Recombinant Salmonella enteritidis PT4 UPF0060 membrane protein ynfA (ynfA)

What is the basic structure and function of Salmonella enteritidis PT4 UPF0060 membrane protein ynfA?

The ynfA protein is a UPF0060 family membrane protein found in Salmonella enteritidis PT4. According to structural analysis, it consists of 108 amino acids with a sequence: mLKTTLLFFVTALCEIIGCFLPWLWLKRGASVWWLLPAAASLALFVWLLTLHPAASGRVYAAYGGVYVCTALLWLRVVDGVRLTVYDWCGALIALCGmLIIVVGWGRT . The protein contains hydrophobic regions typical of membrane-associated proteins, suggesting its integration into bacterial cell membranes. While the precise function remains under investigation, structural homology studies suggest potential roles in membrane integrity, transport processes, or virulence mechanisms that may contribute to Salmonella's survival in hostile environments, similar to other membrane proteins identified in Salmonella strains .

How does ynfA differ from other characterized membrane proteins in Salmonella enteritidis?

Unlike well-characterized virulence-associated proteins such as YafD (which confers resistance to egg albumen through DNA repair mechanisms ), ynfA belongs to the UPF0060 family of proteins with less defined functional characteristics. Comparative analysis with other membrane proteins indicates ynfA has distinct transmembrane topology patterns. While many characterized Salmonella membrane proteins have established roles in adhesion, invasion, or transport processes, ynfA lacks experimentally verified functional annotations. Its unique sequence composition, with high hydrophobic amino acid content and distinctive transmembrane regions, distinguishes it from flagellar proteins like FliC (used in detection systems ) or surface antigens employed in vaccine development .

What experimental evidence supports the current understanding of ynfA's role in Salmonella pathogenesis?

Current experimental evidence for ynfA's specific role in pathogenesis remains limited compared to well-studied virulence factors. Unlike YafD, which has been experimentally linked to egg albumen resistance through gene disruption and overexpression studies , or recombinant flagellin proteins with established roles in host immune response , direct experimental characterization of ynfA's contribution to virulence is still emerging. Research focusing on membrane protein complexes in Salmonella suggests potential associations with bacterial survival mechanisms, but definitive knockout studies or complementation experiments specifically targeting ynfA function in infection models are needed to establish its precise role in pathogenesis.

What are the optimal expression systems for producing recombinant Salmonella enteritidis PT4 UPF0060 membrane protein ynfA?

For membrane proteins like ynfA, expression system selection significantly impacts yield and functionality. The following table summarizes optimal expression systems based on research with similar membrane proteins:

For optimal ynfA expression, E. coli C41/C43 strains with a pET-based vector containing a mild promoter and fusion tags (His6 or MBP) have shown success with similar membrane proteins. Temperature reduction to 18-20°C post-induction and supplementation with membrane-stabilizing additives can significantly improve functional protein yield .

What purification challenges are specific to recombinant ynfA protein, and how can they be addressed?

Purification of membrane proteins like ynfA presents distinct challenges requiring specialized approaches. The primary difficulties include maintaining protein stability during membrane extraction, preventing aggregation, and preserving native conformation. A methodological approach includes:

• Membrane Extraction: A two-step solubilization protocol is recommended, beginning with mild detergents (DDM or LMNG at 1-2% w/v) for 2-3 hours at 4°C with gentle rotation, followed by centrifugation (100,000×g, 1 hour) to remove insoluble material.

• Affinity Purification: For His-tagged ynfA, Ni-NTA chromatography with detergent-containing buffers (typically 0.1% of the solubilization detergent) prevents protein aggregation. Gradual imidazole elution (20-250 mM) rather than step elution improves purity.

• Size Exclusion Chromatography: Critical for removing aggregates and ensuring monodispersity, particularly when structural or functional studies are planned. A Superdex 200 column equilibrated with buffer containing 0.05% detergent produces optimal results.

• Stabilization Additives: Incorporating glycerol (10-15%), cholesterol hemisuccinate (0.01-0.02%), or specific lipids (POPE/POPG at 0.1-0.5 mg/mL) in purification buffers significantly enhances protein stability.

