicators for ion transport

  • Substrate uptake studies using radiolabeled compounds

  • Membrane potential measurements in reconstituted systems

• Protein-protein interaction assays:

  • Pull-down assays with potential partners from S. aureus

  • Surface plasmon resonance for binding kinetics

  • Förster resonance energy transfer (FRET) for in vivo interaction detection

• Phenotypic rescue experiments:

  • Complementation of MW1787 knockouts with wild-type and mutant variants

  • Quantification of restored phenotypes (growth, stress resistance)

  • Competition assays between wild-type and mutant strains

• Stress response measurements:

  • Survival under membrane-targeting antimicrobials

  • Response to osmotic shock conditions

  • Growth in nutrient-limited environments

When developing these assays, researchers should consider that conditional phenotypes might emerge only under specific stress conditions, similar to the small colony variants observed in S. aureus adaptation studies .

What expression systems maximize functional yield of recombinant MW1787?

Optimizing expression systems for functional MW1787 requires balancing yield with protein quality:

• E. coli-based systems:

  • C41(DE3) and C43(DE3) strains engineered for membrane protein expression

  • Tunable promoters (like rhamnose-inducible) for controlled expression rate

  • Low-temperature induction (16-18°C) to improve folding

  • Co-expression with chaperones to enhance proper folding

• Eukaryotic alternatives:

  • Pichia pastoris for higher yields with eukaryotic modifications

  • Insect cell-baculovirus for complex membrane proteins

  • Mammalian expression for highest authenticity but lower yields

• Optimization parameters:

  • Induction conditions (temperature, inducer concentration, timing)

  • Media composition (minimal vs. rich, supplementation strategies)

  • Harvest timing to minimize toxicity effects

  • Scale-up considerations for structural biology applications

Expression in E. coli typically provides sufficient material for biochemical and structural studies, while more complex questions about function might benefit from eukaryotic expression systems .

How can researchers assess MW1787 involvement in S. aureus adaptation to host environments?

Studying MW1787's role in host adaptation requires specialized experimental approaches:

• Experimental evolution studies:

  • Passage S. aureus in relevant host environments (macrophages, serum)

  • Track genomic changes in MW1787 during adaptation

  • Characterize phenotypic changes in adapted strains

  • Perform complementation studies with wild-type MW1787

• Comparative genomics approaches:

  • Analyze MW1787 sequence variation across clinical isolates

  • Correlate sequence variants with virulence or resistance phenotypes

  • Identify selection signatures in the MW1787 gene

• Host-pathogen interaction models:

  • Develop cell culture infection models targeting specific host niches

  • Measure differential expression of MW1787 during infection

  • Assess contribution to survival in human blood or serum

  • Evaluate impact on small colony variant (SCV) formation

• In vivo significance assessment:

  • Animal infection models comparing wild-type and MW1787 mutants

  • Tissue-specific colonization and persistence studies

  • Immune response characterization in the presence/absence of MW1787

The experimental evolution approach described for S. aureus adaptation in macrophages provides a valuable methodological framework, as it revealed how bacteria can develop specific adaptations to survive within host cells, potentially involving membrane proteins like MW1787 .

What are the key considerations for designing MW1787 mutation studies?

Designing effective mutation studies for MW1787 requires careful planning:

• Mutation strategy selection:

  • Alanine scanning for systematic functional mapping

  • Conservation-guided targeting of evolutionary conserved residues

  • Structure-informed mutations targeting predicted functional sites

  • Domain deletion/swapping to assess modular functions

• Expression verification approaches:

  • Western blotting with tag-specific antibodies

  • Flow cytometry for surface expression assessment

  • Immunofluorescence microscopy for localization confirmation

  • Quantitative PCR for transcript level verification

• Phenotypic assay selection:

  • Growth curves under standard and stress conditions

  • Membrane integrity assessments

  • Antibiotic susceptibility testing

  • Host cell interaction assays

When designing mutations, researchers should consider that transmembrane proteins often have specific folding requirements and interactions between TMDs that are essential for stability and function .

How can researchers integrate MW1787 studies with broader S. aureus pathogenesis research?

Integrating MW1787 research with broader pathogenesis studies requires:

• Contextual experimental design:

  • Use clinically relevant strains like MW2 for translational relevance

  • Incorporate MW1787 analysis into existing virulence factor studies

  • Consider strain-specific differences in MW1787 sequence and expression

• Multi-omics integration:

  • Correlate MW1787 expression with transcriptomic profiles during infection

  • Identify co-regulated genes for functional network building

  • Analyze proteomic changes in MW1787 mutants to identify affected pathways

• Collaborative research approaches:

  • Combine MW1787 studies with investigations of novel anti-S. aureus therapies

  • Incorporate MW1787 analysis in experimental evolution studies

  • Evaluate MW1787 in the context of membrane-targeting antimicrobials

The potential role of MW1787 should be considered in light of novel therapeutic approaches against S. aureus, such as engineered proteins (centyrins) that have shown promise in blocking infection processes .

How might MW1787 contribute to antibiotic resistance mechanisms in S. aureus?

As a membrane protein, MW1787 could potentially contribute to antibiotic resistance through several mechanisms:

• Direct involvement:

  • Participation in efflux pump complexes that export antibiotics

  • Alteration of membrane permeability to reduce antibiotic uptake

  • Sensing of antimicrobial compounds and triggering adaptive responses

• Indirect contributions:

  • Stabilization of membrane structure under antibiotic stress

  • Interaction with established resistance determinants

  • Role in small colony variant formation, which is associated with increased antibiotic tolerance

• Experimental approaches to investigate:

  • Antibiotic susceptibility testing of MW1787 mutants

  • Expression analysis under antibiotic challenge

  • Interaction studies with known resistance determinants

  • Evolution experiments under antibiotic selection pressure

The connection between membrane protein function and antibiotic resistance is particularly relevant for methicillin-resistant S. aureus strains like MW2, where MW1787 is encoded .

What role might MW1787 play in S. aureus biofilm formation and persistence?

Investigating MW1787's potential role in biofilm processes requires specialized approaches:

• Biofilm phenotype assessment:

  • Static and flow biofilm quantification assays

  • Confocal microscopy for structural analysis

  • Viability assessment within biofilm communities

  • Dispersal dynamics under various stressors

• Molecular mechanisms to investigate:

  • Contribution to adhesion processes

  • Role in cell-cell communication within biofilms

  • Involvement in extracellular matrix production

  • Function in metabolic adaptation during biofilm growth

• Experimental design considerations:

  • Testing under diverse environmental conditions

  • Evaluation in mixed-species biofilm models

  • Assessment on relevant host tissue surfaces

  • Integration with persister cell formation studies

This research direction could connect to the observed small colony variant phenotypes in adapted S. aureus strains, as SCVs often show enhanced biofilm formation capabilities .