Recombinant Staphylococcus aureus UPF0754 membrane protein SAR1937 (SAR1937)

What is the structural composition of Staphylococcus aureus UPF0754 membrane protein SAR1937?

SAR1937 is a membrane protein from Staphylococcus aureus (strain MRSA252) with 374 amino acids. The protein sequence begins with MNALFIIIFMIVVGAIIGVTNVIAI and continues through several hydrophobic and hydrophilic regions typical of membrane proteins. The complete amino acid sequence indicates multiple transmembrane domains characteristic of integral membrane proteins. The protein is identified in the UniProt database under accession number Q6GFL0 . Its structural features suggest potential roles in membrane transport or cellular signaling pathways, although the specific function remains under investigation in current research.

How is recombinant SAR1937 protein typically stored and what are the optimal conditions for maintaining its stability?

Recombinant SAR1937 protein is optimally stored in Tris-based buffer with 50% glycerol at -20°C for regular storage and -80°C for extended preservation . For working aliquots, storage at 4°C is suitable for up to one week. Repeated freeze-thaw cycles should be avoided as they can compromise protein integrity and biological activity. This is similar to stability patterns observed in other antimicrobial proteins derived from Staphylococcus, which demonstrate optimal stability at pH ranges from 3 to 9 and temperatures up to 50°C . For functional studies, researchers should prepare small working aliquots to minimize freeze-thaw cycles and preserve the native conformation of the protein.

What expression systems are most effective for producing recombinant SAR1937 protein?

While specific expression systems for SAR1937 are not directly detailed in the available literature, comparable staphylococcal membrane proteins are successfully expressed using E. coli BL21(DE3) systems, as demonstrated in parallel research with similar membrane proteins . This bacterial expression system provides high yields and maintains protein functionality. The expression process typically involves cloning the SAR1937 gene into vectors with inducible promoters (like T7), transformation into appropriate E. coli strains, followed by induction with IPTG at optimal densities. Purification protocols generally utilize affinity chromatography methods, taking advantage of fusion tags determined during the production process that optimize for both yield and biological activity preservation.

What are the recommended experimental designs for determining SAR1937 membrane localization?

Researchers investigating SAR1937 membrane localization should employ fluorescent tagging approaches similar to those used for other membrane proteins. By creating SAR1937-GFP fusion constructs, researchers can track protein localization in real-time under various experimental conditions. Based on methodologies applied to other membrane proteins like Atg27-GFP , temperature-sensitive mutants can be particularly valuable in understanding trafficking pathways. For instance, researchers could generate temperature-sensitive strains (similar to vps35ts) where SAR1937 localization is monitored at permissive (26°C) versus non-permissive (37°C) temperatures to identify factors affecting membrane localization.

A comprehensive experimental design would include:

• Construction of SAR1937-GFP fusion protein

• Expression in appropriate staphylococcal strains

• Live-cell imaging at different time points and under various conditions

• Subcellular fractionation followed by Western blotting to confirm localization

• Co-localization studies with known membrane markers

What turbidity reduction assay protocols are most appropriate for assessing the antimicrobial activity of SAR1937?

The turbidity reduction assay for SAR1937 should follow protocols similar to those established for other staphylococcal proteins. Start with exponentially growing S. aureus cultures (OD600 = 0.5) in TSB medium, harvest by centrifugation (16,000 × g, 5 min), wash twice and resuspend in 50 mM NaPi buffer (pH 7.4) to a concentration of ~10^7 CFU/ml . Prepare a test suspension containing 10^6 CFU bacterial cells with varying concentrations of SAR1937 protein (typically starting with 0.1 μM) in a total volume of 5 ml, then incubate at 37°C with shaking at 200 rpm.

At defined time intervals (2, 5, 10, 15, 30, 45, and 60 minutes), collect 50 μl samples and immediately add 0.15 μg of proteinase K to halt the reaction . Plate appropriate dilutions on TSA and incubate at 37°C for 16 hours. Calculate antimicrobial activity as the relative inactivation in log units [log10(N0/Ni)], where N0 represents the initial number of untreated cells and Ni represents the number of residual cells after treatment. All experiments should be performed in triplicate biological replicates to ensure statistical validity.

