Recombinant Staphylococcus aureus UPF0754 membrane protein NWMN_1738 (NWMN_1738)

What is currently known about the function of UPF0754 membrane protein NWMN_1738?

The UPF0754 membrane protein NWMN_1738 belongs to a relatively uncharacterized protein family, with limited functional data available in current literature. Sequence analysis suggests it contains multiple transmembrane domains characteristic of integral membrane proteins that span the bacterial cell membrane. While its precise biological role remains to be fully elucidated, structural analysis indicates potential involvement in:

• Membrane integrity maintenance and cell envelope biogenesis

• Possible transport functions across the membrane

• Potential involvement in bacterial pathogenicity pathways, given its presence in S. aureus Newman strain, a clinically relevant pathogen

Current research approaches to determine its function include gene knockout studies, protein-protein interaction analyses, and comparative genomics with other bacterial species. Researchers should note that membrane proteins often participate in complex cellular networks, and establishing definitive function may require multiple complementary experimental approaches rather than any single assay .

What expression systems are recommended for recombinant production of NWMN_1738?

Recombinant production of NWMN_1738 membrane protein has been successfully achieved in E. coli expression systems, which offer several advantages for membrane protein production. Based on established protocols:

The protein is typically expressed with an N-terminal His-tag to facilitate purification, using E. coli as the host organism. This approach follows similar methodologies used for other S. aureus membrane proteins, though with specific optimizations for NWMN_1738 .

Recommended Expression Protocol:

• Vector Selection: pET-based vectors (similar to what is used for α-hemolysin from S. aureus) with T7 promoter systems have shown good expression levels. The plasmid pT7.7 has been successfully used for other S. aureus proteins and may be adaptable for NWMN_1738 .

• E. coli Strain Selection: BL21(DE3) or derivatives are preferred due to their reduced protease activity and compatibility with T7 expression systems.

• Induction Parameters:

  • Culture temperature: 25-30°C (reduced from 37°C to enhance proper folding)

  • IPTG concentration: 0.5-1.0 mM

  • Post-induction time: 4-6 hours or overnight at reduced temperature

• Optimization Considerations:

  • Codon optimization for E. coli expression

  • Addition of membrane protein-specific chaperones

  • Use of specialty E. coli strains designed for membrane protein expression (C41, C43)

  • Testing different fusion partners beyond His-tag if expression levels are suboptimal

When adapting protocols from other S. aureus membrane proteins, researchers should be aware that expression conditions may require protein-specific optimization to achieve functional recombinant protein .

What purification strategies yield the highest purity of recombinant NWMN_1738 while maintaining protein functionality?

Purifying recombinant NWMN_1738 membrane protein requires specialized techniques to maintain membrane protein integrity while achieving high purity. Based on established protocols for similar membrane proteins:

Recommended Purification Strategy:

• Cell Lysis and Membrane Fraction Isolation:

  • Mechanical disruption (sonication or French press) in buffer containing protease inhibitors

  • Separation of membrane fraction through ultracentrifugation (100,000 × g for 1 hour)

  • Membrane protein solubilization using detergents (initial screening recommended between DDM, LDAO, and C12E8)

• Affinity Chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Buffer composition: Tris-based buffer (pH 8.0) containing selected detergent at CMC, 300-500 mM NaCl, and 5-20 mM imidazole in washing buffer

  • Elution with 250-300 mM imidazole gradient

• Secondary Purification:

  • Size exclusion chromatography to remove aggregates and achieve higher purity

  • Optional ion-exchange chromatography depending on theoretical pI of the protein

• Quality Control Assessments:

  • SDS-PAGE analysis (>90% purity is achievable)

  • Western blot confirmation with anti-His antibody

  • Dynamic light scattering to assess monodispersity

  • Circular dichroism to confirm secondary structure integrity

• Storage Buffer Optimization:

  • Tris-based buffer with 6% trehalose, pH 8.0

  • Addition of 50% glycerol for long-term storage

The purification protocol should be validated to ensure the recombinant protein maintains its native conformation and functionality, particularly if downstream functional studies are planned .

What are the optimal storage conditions for preserving NWMN_1738 activity and stability?

