Recombinant Salmonella typhimurium UPF0060 membrane protein YnfA (ynfA)

What is YnfA and what family of proteins does it belong to?

YnfA is a membrane protein belonging to the Small Multidrug Resistance (SMR) family of efflux transporters. In Salmonella typhimurium, this protein functions as an efflux transporter involved in antimicrobial resistance. The protein consists of 108 amino acids and is characterized by its transmembrane domains that contribute to its transport functionality . The protein's function has been demonstrated through comparative studies with other SMR family transporters, particularly the model transporter EmrE whose functional structure has been used to predict YnfA's three-dimensional configuration .

What is the amino acid sequence of Salmonella typhimurium YnfA protein?

The full amino acid sequence of Salmonella typhimurium YnfA protein (1-108aa) is: MLKTTLLFFVTALCEIIGCFLPWLWLKRGASVWWLLPAAASLALFVWLLTLHPAASGRVY AAYGGVYVCTALLWLRVVDGVRLTVYDWCGALIALCGMLIIVVGWGRT . This sequence is critical for understanding the protein's structural characteristics and for designing recombinant constructs for experimental studies. The sequence reveals the hydrophobic nature of many amino acid residues, consistent with its role as a membrane protein.

How is recombinant YnfA typically produced for research purposes?

Recombinant Full Length Salmonella typhimurium UPF0060 membrane protein YnfA is typically expressed in E. coli expression systems with an N-terminal His tag to facilitate purification. The protein preparation is generally supplied as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE . For reconstitution, it is recommended to briefly centrifuge the vial before opening and reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, the addition of 5-50% glycerol and aliquoting for storage at -20°C/-80°C is recommended .

How has the structure of YnfA been characterized in research settings?

The structure of YnfA has been characterized using computational techniques based on the already solved functional structure of EmrE, another SMR family transporter. This involves a combination of homology modeling and comparative structural analysis . Researchers have used this approach to predict the three-dimensional structure of YnfA, which helps in understanding its functional mechanisms. Additionally, knowledge derived from mutational studies in EmrE has been leveraged to identify crucial amino acid residues in YnfA through targeted mutagenesis experiments .

What computational methods are most effective for predicting YnfA's structural properties?

Computational methods leveraging homology modeling have proven effective for predicting YnfA's structural properties. The approach typically begins with utilizing the previously solved structure of EmrE as a template, followed by in silico analysis to further characterize YnfA and its homologs in different Gram-negative bacteria . This computational workflow combines sequence alignment, structural modeling, and functional prediction to generate insights into the protein's configuration. The approach is particularly valuable for membrane proteins like YnfA, which are notoriously difficult to crystallize for direct structural determination.

How can researchers experimentally demonstrate YnfA's role as an efflux transporter?

Researchers can demonstrate YnfA's role as an efflux transporter through several complementary experimental approaches:

• Gene Knockout Studies: Creating a ΔYnfA knockout mutant (e.g., SFL2640) and comparing its antimicrobial susceptibility to wild-type strains (e.g., SFL2608) and complemented strains (e.g., SFL2643/YnfAComp) .

• MIC Assays: Using microtiter plate dilution methods to determine Minimum Inhibitory Concentration (MIC₉₀) values for various antimicrobials against wild-type, knockout, and complemented strains .

• Drug Sensitivity Assays: Spotting 10-fold dilutions of bacterial cultures on plates containing antimicrobial compounds at specific concentrations to observe growth differences .

• Fluorescence-based Transport Assays: Utilizing fluorescent substrates like ethidium bromide (EtBr) and acriflavine to directly measure transport activity .

These methodologies collectively provide compelling evidence of YnfA's function in antimicrobial resistance through efflux activity.

What substrates are commonly used to assess YnfA transport activity?

Ethidium bromide (EtBr) and acriflavine are two common substrates used to assess YnfA transport activity . These compounds are particularly useful for fluorescence-based transport assays due to their fluorescent properties. Research has shown that wild-type YnfA protein (SFL2643) demonstrates a 2-fold greater resistance to EtBr and 4-fold greater resistance to acriflavine compared to control strains . These substrates are valuable tools for assessing the functional implications of mutations in YnfA and for evaluating its role in antimicrobial resistance.