What are the optimal conditions for conducting minimum inhibitory concentration (MIC) assays with SAR1937?

The optimal MIC determination for SAR1937 should follow the conventional broth microdilution technique in tryptic soy broth (TSB) according to Clinical and Laboratory Standards Institute (CLSI) guidelines . Prepare two-fold serial dilutions of purified SAR1937 protein in a 96-well microtiter plate. Add 10^5 CFU/well of the target staphylococcal strains to each well containing the diluted protein. Include appropriate controls: positive control (bacteria without protein), negative control (media only), and comparative control (a well-characterized antimicrobial agent).

Incubate the plates at 37°C for 24 hours and determine the MIC as the lowest protein concentration that completely inhibits visible bacterial growth . For rigorous analysis, perform three independent biological replicates and report the mode value as the final MIC. This methodology allows for standardized comparison of SAR1937's antimicrobial activity against other antimicrobial agents and across different bacterial strains, particularly clinical isolates of S. aureus including methicillin-resistant strains.

How can researchers effectively evaluate the membrane transport mechanisms associated with SAR1937?

To evaluate SAR1937 membrane transport mechanisms, researchers should implement a multi-faceted approach combining genetic and biochemical techniques. Start by generating conditional mutants with temperature-sensitive phenotypes similar to the vps35ts system used for other membrane proteins . This allows controlled disruption of potential trafficking pathways. Additionally, systematically delete genes involved in membrane protein recycling (such as snx4Δ, vps35Δ) and assess their impact on SAR1937 localization and function.

For biochemical characterization, utilize liposome reconstitution assays to directly measure transport activity. Purified SAR1937 can be reconstituted into liposomes with fluorescent substrates to monitor potential transport across membranes. Complement these approaches with:

• Site-directed mutagenesis of key residues in predicted functional domains

• Crosslinking studies to identify interaction partners

• Proteoliposome-based electrophysiology to characterize channel properties (if applicable)

• Molecular dynamics simulations to predict substrate binding sites and transport mechanisms

Combined, these approaches would provide comprehensive insights into the transport mechanisms of SAR1937 that go beyond simple localization studies.

What techniques should be employed to assess the role of SAR1937 in biofilm formation and how does this compare to other staphylococcal membrane proteins?

Investigation of SAR1937's role in biofilm formation requires both prevention and disruption studies, similar to methodologies used for other staphylococcal proteins. For prevention assays, grow S. aureus strain 15981 in TSB supplemented with 0.25% glucose (TSBg) in the presence of different concentrations (0.002–2.5 μM) of SAR1937 . After 24 hours incubation at 37°C, remove the planktonic phase, wash gently with 50 mM NaPi buffer (pH 7.4), and quantify biofilm biomass using crystal violet staining (0.1% w/v for 15 minutes) .

For disruption assays, first establish 8-hour preformed biofilms of S. aureus 15981, then treat with SAR1937 at concentrations 10 times higher than the minimal concentration that prevented biofilm formation . Compare the efficacy of SAR1937 with other known biofilm-disrupting agents using the following parameters:

Additionally, employ confocal laser scanning microscopy with fluorescently labeled bacteria (e.g., S. aureus 15981 pSB2019 expressing GFP) to visualize biofilm architecture before and after SAR1937 treatment .

What are the recommended approaches for investigating SAR1937 interactions with host cell membranes in infection models?

To investigate SAR1937 interactions with host cell membranes, utilize both in vitro and ex vivo models similar to those established for other staphylococcal proteins. Begin with a Franz Cell diffusion system using pig skin explants to assess protein penetration capabilities . Label SAR1937 with fluorescent markers (such as fluorescein) and analyze its distribution across different skin layers after 6 hours of application using fluorescence spectroscopy (excitation/emission 494/518 nm) .