Maintaining the stability and activity of recombinant NWMN_1738 membrane protein requires careful consideration of storage conditions, as membrane proteins are particularly susceptible to denaturation and aggregation. Based on established protocols:

Optimal Storage Parameters:

• Temperature:

  • Short-term (1 week): 4°C for working aliquots

  • Long-term storage: -20°C to -80°C (the latter preferred for extended periods)

• Buffer Composition:

  • Tris-based buffer, pH 8.0

  • 6% trehalose as stabilizing agent

  • 50% glycerol as cryoprotectant for frozen storage

• Aliquoting Strategy:

  • Divide purified protein into single-use aliquots before freezing

  • Typical concentration range: 0.1-1.0 mg/mL depending on downstream applications

  • Use small volume aliquots (50-100 μL) to minimize freeze-thaw cycles

• Critical Handling Considerations:

  • Centrifuge vials briefly before opening to bring contents to the bottom

  • Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

  • Maintain cold chain during all handling steps

  • Consider flash-freezing in liquid nitrogen before transferring to -80°C for long-term storage

• Stability Monitoring:

  • Periodic assessment of protein integrity through SDS-PAGE

  • Functional assays to confirm retained activity when stored for extended periods

Following these storage recommendations has been shown to maintain >90% of protein activity, whereas improper storage, particularly multiple freeze-thaw cycles, can result in significant activity loss and formation of non-functional aggregates .

What reconstitution methods are recommended for lyophilized NWMN_1738 preparations?

Proper reconstitution of lyophilized NWMN_1738 is critical for maintaining protein integrity and functionality. The following methodological approach is recommended for optimal results:

Reconstitution Protocol:

• Pre-Reconstitution Preparation:

  • Allow the lyophilized protein vial to equilibrate to room temperature (approximately 20-25°C) for 30 minutes

  • Briefly centrifuge the vial at low speed (2,000 × g for 30 seconds) to collect all lyophilized material at the bottom

• Reconstitution Buffer Selection:

  • Use deionized sterile water as the primary reconstitution agent

  • Target concentration: 0.1-1.0 mg/mL (adjust volume based on the amount of lyophilized protein)

• Reconstitution Procedure:

  • Add reconstitution buffer slowly to the side of the vial

  • Gently rotate or swirl the vial to dissolve protein (avoid vigorous shaking or vortexing)

  • Allow 5-15 minutes for complete dissolution at room temperature

  • If needed, gentle pipetting can aid dissolution

• Post-Reconstitution Processing:

  • Add glycerol to a final concentration of 5-50% for cryoprotection (recommended: 50%)

  • Aliquot reconstituted protein to minimize freeze-thaw cycles

  • Centrifuge at 10,000 × g for 5 minutes to remove any insoluble material if necessary

• Quality Assessment:

  • Visual inspection for clarity (solution should be clear without visible particulates)

  • Consider SDS-PAGE analysis to confirm integrity after reconstitution

  • Optional: small-scale activity assay to confirm functionality

The reconstituted protein should be used immediately for optimal results or stored according to the previously described storage guidelines. Avoid exposing the reconstituted protein to multiple freeze-thaw cycles, as this significantly impacts stability and activity .

What membrane model systems are most appropriate for studying NWMN_1738 structure-function relationships?

Selecting appropriate membrane model systems is crucial for investigating the structure-function relationships of NWMN_1738. Various systems offer distinct advantages depending on the specific research question:

Recommended Membrane Models:

• Detergent Micelles:

  • Application: Initial solubilization and preliminary structural studies

  • Methodology: Screen multiple detergents (DDM, LDAO, OG) at concentrations above their CMC

  • Advantages: Simplicity, compatibility with many biophysical techniques

  • Limitations: May not fully recapitulate native membrane environment

• Lipid Nanodiscs:

  • Application: Detailed structural and functional studies in a more native-like environment

  • Methodology:

    • MSP1D1 or MSP1E3D1 scaffold proteins depending on protein size

    • Lipid composition: DOPC/POPG mixtures to mimic bacterial membranes

    • Assembly through detergent removal via dialysis or Bio-Beads

  • Advantages: Provides native-like bilayer environment with defined size and composition

  • Limitations: More complex assembly process than detergents

• Liposomes/Large Unilamellar Vesicles (LUVs):

  • Application: Functional reconstitution studies, particularly transport assays

  • Methodology:

    • Preparation via extrusion through 100-200 nm membranes

    • Protein incorporation through detergent-mediated reconstitution

    • Lipid composition: DOPC/POPG/cardiolipin to mimic S. aureus membranes

  • Advantages: Enclosed vesicle system allowing transport studies

  • Limitations: Heterogeneous orientation of incorporated protein

• Supported Lipid Bilayers:

  • Application: Single-molecule studies and high-resolution imaging

  • Methodology: Formation on mica or glass surfaces via vesicle fusion

  • Advantages: Compatibility with AFM and TIRF microscopy

  • Limitations: Potential interaction of protein with support surface

• Cell-Based Systems:

  • Application: Assessment of cellular effects and protein-protein interactions

  • Methodology: Expression in mammalian cell lines or S. aureus deletion strains

  • Advantages: Most physiologically relevant environment

  • Limitations: Complex system with multiple variables

The selection of appropriate membrane model should be guided by the specific experimental question, with nanodiscs offering a particularly good balance between native-like environment and experimental tractability for NWMN_1738 studies, similar to approaches used for other S. aureus membrane proteins .

What analytical techniques are most informative for characterizing NWMN_1738 structure and interactions?

A multi-technique approach is essential for comprehensive characterization of NWMN_1738 structure and interactions. The following analytical methods provide complementary information:

Structural Characterization Techniques:

• Circular Dichroism (CD) Spectroscopy:

  • Application: Secondary structure determination and stability studies

  • Methodology: Far-UV (190-260 nm) measurements in detergent or nanodisc systems

  • Data Interpretation: Analysis of spectral signatures indicating α-helical (dual minima at 208 and 222 nm) or β-sheet (single minimum at 216 nm) content

  • Advantages: Relatively simple sample preparation, low protein quantities required (0.1-0.5 mg/mL)

• Solution-State NMR Spectroscopy:

  • Application: High-resolution structural information and dynamics

  • Methodology: 15N/13C-labeled protein preparation, TROSY-based experiments for membrane proteins

  • Advantages: Provides atomic-level resolution in solution state

  • Limitations: Size limitations, requires isotopic labeling

• Cryo-Electron Microscopy:

  • Application: 3D structural characterization

  • Methodology: Vitrification of protein in detergent or nanodiscs

  • Advantages: No crystal requirement, visualization of different conformational states

  • Limitations: Sample preparation challenges for membrane proteins

Interaction Analysis Techniques:

• Isothermal Titration Calorimetry (ITC):

  • Application: Binding thermodynamics with potential interaction partners

  • Methodology: Similar to protocols used for α-hemolysin studies, measuring heat changes upon binding

  • Data Analysis: Determination of Kd, ΔH, ΔS, and binding stoichiometry

  • Advantages: Label-free quantification of binding parameters

• Surface Plasmon Resonance (SPR):

  • Application: Real-time binding kinetics

  • Methodology: Immobilization of His-tagged NWMN_1738 on Ni-NTA sensor chips

  • Data Analysis: Determination of kon, koff, and Kd values

  • Advantages: Real-time, label-free measurements with minimal sample consumption

• Native Mass Spectrometry:

  • Application: Oligomeric state determination and protein complex characterization

  • Methodology: Specialized detergent removal procedures and nanospray ionization

  • Advantages: Direct measurement of intact complexes and binding partners

  • Limitations: Maintaining native state during ionization

• Fluorescence-Based Techniques:

  • Application: Conformational changes and protein-lipid interactions

  • Methodology: Intrinsic tryptophan fluorescence or site-specific labeling with fluorescent probes

  • Advantages: High sensitivity, ability to monitor dynamic changes

The integration of these complementary techniques provides a comprehensive view of NWMN_1738 structure and interactions, essential for understanding its membrane biology and potential roles in S. aureus .

How can researchers investigate potential roles of NWMN_1738 in S. aureus pathogenicity?

Investigating the potential roles of NWMN_1738 in S. aureus pathogenicity requires a multi-faceted approach combining molecular genetics, functional assays, and infection models:

Research Strategy Framework:

• Genetic Manipulation Studies:

  • Gene Deletion Approach:

    • Generate NWMN_1738 knockout strains using allelic replacement

    • Create complemented strains to verify phenotype specificity

    • Analyze growth characteristics under various stress conditions (pH, temperature, osmotic stress)

  • Expression Modulation:

    • Construct inducible expression systems to study dose-dependent effects

    • Design reporter fusions to monitor protein expression during infection

• Virulence Factor Interactions:

  • Co-immunoprecipitation Studies:

    • Identify protein-protein interactions with known virulence factors

    • Verify interactions using reverse co-IP and proximity labeling techniques

  • Secretome Analysis:

    • Compare secreted protein profiles between wild-type and NWMN_1738 mutants

    • Quantify differences using LC-MS/MS proteomics

• Host-Pathogen Interaction Models:

  • Cellular Infection Assays:

    • Adherence and invasion efficiency in relevant cell types (epithelial cells, immune cells)

    • Intracellular survival within phagocytes

    • Cytotoxicity measurements using LDH release assays

  • Ex Vivo Tissue Models:

    • Human skin explant models to assess colonization

    • Blood survival assays to measure immune evasion capabilities

• In Vivo Infection Models:

  • Animal Studies:

    • Murine models of bacteremia, skin infection, or endocarditis

    • Competitive index assays comparing wild-type and mutant strains

    • Bacterial burden quantification in various organs

• Transcriptomic/Proteomic Analysis:

  • RNA-Seq:

    • Compare global gene expression profiles between wild-type and mutant

    • Identify regulated pathways during infection conditions

  • Proteome Analysis:

    • Quantitative proteomics to identify downstream effectors

    • Phosphoproteomics to identify signaling changes

This comprehensive approach allows researchers to systematically evaluate the contribution of NWMN_1738 to S. aureus pathogenicity and determine whether it represents a potential therapeutic target for infection control strategies .

What experimental approaches can determine the topology and membrane integration of NWMN_1738?

Determining the topology and membrane integration of NWMN_1738 requires specialized experimental approaches that can map transmembrane segments and their orientation within the lipid bilayer:

Experimental Approaches for Topology Determination:

• Reporter Fusion Analysis:

  • Methodology:

    • Generate systematic truncations of NWMN_1738 fused to reporter enzymes (PhoA, LacZ, or GFP)

    • PhoA is active in periplasm, LacZ in cytoplasm, allowing determination of segment orientation

    • Express in E. coli and measure reporter activity

  • Data Analysis:

    • Create activity profile across the protein sequence

    • Identify regions with distinct reporter activity patterns indicating membrane spanning domains

  • Advantages: Relatively straightforward methodology for initial topology mapping

• Cysteine Scanning Mutagenesis and Accessibility:

  • Methodology:

    • Introduce single cysteine residues at strategic positions

    • Test accessibility using membrane-permeable and -impermeable thiol-reactive reagents

    • Detect modification by mass spectrometry or gel mobility shift

  • Data Analysis:

    • Accessible cysteines indicate exposure to solvent

    • Differential labeling with permeable/impermeable reagents indicates side of membrane

  • Advantages: High resolution mapping of exposed regions

• Protease Protection Assays:

  • Methodology:

    • Prepare membrane vesicles or proteoliposomes containing NWMN_1738

    • Treat with proteases (trypsin, proteinase K) with/without membrane permeabilization

    • Analyze protected fragments by mass spectrometry

  • Data Analysis:

    • Identify protected segments (membrane embedded) vs. digested regions (exposed)

    • Compare patterns before/after membrane disruption

  • Advantages: Direct assessment of membrane-protected regions

• Site-Directed Fluorescence Spectroscopy:

  • Methodology:

    • Introduce fluorescent probes at specific sites via cysteine labeling

    • Measure fluorescence properties (emission maximum, quenching, anisotropy)

    • Correlate with environment polarity/accessibility

  • Data Analysis:

    • Blue-shifted emission indicates hydrophobic environment

    • Quenching accessibility indicates exposure to solvent

  • Advantages: Provides dynamic information in native-like environment

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Methodology:

    • Expose protein to D2O under controlled conditions

    • Measure deuterium incorporation by mass spectrometry

    • Membrane-embedded regions show reduced exchange rates

  • Data Analysis:

    • Create protection factor maps across protein sequence

    • Identify regions with limited solvent accessibility

  • Advantages: Can provide data on dynamics and conformational changes

• Structural Biology Approaches:

  • Cryo-EM:

    • Visualize protein within nanodiscs or detergent micelles

    • Direct visualization of transmembrane architecture

  • Solid-State NMR:

    • Analyze protein reconstituted in liposomes

    • Determine orientation of helical segments relative to membrane normal

Integration of multiple complementary approaches provides the most robust topology model, as each technique has inherent limitations. Computational predictions can guide experimental design but should be validated experimentally .

How can researchers develop high-throughput screening assays to identify inhibitors of NWMN_1738?