Which amino acid residues are critical for YnfA function based on mutagenesis studies?

Mutagenesis studies have identified several critical amino acid residues essential for YnfA function. Specifically, mutations E15A, G18A, and Y60A have been shown to significantly decrease resistance to antimicrobial compounds like ethidium bromide and acriflavine . This is evidenced by experimental results showing that YnfA mutants SFL2653 (E15A), SFL2654 (G18A), and SFL2657 (Y60A) displayed decreased resistance to these compounds compared to the wild-type YnfA protein . In contrast, mutations to other residues—including FF-LL (SFL2652), WLL-QVV (SFL2655), GGV-AAA (SFL2656), Y63A (SFL2658), Y67A (SFL2659), and Y86A (SFL2660)—showed no significant changes in resistance profiles .

How should researchers design site-directed mutagenesis experiments to study YnfA function?

Researchers should design site-directed mutagenesis experiments for YnfA by first conducting comparative analysis with well-characterized homologs like EmrE to identify potentially critical residues. The experimental design should include:

• Selection of Target Residues: Focus on conserved amino acids, particularly those in transmembrane domains or binding pockets, such as E15, G18, and Y60 .

• Mutation Strategy: Consider both conservative and non-conservative substitutions to evaluate the importance of specific physicochemical properties.

• Controls: Include both wild-type protein and empty vector controls for accurate comparison.

• Functional Assays: Implement multiple complementary assays including:

  • Resistance profiling using MIC assays

  • Transport activity measurements with fluorescent substrates

  • Growth curve analysis to rule out general growth defects

• Validation: Confirm protein expression levels to ensure observed phenotypes are due to functional changes rather than expression differences.

This systematic approach enables the identification of residues critical for substrate recognition, transport activity, and antimicrobial resistance.

What expression systems are optimal for producing functional recombinant YnfA?

E. coli expression systems have proven effective for producing functional recombinant YnfA protein . When designing expression systems for membrane proteins like YnfA, researchers should consider:

• Expression Vectors: Vectors with controllable promoters (like pBAD) that allow fine-tuning of expression levels to prevent protein aggregation.

• Fusion Tags: N-terminal His tags have been successfully used for YnfA purification, enhancing protein recovery while maintaining functionality .

• Host Strains: Specialized E. coli strains designed for membrane protein expression may improve yields and proper folding.

• Growth Conditions: Lower temperatures (16-30°C) and reduced inducer concentrations often improve functional membrane protein yields by slowing production and allowing proper membrane insertion.

• Media Composition: Rich media supplemented with glycerol can support higher membrane protein expression levels.

Optimizing these parameters is essential for obtaining sufficient quantities of properly folded, functional YnfA for subsequent structural and functional studies.

What are the challenges in membrane insertion of YnfA and how can they be overcome?

Membrane protein insertion presents several challenges that researchers must address:

• Prevention of Premature Folding: Membrane proteins like YnfA risk premature folding or aggregation in the cytosol before reaching their target membrane .

• Insertion Mechanism: In natural systems, membrane proteins utilize complex cellular machinery including:

  • Signal recognition particle (SRP) for targeting

  • FtsY receptor for membrane localization

  • SecYEG translocon or YidC insertase for membrane integration

• Lipid Environment: The composition of the target membrane, particularly the presence of negatively charged phospholipids like phosphatidylglycerol (PG), significantly affects insertion efficiency .

To overcome these challenges in experimental settings, researchers can:

• Utilize cell-free expression systems coupled with liposomes

• Incorporate detergents or lipid nanodiscs to maintain protein solubility

• Optimize lipid composition in reconstitution experiments, ensuring presence of negatively charged phospholipids

• Consider co-expression with chaperones to prevent aggregation

These strategies can significantly improve the functional reconstitution of YnfA in experimental systems.