For cellular interaction studies, establish an in vitro wound infection model using human keratinocyte HaCaT cell monolayers . Grow HaCaT monolayers in 16-well E-plates, create a mechanical wound with a sterile plastic tip, and introduce S. aureus 15981. Monitor real-time cellular responses with and without SAR1937 treatment, including:

• Cell viability using MTT or XTT assays

• Membrane integrity via LDH release assays

• Inflammatory responses through cytokine profiling (IL-6, IL-8, TNF-α)

• Bacterial adherence and internalization using gentamicin protection assays

• Real-time cellular impedance measurements to quantify cell-SAR1937 interactions

These approaches will provide mechanistic insights into how SAR1937 interacts with host cell membranes during infection and potential therapeutic applications.

How should researchers interpret contradicting results between in vitro and ex vivo studies involving SAR1937?

When confronted with contradictory results between in vitro and ex vivo studies of SAR1937, researchers should implement a systematic analytical approach. First, analyze the specific experimental conditions that might explain the discrepancies, including differences in protein concentration, buffer composition, temperature, and experimental timeframes. For example, SAR1937 may demonstrate different activity profiles in simplified in vitro systems compared to the complex environment of ex vivo skin models .

Researchers should consider the following factors when reconciling contradictory results:

• Protein stability under different experimental conditions - test SAR1937 stability at various temperatures (20-50°C) and pH ranges (3-9) to determine if environmental factors in ex vivo systems might compromise activity

• Matrix effects - assess whether components in ex vivo systems (e.g., extracellular matrix proteins, lipids) interact with SAR1937 using pull-down assays and co-immunoprecipitation studies

• Biological variability - increase biological replicates and perform power analysis to determine if apparent contradictions fall within expected statistical variations

• Methodological limitations - develop hybrid systems that bridge the complexity gap between in vitro and ex vivo models, such as 3D cell culture models with controlled matrix composition

• Fractional factorial design experiments to systematically isolate variables that cause result discrepancies

The most valuable interpretations will acknowledge limitations of both systems and prioritize results that can be reproduced across multiple experimental platforms.

What statistical approaches are most appropriate for analyzing SAR1937 activity across different staphylococcal strains?

When analyzing SAR1937 activity across multiple staphylococcal strains, researchers should employ robust statistical methods that account for strain variability and experimental conditions. For MIC analyses across different strains, non-parametric tests like the Kruskal-Wallis test followed by Dunn's multiple comparison are preferred since MIC data often do not follow normal distributions .

For time-kill kinetics studies, two-way ANOVA with repeated measures is appropriate to analyze the effects of both strain and time on bacterial survival. When comparing biofilm inhibition or disruption across strains, researchers should normalize data to untreated controls for each strain before performing statistical analyses to account for inherent differences in biofilm-forming capacity.

A comprehensive statistical approach should include:

Additionally, researchers should report effect sizes alongside p-values to provide a more complete picture of SAR1937's biological significance across different staphylococcal strains.

What strategies can researchers employ when SAR1937 shows inconsistent activity in antibiofilm assays?

When SAR1937 demonstrates inconsistent activity in antibiofilm assays, researchers should implement a systematic troubleshooting approach. First, standardize the biofilm growth conditions by ensuring consistent inoculum preparation (106 CFU/ml from exponential phase cultures) and growth media composition (TSB supplemented with precisely 0.25% w/v D-(+)-glucose) . Small variations in glucose concentration can significantly impact biofilm formation.

Additional troubleshooting strategies include:

• Protein quality assessment: Verify SAR1937 purity using SDS-PAGE and activity using standardized turbidity reduction assays before each experiment

• Technical standardization: Ensure consistent washing steps (two gentle washes with sterile 50 mM NaPi buffer, pH 7.4) and staining procedures (15 minutes with 0.1% w/v crystal violet)

• Environmental control: Monitor and maintain consistent temperature (37°C) and humidity during incubation periods

• Strain verification: Confirm the identity and genetic stability of S. aureus strains used in experiments through whole-genome sequencing or strain-specific PCR

• Medium batch variability: Prepare larger batches of media from the same components to reduce experimental variability

• Statistical robustness: Increase the number of biological replicates (minimum n=6) and incorporate technical replicates within each biological replicate

If inconsistencies persist, researchers should consider developing a standardized reference biofilm system using well-characterized S. aureus strains like 15981 or MRSA252 against which all experiments can be normalized.