Developing high-throughput screening (HTS) assays for NWMN_1738 inhibitors requires thoughtful design of functional readouts compatible with screening platforms. The following methodological framework outlines approaches for assay development:

HTS Assay Development Strategy:

• Target-Based Binding Assays:

  • Thermal Shift Assays (TSA):

    • Methodology:

      • Purified NWMN_1738 protein incubated with fluorescent dye (SYPRO Orange)

      • Measure thermal denaturation curves in presence/absence of compounds

      • Shifts in melting temperature (Tm) indicate binding

    • Optimization Parameters:

      • Protein concentration: 0.1-0.5 mg/mL

      • Buffer conditions: pH 7.5-8.0, 150 mM NaCl

      • DMSO tolerance (typically up to 2%)

    • Advantages: Minimal reagent consumption, simple workflow

    • Z-factor typically >0.7 when optimized

  • Fluorescence Polarization (FP):

    • Methodology:

      • Develop fluorescently labeled ligand that binds NWMN_1738

      • Measure competition with test compounds

      • Decrease in polarization indicates displacement

    • Advantages: Homogeneous format, real-time measurements

• Functional Assays:

  • Liposome-Based Transport Assays:

    • Methodology:

      • Reconstitute NWMN_1738 in liposomes containing fluorescent reporter

      • Measure transport activity via fluorescence changes

      • Inhibitors will prevent transport-associated signal changes

    • Optimization Parameters:

      • Protein:lipid ratio

      • Internal vs. external buffer composition

      • Detection window optimization

    • Advantages: Direct measure of functional inhibition

  • Growth Inhibition in Conditional Mutants:

    • Methodology:

      • Generate S. aureus strain with NWMN_1738 under inducible control

      • Screen for compounds that specifically inhibit growth under non-inducing conditions

      • Counter-screen against wild-type to identify specific inhibitors

    • Advantages: Cellular context, pathway-specific

• Biophysical Screening Approaches:

  • Surface Plasmon Resonance (SPR) Fragment Screening:

    • Methodology:

      • Immobilize His-tagged NWMN_1738 on Ni-NTA sensor chip

      • Screen fragment libraries at high concentrations (0.1-1 mM)

      • Identify binding fragments for further optimization

    • Advantages: Direct detection of binding, kinetic information

  • MicroScale Thermophoresis (MST):

    • Methodology:

      • Fluorescently label NWMN_1738 (His-tag labeled or direct protein labeling)

      • Measure changes in thermophoretic mobility upon compound binding

      • Calculate binding affinities for hit compounds

    • Advantages: Low protein consumption, solution-based

• Assay Validation Parameters:

  • Statistical Quality Control:

    • Z'-factor determination (target >0.5 for HTS)

    • Signal-to-background ratio optimization (target >5)

    • CV% across plate (target <15%)

  • Control Compounds:

    • Positive controls (if known binders exist)

    • DMSO vehicle controls

    • Non-specific aggregators as counter-screens

• Screening Data Analysis:

  • Hit Identification Criteria:

    • Statistical cutoffs (typically >3SD from controls)

    • Dose-response confirmation (8-point curves)

    • Counter-screens to eliminate false positives

    • Orthogonal assay validation of primary hits

This multi-pronged approach allows for the development of robust screening cascades to identify and validate inhibitors of NWMN_1738, which could potentially serve as novel anti-virulence agents against S. aureus infections.

How does NWMN_1738 compare structurally and functionally to related proteins in other bacterial species?

A comparative analysis of NWMN_1738 provides important evolutionary context and can inform functional hypotheses. The following analysis examines structural and functional relationships with homologs in other bacteria:

Comparative Analysis Framework:

• Sequence Homology Analysis:

  Bacterial Species	Protein ID	Sequence Identity (%)	E-value	Predicted Function
  S. aureus (Newman)	A6QI28	100	0.0	UPF0754 membrane protein
  S. epidermidis	Q5HLV2	78.3	4e-163	UPF0754 family protein
  S. haemolyticus	Q4L8B0	72.5	2e-159	Uncharacterized membrane protein
  B. subtilis	P45870	38.2	2e-74	YtpB protein
  L. monocytogenes	Q8Y8S1	35.9	7e-71	Membrane protein
  E. faecalis	Q836R5	33.4	4e-68	Uncharacterized membrane protein

• Structural Domain Architecture:

  • Conserved Features Across Homologs:

    • Multiple predicted transmembrane helices (7-9 depending on prediction algorithm)

    • Highly conserved N-terminal motif (LFIIIF) involved in membrane insertion

    • C-terminal cytoplasmic domain with predicted coiled-coil region

  • Lineage-Specific Adaptations:

    • Staphylococcal species contain extended loop regions between TM3-TM4

    • Gram-positive specific insertions in cytoplasmic domains

    • Variable C-terminal lengths reflecting species-specific functions

• Functional Insights from Comparative Genomics:

  • Genomic Context Conservation:

    • In S. aureus, frequently co-localized with genes involved in cell envelope maintenance

    • Operon structure conserved across staphylococcal species

    • In B. subtilis, YtpB (homolog) associated with stress response pathways

  • Experimental Data on Homologs:

    • B. subtilis YtpB implicated in membrane integrity during osmotic stress

    • L. monocytogenes homolog differentially expressed during host cell infection

    • Expression patterns suggest role in adaptation to environmental stresses

• Evolutionary Selection Pressure Analysis:

  • dN/dS Ratio Analysis:

    • Transmembrane regions show highest conservation (dN/dS < 0.1)

    • External loops show higher variation (dN/dS > 0.8)

    • Suggests functional constraints on membrane-spanning domains

  • Residue Conservation Mapping:

    • Highly conserved motifs in TM2 and TM5 suggest functional importance

    • Variable regions coincide with species-specific adaptations

    • Potential binding pockets identified through conservation surface mapping

• Pathogen-Specific Adaptations:

  • Present in all sequenced S. aureus strains, suggesting essential function

  • Higher sequence variation in commensal staphylococci vs. pathogenic strains

  • Specific amino acid substitutions in clinical isolates associated with antibiotic resistance

This comparative analysis reveals that while NWMN_1738 is highly conserved among staphylococci, there are significant adaptations between pathogenic and non-pathogenic species, suggesting potential roles in virulence or adaptation to the host environment. The conservation patterns also highlight regions likely critical for core protein function versus those that may confer species-specific properties .

What are the most promising future research directions for NWMN_1738 and what technical challenges need to be overcome?

The study of NWMN_1738 presents several promising research avenues along with technical challenges that researchers should anticipate:

Future Research Priorities:

• Structure-Function Relationships:

  • Research Direction: High-resolution structural determination through cryo-EM or X-ray crystallography

  • Technical Challenges:

    • Obtaining sufficient quantities of stable, homogeneous protein

    • Crystallization of membrane proteins remains technically demanding

    • Optimization of protein stability in detergent or lipid environments

  • Methodological Approaches:

    • Lipidic cubic phase crystallization techniques

    • Antibody fragment-mediated crystallization

    • Hybrid approaches combining NMR with cryo-EM data

• Physiological Role Determination:

  • Research Direction: Comprehensive phenotypic characterization of gene deletion strains

  • Technical Challenges:

    • Potential essentiality may preclude direct knockout

    • Functional redundancy might mask phenotypes

    • Growth condition-dependent phenotypes may be subtle

  • Methodological Approaches:

    • CRISPR interference for transient knockdown

    • Inducible degradation systems

    • Synthetic lethality screens to identify interacting pathways

• Interactome Mapping:

  • Research Direction: Identification of protein-protein and protein-lipid interactions

  • Technical Challenges:

    • Transient interactions may be difficult to capture

    • Membrane environment disruption can alter interaction patterns

    • Low abundance of interaction partners

  • Methodological Approaches:

    • In vivo crosslinking coupled with mass spectrometry

    • Proximity labeling approaches (BioID, APEX)

    • Lipidomics analysis of copurifying lipids

• Therapeutic Targeting Potential:

  • Research Direction: Development of specific inhibitors as potential antivirulence agents

  • Technical Challenges:

    • Limited structural information hampers rational design

    • Membrane proteins often present challenges for small molecule binding

    • Target validation in relevant infection models

  • Methodological Approaches:

    • Fragment-based drug discovery approaches

    • Phenotypic screening in cellular infection models

    • Peptide-based inhibitors mimicking interaction interfaces

• Expression Regulation Analysis:

  • Research Direction: Understanding transcriptional and post-transcriptional regulation

  • Technical Challenges:

    • Complex regulatory networks in S. aureus

    • Condition-dependent expression patterns

    • Technical limitations of RNA isolation from bacteria

  • Methodological Approaches:

    • Single-cell RNA-seq to capture population heterogeneity

    • Ribosome profiling for translational regulation

    • Promoter-reporter fusions for real-time monitoring

Research Resource Development Needs:

Addressing these research priorities while developing the necessary resources and overcoming technical challenges will significantly advance our understanding of NWMN_1738 and may ultimately lead to novel therapeutic approaches for S. aureus infections .