This approach typically yields 0.2-0.5 mg of purified protein per liter of bacterial culture, with >90% purity as assessed by SDS-PAGE and Western blotting .

How can researchers verify the structural integrity and functionality of purified recombinant ynfA?

Verification of structural integrity and functionality for membrane proteins like ynfA requires multiple complementary approaches:

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy: For secondary structure confirmation, expecting predominant α-helical content (negative peaks at 208 and 222 nm) characteristic of transmembrane proteins.

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): To verify monodispersity and determine protein-detergent complex size.

• Thermal Shift Assays: Using differential scanning fluorimetry with membrane protein-compatible dyes to assess stability under various buffer conditions.

• Limited Proteolysis: Properly folded membrane proteins show distinctive proteolytic fragment patterns when subjected to controlled digestion.

Functional Verification:

• Liposome Reconstitution: Incorporation into artificial liposomes to assess membrane integration capacity.

• Cross-linking Studies: To identify potential interaction partners when reintroduced to bacterial membrane fractions.

• Electrophysiology: If transport function is suspected, patch-clamp analysis of proteoliposomes can reveal channel or transporter activity.

• Antibody Recognition: Conformational antibodies that recognize native epitopes can confirm proper folding.

These multiple lines of evidence provide comprehensive validation of recombinant ynfA quality before proceeding to downstream applications .

How can recombinant ynfA be utilized in developing detection methods for Salmonella enteritidis?

Recombinant ynfA offers significant potential for developing specific detection methods for Salmonella enteritidis, particularly in ELISA-based systems similar to those developed using other Salmonella proteins. A methodological approach includes:

• Antibody Development: Using purified recombinant ynfA as an immunogen in mice or rabbits to generate polyclonal antisera specific to this membrane protein. Antibody production typically follows a 4-6 week immunization schedule with 3-4 booster injections of 50-100 μg protein with appropriate adjuvants .

• ELISA System Development: Establishing an indirect ELISA system where plates are coated with either:

  • Purified recombinant ynfA (direct detection of anti-Salmonella antibodies)

  • Anti-ynfA antibodies (sandwich ELISA for detecting Salmonella in samples)

• Optimization Parameters: Critical optimization factors include:

  • Coating concentration (typically 1-5 μg/mL)

  • Blocking buffer composition (1-3% BSA or milk proteins)

  • Sample preparation methods (enrichment protocols)

  • Detection antibody dilutions (1:1000-1:5000)

  • Incubation times and temperatures

• Validation Metrics: System performance should be validated for:

  • Sensitivity (detection limit of 10³-10⁴ CFU/mL)

  • Specificity (cross-reactivity testing with related Salmonella serotypes and other Enterobacteriaceae)

  • Reproducibility (intra- and inter-assay coefficient of variation <10%)

This approach has demonstrated success with other Salmonella antigens, with studies showing detection limits as low as 10⁴ CFU/mL for similar recombinant protein-based detection systems .

What is the potential of ynfA as a vaccine candidate against Salmonella enteritidis infection?

Research on bacterial membrane proteins suggests ynfA may have potential as a component in vaccine development, though significant investigation is still required. The methodological assessment of ynfA's vaccine potential would include:

• Antigenicity Evaluation:

  • In silico epitope prediction using algorithms like BepiPred and IEDB

  • Experimental mapping of B-cell and T-cell epitopes through peptide arrays

  • Assessment of conservation across Salmonella strains to ensure broad protection

• Immunization Studies:

  • Administration routes (subcutaneous, intraperitoneal, oral)

  • Adjuvant selection (aluminum salts, oil-in-water emulsions, bacterial components)

  • Prime-boost strategies with optimal intervals

• Immune Response Characterization:

  • Antibody isotype profiling (IgG1, IgG2a/IgG2c ratios as Th1/Th2 indicators)

  • Cytokine production patterns from restimulated splenocytes

  • Memory cell generation assessment

• Protection Assessment:

  • Challenge studies with virulent Salmonella enteritidis

  • Bacterial load quantification in tissues

  • Survival rates and clinical scoring

Based on studies with other recombinant Salmonella antigens, subunit vaccines containing membrane proteins can induce significant protection, with recombinant flagellin demonstrating 70-90% protection in mouse models . Similar approaches with O-antigen components have shown protection levels of 83% in heterologous systems , suggesting ynfA might contribute to protective immunity, particularly if incorporated into a multi-component vaccine formulation.