How does YnfA contribute to antimicrobial resistance in Salmonella and other bacteria?

YnfA contributes to antimicrobial resistance through its function as an efflux transporter, actively pumping antimicrobial compounds out of bacterial cells. Research has demonstrated that deletion of the ynfA gene in Salmonella results in significantly increased sensitivity to various antimicrobial compounds . Specifically, the ynfA knockout mutant (SFL2640) shows lower MIC₉₀ values compared to wild-type (SFL2608) and complemented strains (SFL2643), indicating increased susceptibility to antimicrobials . This efflux function appears to be conserved among YnfA homologs in different Gram-negative bacteria, making it a potentially important contributor to intrinsic antimicrobial resistance across multiple species .

What is the substrate specificity profile of YnfA compared to other efflux transporters?

YnfA exhibits substrate specificity typical of SMR family transporters, with demonstrated activity against compounds including ethidium bromide and acriflavine . Experimental evidence shows that wild-type YnfA confers 2-fold greater resistance to ethidium bromide and 4-fold greater resistance to acriflavine compared to control strains . This substrate profile overlaps with other SMR family transporters like EmrE, suggesting conserved structural features involved in substrate recognition.

The table below summarizes YnfA's comparative resistance profile against selected compounds:

This substrate profile positions YnfA as a multidrug efflux pump with potential clinical significance in antimicrobial resistance.

How does YnfA compare structurally and functionally to the model transporter EmrE?

YnfA shares significant structural and functional similarities with EmrE, a well-characterized model transporter in the SMR family. Both proteins function as efflux transporters that contribute to antimicrobial resistance . The structural comparison between YnfA and EmrE has been instrumental in predicting YnfA's three-dimensional configuration through computational techniques .

Key comparative aspects include:

• Structural Homology: The three-dimensional structure of YnfA has been predicted based on EmrE's solved functional structure, indicating significant structural conservation .

• Functional Conservation: Both transporters confer resistance to similar substrates, including ethidium bromide and acriflavine .

• Critical Residues: Mutational studies in EmrE have guided the identification of functionally important amino acid residues in YnfA, suggesting conservation of key structural elements involved in transport activity .

• Substrate Specificity: While there is overlap in substrate profile, there may be differences in specificity or efficiency that reflect evolutionary adaptations to different bacterial environments.

This comparative approach has proven valuable for understanding YnfA's structure-function relationships and for guiding experimental design.

What can we learn about YnfA from homologs in other Gram-negative bacteria?

Studying YnfA homologs across different Gram-negative bacteria provides valuable insights into:

• Evolutionary Conservation: The degree of sequence conservation indicates functionally critical regions that have been maintained through evolutionary pressure.

• Functional Adaptations: Variations in sequence or structure among homologs may reflect adaptations to different bacterial environments or substrate profiles.

• Resistance Patterns: Comparing antimicrobial resistance profiles associated with YnfA homologs in different bacterial species can reveal the breadth of its contribution to clinical resistance.

• Structural Predictions: Computational in silico analysis of YnfA homologs in different Gram-negative bacteria has been used to further characterize the transporter and refine structural models .

• Inhibitor Development: Conserved features across homologs represent potential targets for developing broad-spectrum efflux pump inhibitors to combat antimicrobial resistance.

This comparative analysis across bacterial species provides a more comprehensive understanding of YnfA's biological significance and potential as a therapeutic target.

How can researchers investigate the role of YnfA in clinical antimicrobial resistance?

To investigate YnfA's role in clinical antimicrobial resistance, researchers should employ a multi-faceted approach:

• Clinical Isolate Screening: Analyze ynfA gene expression in clinical isolates showing multidrug resistance, comparing expression levels between resistant and susceptible strains.

• Genetic Manipulation in Clinical Isolates: Generate ynfA knockouts in clinically relevant strains to assess the impact on antimicrobial susceptibility profiles.

• Synergy Testing: Evaluate potential synergy between conventional antibiotics and experimental YnfA inhibitors to determine if efflux inhibition can restore antibiotic efficacy.

