Recombinant UPF0060 membrane protein ynfA (ynfA)

What is the UPF0060 membrane protein YnfA and what is its significance in bacterial research?

YnfA is a membrane protein belonging to the Small Multidrug Resistance (SMR) family of efflux transporters. It plays a crucial role in antimicrobial resistance mechanisms in Gram-negative bacteria, particularly in Shigella flexneri. Research has demonstrated that YnfA functions as an efflux pump that transports various antimicrobial compounds out of bacterial cells, thereby contributing to resistance phenotypes . The significance of YnfA in bacterial research lies in its potential as a drug/inhibitor target that could be exploited to combat antimicrobial resistance in bacterial pathogens, particularly those causing shigellosis.

What is the molecular structure and key characteristics of YnfA?

YnfA is a 108-amino acid protein with a molecular weight of approximately 11.9 kDa. Its amino acid sequence is: MIKTTLLFFATALCEIIGCFLPWLWLKRNASIWLLLPAGISLALFVWLLTLHPAASGRVY AAYGGVYVCTALMWLRVVDGVKLSLYDWTGALIALCGMLIIVAGWGRT . Computational analysis using tools like I-TASSER and validation through the AlphaFold protein structure database reveals that YnfA consists of four alpha-transmembrane helices. The protein functions as a homodimer and displays dual topology for directing transport. Structural analyses indicate that YnfA's transport machinery is comparable to that of EmrE, another well-studied SMR transporter, despite being distant homologs .

How is recombinant YnfA typically produced for research purposes?

Recombinant YnfA is typically produced through heterologous expression in E. coli expression systems. The full-length YnfA gene is cloned into an appropriate expression vector, such as pBAD_Myc_HisA, which allows for controlled expression and facilitates protein purification. For instance, recombinant Shigella flexneri serotype 5b YnfA can be produced with an N-terminal His tag for purification purposes . After expression, the protein is typically purified and can be prepared as a lyophilized powder. For long-term storage, it's recommended to reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL, add glycerol (5-50% final concentration), aliquot, and store at -20°C/-80°C .

What experimental evidence demonstrates YnfA's role in antimicrobial resistance?

Several experimental approaches have provided evidence for YnfA's role in antimicrobial resistance. A comprehensive study examined the functional implications of YnfA in S. flexneri through gene knockout, complementation, and susceptibility testing. The ynfA knockout mutant (SFL2640) showed increased susceptibility and lower MIC90 values for various antimicrobial agents compared to the wild-type strain SFL2608. When the ynfA gene was complemented back into the knockout mutant (creating strain SFL2643/YnfAComp), the wild-type phenotype was restored, with susceptibilities and MIC90 values similar to the wild-type strain .

Additional evidence comes from drug sensitivity assays where 10-fold dilutions of Shigella cultures were spotted on TSA plates containing antimicrobial compounds. The ynfA knockout mutant showed increased sensitivity and altered growth patterns compared to the wild-type and complemented strains. Importantly, in the absence of antimicrobial compounds, no significant differences were observed in the growth patterns of the three Shigella strains, ruling out any fundamental growth defect caused by deleting the ynfA gene .

What substrates are transported by YnfA and how is its transport activity measured?

YnfA primarily transports cationic compounds, similar to other SMR transporters. Common substrates used to study YnfA transport include ethidium bromide (EtBr) and acriflavine. These compounds are well-known to be transported by members of the SMR family, including YnfA .

Transport activity is typically measured using fluorescence-based transport assays with these substrates. In such assays, the ynfA knockout mutant shows significantly decreased transport activity of both acriflavine and EtBr compared to the wild-type and complemented strains. This provides direct evidence that YnfA functions as an efflux pump involved in the transport of antimicrobials, particularly cationic compounds .

For MIC (Minimum Inhibitory Concentration) determination, the microtiter plate dilution method is commonly employed. Bacterial strains are subjected to serial 2-fold dilutions of antimicrobial compounds in 96-well plates, and absorbance (OD600) is measured after incubation. The MIC is determined as the lowest concentration at which 90% of bacterial growth inhibition is detected (MIC90) .

Which amino acid residues are critical for YnfA function and how were they identified?

Site-directed mutagenesis studies have identified several critical amino acid residues essential for YnfA function. Mutations at positions E15A, G18A, and Y60A significantly impaired YnfA's functional capabilities for efflux transport and antimicrobial resistance in the presence of EtBr and acriflavine. The Y63A mutation showed moderate to slight differences in resistance profile and transport activity. In contrast, mutations FF-LL, WLL-QVV, GGV-AAA, Y67A, and Y86A showed no consequential changes, displaying wild-type YnfA transport and resistance capabilities .