How does ynfA contribute to understanding bacterial membrane protein evolution and functional adaptation?

Studying ynfA provides valuable insights into bacterial membrane protein evolution and adaptation mechanisms. A comprehensive research approach includes:

This research approach has revealed that membrane proteins like those in the UPF0060 family often show accelerated evolution in surface-exposed regions while maintaining structural integrity of transmembrane domains, a pattern consistent with immune evasion strategies while preserving essential functions .

What methodological approaches can resolve the structure-function relationship of ynfA in cell membrane contexts?

Determining the structure-function relationship of ynfA requires sophisticated methodological approaches that preserve the native membrane environment:

• Cryo-Electron Microscopy: For membrane proteins like ynfA, cryo-EM in nanodiscs or amphipols provides near-native structural determination:

  • Sample preparation using MSP (membrane scaffold protein) nanodiscs with defined lipid compositions

  • Vitrification optimization to prevent ice crystal formation

  • Data collection with direct electron detectors at 300 kV

  • Processing workflow including motion correction, CTF estimation, particle picking, and 3D reconstruction

  • Resolution enhancement through focus gradient and symmetry averaging

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

  • Differential solvent accessibility mapping in detergent micelles versus lipid bilayers

  • Time-course experiments (10 sec to 24 hours) to identify dynamic regions

  • Pepsin digestion optimization for hydrophobic membrane proteins

  • MS/MS sequencing for precise localization of deuterium incorporation

• Site-Directed Spin Labeling with EPR Spectroscopy:

  • Systematic introduction of cysteine residues for spin label attachment

  • Accessibility and mobility measurements in various membrane mimetics

  • Distance measurements between labeled residues using DEER spectroscopy

  • Triangulation for topological mapping within the membrane

• In Situ Cross-linking Mass Spectrometry:

  • Photo-activatable lipid probes to map protein-lipid interfaces

  • Chemical cross-linkers with varying spacer lengths to identify interaction partners

  • Cross-linked product identification by tandem mass spectrometry

  • Computational integration with structural models

These approaches have successfully resolved structure-function relationships for other bacterial membrane proteins, revealing how specific structural features contribute to transport, signaling, or virulence functions .

How can researchers address challenges in studying potential protein-protein interactions involving ynfA?

Studying protein-protein interactions (PPIs) for membrane proteins like ynfA presents unique challenges requiring specialized approaches:

• Membrane-Compatible Interaction Assays:

  • Modified Split-Ubiquitin Yeast Two-Hybrid: Adapted for membrane proteins with C-terminal ubiquitin fragment fusions

  • MYTH (Membrane Yeast Two-Hybrid): Specifically designed for identifying partners of integral membrane proteins

  • BRET/FRET Analysis: Using fluorescent protein fusions in live bacterial cells to detect interactions in their native environment

  • Protein-fragment Complementation Assays: With membrane-permeable substrates for interaction detection

• Co-purification Strategies:

  • Tandem Affinity Purification: Using dual tags (e.g., His-FLAG) with optimized detergent mixtures

  • Complexome Profiling: Blue native electrophoresis followed by mass spectrometry to identify native complexes

  • Chemical Cross-linking: With membrane-permeable, cleavable cross-linkers followed by targeted purification

• In Situ Validation:

  • Proximity Labeling: BioID or APEX2 fusion proteins that biotinylate proximal proteins in living cells

  • Co-localization Microscopy: Immunofluorescence with super-resolution techniques (STORM/PALM)

  • Bacterial Two-Hybrid: Modified for membrane protein interaction detection in prokaryotic systems

• Data Integration Workflow:

  • Computational prediction of interaction interfaces using evolutionary coupling analysis

  • Validation with targeted mutagenesis of predicted interface residues

  • Functional assays to assess biological relevance of identified interactions

  • Network analysis to position ynfA within the bacterial interactome

These methodologies have successfully identified interaction partners for other bacterial membrane proteins, revealing complex networks involved in virulence, antimicrobial resistance, and stress adaptation .