• Biofilm Studies: Investigate YnfA's role in antimicrobial resistance within biofilm communities, which often show enhanced resistance compared to planktonic cells.

• In vivo Infection Models: Use animal infection models with wild-type and ΔynfA strains to assess the impact on virulence and in vivo antimicrobial efficacy.

• Transcriptional Analysis: Identify conditions that upregulate ynfA expression during infection or antimicrobial exposure using RNA-seq or qPCR.

This comprehensive approach can establish the clinical relevance of YnfA in antimicrobial resistance and evaluate its potential as a therapeutic target.

What strategies can be employed to develop inhibitors targeting YnfA efflux function?

Developing inhibitors targeting YnfA efflux function requires a systematic approach:

• Structure-Based Design: Utilize the predicted three-dimensional structure of YnfA, based on computational analysis and homology with EmrE, to identify potential binding pockets for inhibitor design .

• High-Throughput Screening: Develop fluorescence-based assays using substrates like ethidium bromide to screen chemical libraries for compounds that inhibit YnfA-mediated efflux .

• Rational Design Based on Mutagenesis: Target inhibitor development to interact with critical residues identified through mutagenesis studies, particularly E15, G18, and Y60, which have been shown to be essential for YnfA function .

• Peptidomimetic Approach: Design peptides that mimic transmembrane segments of YnfA to disrupt oligomerization or conformational changes necessary for transport.

• Combination Therapy Evaluation: Test potential inhibitors in combination with conventional antibiotics to identify synergistic combinations that overcome resistance.

• Cross-Resistance Evaluation: Assess inhibitor efficacy against multiple SMR family transporters to develop broad-spectrum efflux inhibitors.

This multifaceted approach could lead to novel therapeutic strategies to combat antimicrobial resistance by targeting YnfA and related efflux systems.

What are the most promising future research directions for YnfA studies?

The most promising future research directions for YnfA studies include:

• Comprehensive Structural Characterization: Moving beyond computational predictions to experimental determination of YnfA's structure using techniques like cryo-electron microscopy or X-ray crystallography.

• Transport Mechanism Elucidation: Detailed investigation of the conformational changes and energy coupling mechanisms involved in YnfA-mediated transport.

• Clinical Significance Assessment: Evaluating the contribution of YnfA to clinical antimicrobial resistance across different bacterial pathogens and infection types.

• Inhibitor Development: Design and optimization of specific inhibitors targeting YnfA as potential adjuvants to conventional antimicrobial therapy.

• Regulatory Network Analysis: Understanding how ynfA expression is regulated in response to environmental stressors, particularly antimicrobial exposure.

• Broader Substrate Profiling: Comprehensive characterization of YnfA's substrate range beyond the currently identified compounds like ethidium bromide and acriflavine .

These research directions could significantly advance our understanding of YnfA's biological significance and its potential as a therapeutic target for combating antimicrobial resistance.

How might targeting YnfA contribute to overcoming antimicrobial resistance in clinical settings?

Targeting YnfA could contribute to overcoming antimicrobial resistance through several mechanisms:

• Efflux Inhibition: Specific inhibitors of YnfA could prevent the efflux of antimicrobial compounds, effectively increasing their intracellular concentration and restoring efficacy against resistant strains .

• Combination Therapy: YnfA inhibitors could be developed as adjuvants to be used in combination with conventional antibiotics, potentially lowering the required antibiotic concentrations and reducing selection pressure for resistance.

• Multi-Target Approach: Inhibitors designed to target conserved features of SMR family transporters might simultaneously inhibit multiple efflux systems, providing a broader spectrum of resistance reversal.

• Biofilm Penetration: Efflux pump inhibition might enhance antibiotic penetration into bacterial biofilms, which are particularly recalcitrant to antimicrobial treatment.

• Virulence Modulation: Beyond direct effects on antimicrobial resistance, targeting YnfA might affect bacterial fitness or virulence, particularly if this transporter plays additional roles in bacterial physiology.