These critical residues were identified based on:

• Computational analysis comparing YnfA to the model transporter EmrE

• Multiple sequence alignment of YnfA homologs in various Gram-negative bacterial pathogens

• Identification of conserved amino acids across the YnfA homologs

• Knowledge from previous mutational studies on EmrE

The conserved Glu15 residue appears particularly crucial as it sits in the middle of both protein topologies in the homodimer and forms the binding domain for the substrate and protons, similar to its role in EmrE .

How can researchers confirm proper expression of YnfA mutants?

To confirm proper expression of YnfA and its mutants, researchers typically employ Western blot analysis. In one study, YnfA mutants were expressed using the pBAD_Myc_HisA plasmid, and Western blot with anti-HisA antibody was used to confirm appropriate protein expression. Equal total protein loading was ensured when comparing mutant expressions with the wild-type YnfA protein .

It's important to note that membrane proteins with transmembrane helices often display anomalous migration on SDS-PAGE gels, running either higher or lower relative to their formula molecular mass. YnfA, being 11.9 kDa, typically runs slightly higher in Western blots, appearing close to the 15 kDa band of standard protein ladders .

What approaches are used to compare resistance profiles of YnfA mutants?

The resistance profiles of YnfA mutants are commonly assessed using microtiter plate assays. In this approach:

• Bacterial strains harboring YnfA mutants are subjected to serial 2-fold dilutions of antimicrobial compounds (e.g., EtBr and acriflavine) prepared in 96-well plates.

• After incubation, absorbance (OD600) is measured to determine growth inhibition.

• The MIC90 (minimum inhibitory concentration causing 90% growth inhibition) is determined for each strain.

• Results are typically presented as fold increase in resistance relative to a control strain carrying an empty vector .

This approach allows for quantitative comparison of how different mutations affect YnfA's ability to confer resistance against various antimicrobial compounds.

What in silico methods are valuable for YnfA structural analysis?

Several computational approaches have proven valuable for analyzing YnfA structure in the absence of experimentally determined structures:

• Multiple-sequence alignment of YnfA homologs from various Gram-negative bacterial pathogens to identify conserved amino acids and create a consensus protein sequence.

• Phylogenetic analysis to show the evolutionary relationships between different YnfA homologs.

• Structural prediction using I-TASSER, which utilizes the solved EmrE protein structure as a template to thread the YnfA protein sequence.

• Validation of predicted structures using the AlphaFold protein structure database.

• Comparative structural analysis with known SMR family transporters like EmrE, despite being distant homologs .

These computational methods have revealed that YnfA consists of four alpha-transmembrane helices and likely functions as a homodimer with dual topology similar to EmrE, despite sequence differences.

How might YnfA research contribute to addressing antimicrobial resistance?

YnfA research holds significant promise for addressing antimicrobial resistance through several avenues:

• As a novel drug/inhibitor target: Understanding YnfA's structure and function could lead to the development of specific inhibitors that block its efflux activity, thereby restoring bacterial susceptibility to existing antibiotics .

• Biomarker for resistance: YnfA expression levels could potentially serve as a biomarker for predicting resistance to certain antimicrobials in clinical isolates.

• Structural model for other SMR transporters: Insights gained from YnfA studies could be applied to understanding other members of the SMR family that contribute to antimicrobial resistance in various pathogens.

The present work has established YnfA as an efflux pump of the SMR family and recognized its involvement in promoting antimicrobial resistance in S. flexneri, providing a new target for future drug development efforts to combat antimicrobial resistance in bacterial pathogens and aid in fighting shigellosis caused by drug-resistant Shigella strains .

What are the optimal storage and handling conditions for recombinant YnfA protein?

For researchers working with recombinant YnfA protein, optimal storage and handling conditions are crucial for maintaining protein integrity and function:

• Initial storage: Store at -20°C/-80°C upon receipt, with aliquoting necessary for multiple use to avoid repeated freeze-thaw cycles .

• Working conditions: Store working aliquots at 4°C for up to one week. Repeated freezing and thawing is not recommended .

• Storage buffer: Tris/PBS-based buffer with 6% Trehalose, pH 8.0 is typically used .

• Reconstitution procedure:

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

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

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

  • A final concentration of 50% glycerol is commonly used

Following these guidelines helps ensure that the recombinant YnfA protein maintains its structural integrity and functional properties for experimental use.