What are the critical considerations when designing gene knockout or complementation studies to elucidate ynfA function?

Designing rigorous gene knockout or complementation studies for membrane proteins like ynfA requires careful methodological planning:

• Knockout Strategy Selection:

  • Clean Deletion: Using λ-Red recombineering for scarless removal of the gene without polar effects on downstream genes

  • Insertional Inactivation: For initial studies, with consideration of transcriptional effects on operonic genes

  • Conditional Systems: Employing tetracycline-repressible promoters or temperature-sensitive alleles for essential genes

  • CRISPR-Cas9 Approaches: For precise genome editing with minimal off-target effects

• Phenotypic Characterization Matrix:

• Complementation Design:

  • Expression Level Control: Using native promoter versus inducible systems

  • Integration Options: Chromosomal integration at neutral sites versus plasmid-based expression

  • Domain Analysis: Complementation with truncated or chimeric proteins to map functional domains

  • Tagged Versions: Including functionality verification of tagged proteins used for localization studies

• Genetic Background Considerations:

  • Generation of mutations in multiple Salmonella strains to assess strain-specific effects

  • Construction of double mutants with related genes to identify functional redundancy

  • Complementation with orthologs from other species to assess functional conservation

This systematic approach has been successfully employed for other Salmonella membrane proteins, such as YafD, where gene disruption directly correlated with reduced survival in egg albumen , providing clear evidence for functional roles in specific environments.

How should researchers interpret conflicting data regarding membrane protein function across different experimental systems?

Interpretation of conflicting data for membrane proteins like ynfA requires systematic analytical approaches:

• System-Specific Variable Analysis: Create a comprehensive comparison table documenting all experimental variables:

• Methodological Reconciliation Strategy:

  • Establish a hierarchy of evidence based on system proximity to native conditions

  • Implement orthogonal validation approaches for key findings

  • Determine if conflicts represent different aspects of multifunctional proteins

  • Consider context-dependency of membrane protein function based on lipid environment

• Quantitative Comparison Framework:

  • Normalize functional data to protein expression levels

  • Establish dose-response relationships where applicable

  • Apply statistical methods appropriate for each experimental system

  • Use Bayesian approaches to integrate evidence from multiple sources

• Resolution Experiments:

  • Design experiments specifically targeting discrepancies

  • Systematically isolate variables creating conflicting results

  • Employ chimeric constructs to identify domains responsible for system-specific functions

  • Develop unified assay conditions applicable across experimental systems

This approach has successfully resolved conflicting data for membrane proteins like bacterial transporters and receptors, where function often depends critically on lipid environment, protein density, and post-translational modifications .

What bioinformatic approaches can predict functional domains and interaction networks for poorly characterized proteins like ynfA?

Advanced bioinformatic approaches offer powerful insights into poorly characterized membrane proteins like ynfA:

• Integrated Sequence-Structure Prediction Pipeline:

  • Multiple sequence alignment with diverse homologs (minimum 100 sequences)

  • Conservation analysis using Jensen-Shannon divergence to identify functionally constrained residues

  • Transmembrane topology prediction using consensus from multiple algorithms (TMHMM, MEMSAT, Phobius)

  • Ab initio structure prediction using AlphaFold2 or RoseTTAFold with membrane-specific parameters

  • Molecular dynamics simulations in explicit membrane environments to refine models

• Functional Domain Identification:

  • Motif scanning against specialized membrane protein databases (PROSITE, Pfam-A)

  • Hidden Markov Model construction from related proteins with known functions

  • Identification of functionally coupled genes through phylogenetic profiling

  • Analysis of genomic context and gene neighborhood conservation

• Interaction Network Prediction:

  • Coevolutionary analysis using direct coupling analysis (DCA) or GREMLIN

  • Text mining of literature for implicit association with other proteins

  • Integration with experimental interactome datasets

  • Protein-protein docking simulations with membrane constraints

• Functional Annotation Transfer:

  • Identification of distant homologs with known functions using profile-profile alignments

  • Structure-based function prediction through binding site comparison

  • Metabolic context analysis through pathway reconstruction

  • Gene expression correlation analysis across diverse conditions

These methods have successfully identified functional domains in previously uncharacterized membrane proteins, revealing unexpected functions and interaction partners. For proteins in the UPF0060 family, these approaches have suggested potential roles in stress response pathways and membrane organization .

How can researchers distinguish between direct and indirect effects when analyzing phenotypes of ynfA mutants?

Distinguishing direct from indirect effects in ynfA mutant phenotypes requires systematic experimental design and careful data analysis:

• Temporal Resolution Analysis:

  • Time-course experiments capturing immediate versus delayed effects after gene deletion

  • Inducible expression systems allowing precise timing of complementation

  • Pulse-chase approaches to track direct molecular consequences

  • Real-time monitoring of cellular responses using biosensors or reporters

• Molecular Signature Identification:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics) to establish causality chains

  • Phosphoproteomics to identify rapid signaling changes preceding transcriptional responses

  • Chromatin immunoprecipitation to detect direct regulatory interactions

  • Metabolic flux analysis to distinguish primary from adaptive metabolic changes

• Genetic Interaction Mapping:

  • Synthetic genetic array analysis to identify functional relationships

  • Suppressor screening to identify compensatory mutations

  • Epistasis analysis with genes in putative related pathways

  • Construction of allelic series with varying functional defects

• Targeted Validation Experiments:

  • Site-directed mutagenesis of specific functional residues versus complete gene deletion

  • Domain-specific complementation to isolate functions

  • Heterologous expression of ynfA in different bacterial species to identify context-dependent effects

  • In vitro reconstitution of purified components to demonstrate direct biochemical activities

By implementing this systematic approach, researchers can distinguish primary effects directly attributable to ynfA from secondary consequences of disrupting membrane integrity or triggering compensatory pathways. Studies with other bacterial membrane proteins have shown that immediate effects on membrane potential or permeability are often direct consequences, while transcriptional changes and growth phenotypes frequently represent adaptive responses .

What emerging technologies hold the most promise for advancing our understanding of ynfA function?

Several cutting-edge technologies are poised to significantly advance understanding of membrane proteins like ynfA:

• Advanced Structural Biology Approaches:

  • Cryo-Electron Tomography: For visualizing ynfA in its native membrane context with 10-20Å resolution

  • Microcrystal Electron Diffraction (MicroED): Allowing structure determination from nanocrystals of membrane proteins

  • Serial Femtosecond Crystallography: Using X-ray free electron lasers for room-temperature structures without radiation damage

  • Integrative Structural Biology: Combining multiple experimental data sources with computational modeling

• Single-Molecule Techniques:

  • Single-Molecule FRET: For real-time conformational dynamics in native-like membrane environments

  • High-Speed Atomic Force Microscopy: Capturing topographical changes and interactions at nanometer resolution

  • Nanopore Recording: For functional characterization if ynfA has channel or transport properties

  • Single-Particle Tracking: In live bacteria to determine dynamic localization patterns

• Genetic and Cellular Technologies:

  • CRISPRi/CRISPRa: For tunable repression or activation rather than binary knockout

  • Base Editing and Prime Editing: For precise introduction of point mutations without double-strand breaks

  • Expanded Genetic Code: Incorporating non-canonical amino acids for site-specific probes

  • Synthetic Cell Platforms: Reconstituting minimal systems to identify essential interaction partners

• Computational Advances:

  • Deep Learning Approaches: For improved structure prediction, especially for dynamic regions

  • Enhanced Sampling MD Simulations: For mapping conformational landscapes in membrane environments

  • Quantum Mechanics/Molecular Mechanics: For understanding specific interaction energetics

  • Network Medicine Approaches: Positioning ynfA within the broader cellular interaction landscape

These technologies will likely reveal dynamic aspects of ynfA function, membrane-protein-lipid interactions, and integration into cellular signaling networks that are currently inaccessible with conventional approaches .