How can researchers address challenges in maintaining SAR1937 stability during purification and experimental procedures?

Maintaining SAR1937 stability during purification and experiments represents a significant challenge due to its membrane protein nature. Researchers should implement a comprehensive stability optimization approach beginning with expression system selection. For purification, incorporate stability-enhancing additives such as 50% glycerol in storage buffers and optimize the Tris-based buffer system to maintain proper protein folding .

Implement the following stability-enhancing strategies:

• Temperature management: Process all purification steps at 4°C and maintain a cold chain throughout the experimental workflow

• Protease inhibitors: Include a complete protease inhibitor cocktail during cell lysis and initial purification steps

• Reducing agents: Add appropriate reducing agents (DTT or β-mercaptoethanol) to prevent oxidation of cysteine residues

• Detergent selection: Screen multiple detergents (DDM, CHAPS, Triton X-100) at various concentrations to identify optimal solubilization conditions

• Buffer optimization: Perform thermal shift assays to identify buffer compositions that maximize protein stability

• Aliquoting strategy: Prepare single-use aliquots to avoid freeze-thaw cycles which are particularly detrimental to membrane proteins

• Quality control: Implement regular quality control checkpoints using activity assays, circular dichroism, or fluorescence spectroscopy to monitor protein folding

For specific experiments that require extended incubation times, consider employing on-demand protein production systems or developing stabilized SAR1937 variants through protein engineering approaches.

What novel experimental approaches could reveal unexplored functions of SAR1937 in staphylococcal physiology?

To uncover novel functions of SAR1937, researchers should consider implementing cutting-edge experimental approaches that go beyond traditional assays. CRISPR interference (CRISPRi) systems specifically adapted for S. aureus would allow for precise temporal control of SAR1937 expression, revealing phenotypes that might be masked in complete knockout studies due to compensatory mechanisms.

Innovative approaches to explore SAR1937 function include:

• Proximity-dependent biotin identification (BioID) coupled with mass spectrometry to identify SAR1937 protein interaction networks in living cells

• Single-cell transcriptomics to identify cellular subpopulations with differential responses to SAR1937 modulation

• Metabolomic profiling using LC-MS/MS to detect metabolic shifts associated with SAR1937 function or dysfunction

• Synthetic genetic array analysis to identify genetic interactions and place SAR1937 within functional pathways

• Cryo-electron microscopy to determine high-resolution structure of SAR1937 within native membrane environments

• Microfluidic-based phenotypic screening to identify conditions where SAR1937 becomes essential for survival

• Exploration of SAR1937 roles in phospholipid regulation pathways, building on knowledge from other membrane proteins that require PI3P for proper trafficking

These approaches could potentially connect SAR1937 to unexplored aspects of staphylococcal membrane homeostasis, virulence regulation, or antibiotic resistance mechanisms.

How might researchers leverage SAR1937 knowledge to develop novel antimicrobial strategies against Staphylococcus aureus?

Leveraging SAR1937 knowledge for antimicrobial development requires a translational research approach that builds upon fundamental understanding of the protein's structure and function. Researchers could employ structure-based drug design to identify small molecules that selectively bind to SAR1937, potentially disrupting essential membrane functions. This approach would begin with high-resolution structural determination through X-ray crystallography or cryo-EM.

Promising translational research strategies include:

• Development of antibody-antibiotic conjugates targeting SAR1937 extracellular epitopes for selective delivery of antimicrobial compounds

• Design of peptidomimetics that compete with SAR1937 for essential interaction partners

• Exploitation of SAR1937's membrane localization to create membrane-disrupting agents that specifically recognize and bind to this protein

• Creation of PROTAC (proteolysis targeting chimera) molecules that target SAR1937 for degradation by the bacterial proteolytic machinery

• Implementation of anti-virulence strategies that don't kill bacteria but neutralize pathogenicity by modulating SAR1937 function

These approaches could be particularly valuable for addressing methicillin-resistant S. aureus (MRSA) infections, where conventional antibiotics are increasingly ineffective . By targeting a membrane protein that may be essential for bacterial virulence rather than growth, these strategies could potentially reduce selection pressure for resistance development.