What are the most promising strategies for targeting bacterial membrane proteins like ynfA for antimicrobial development?

Targeting bacterial membrane proteins for antimicrobial development presents unique opportunities that can be applied to proteins like ynfA:

• Structure-Based Drug Design Approaches:

  • Fragment-Based Screening: Using specialized membrane-mimetic systems compatible with biophysical screening methods

  • In Silico Docking: With membrane protein-specific scoring functions that account for lipid environment

  • Peptide Mimetics: Designed to interfere with protein-protein interactions within the membrane

  • Allosteric Modulators: Targeting regulatory sites rather than active sites for increased specificity

• Functional Inhibition Strategies:

  • Conformation-Specific Antibodies: Locking proteins in inactive states

  • Peptidomimetics: Based on interaction interfaces that are essential for function

  • Nanobodies or Affimers: Smaller than antibodies with potential to access cryptic binding sites

  • Antisense Oligonucleotides: For selective inhibition of expression in accessible systems

• Novel Delivery Approaches:

  • Fusogenic Liposomes: For direct delivery of inhibitors to bacterial membranes

  • Bacteriophage-Based Delivery: For species-specific targeting

  • Membrane-Penetrating Peptides: As carriers for otherwise impermeable compounds

  • Trojan Horse Strategies: Coupling to essential nutrient uptake systems

• Combination Approaches:

  • Membrane Sensitizers: Compounds that increase permeability to enhance access to targets

  • Efflux Inhibitor Combinations: To prevent extrusion of membrane-targeting agents

  • Biofilm Dispersal Agents: Combined with membrane protein inhibitors

  • Immune Potentiators: Enhancing host defense while targeting bacterial membrane proteins

These strategies are particularly relevant for proteins like ynfA that may contribute to bacterial survival in hostile environments, potentially including resistance to host defenses or conventional antimicrobials .

How might systems biology approaches integrate ynfA function into broader understanding of Salmonella virulence networks?

Systems biology approaches offer powerful frameworks for integrating ynfA into comprehensive Salmonella virulence networks:

• Multi-Omics Integration Framework:

  • Condition-Specific Transcriptomics: Mapping ynfA expression patterns across infection-relevant conditions

  • Spatial Proteomics: Determining subcellular localization changes during infection stages

  • Phosphoproteomics: Identifying post-translational modifications in response to host signals

  • Metabolic Profiling: Connecting membrane protein function to metabolic adaptations

• Network Biology Approaches:

  • Protein-Protein Interaction Networks: Positioning ynfA within membrane protein complexes

  • Gene Regulatory Networks: Identifying transcription factors controlling ynfA expression

  • Metabolic Networks: Assessing impact of ynfA on bacterial metabolism during infection

  • Host-Pathogen Interaction Maps: Connecting bacterial membrane proteins to host targets

• Mathematical Modeling Strategies:

  • Ordinary Differential Equation Models: For temporal dynamics of regulatory networks

  • Flux Balance Analysis: To predict metabolic consequences of ynfA perturbation

  • Agent-Based Models: For multicellular infection dynamics

  • Bayesian Networks: Integrating diverse data types with uncertainty quantification

• Comparative Systems Analysis:

  • Pan-Genome Analysis: Examining ynfA conservation and variation across Salmonella strains

  • Cross-Species Network Comparison: With related pathogens to identify conserved modules

  • Virulence Factor Co-evolution Analysis: Identifying coordinated evolution with other factors

  • Host Response Integration: Correlating bacterial network states with host defense networks

This systems approach can reveal emergent properties not apparent from reductionist studies, positioning ynfA within complex adaptive responses during infection. Similar approaches have successfully integrated other bacterial membrane proteins into comprehensive virulence networks, revealing unexpected connections to stress responses, metabolic adaptation, and host immune evasion .