Recombinant Escherichia fergusonii UPF0060 membrane protein ynfA (ynfA)

What is ynfA protein and what is its classification?

ynfA is classified as a UPF0060 membrane protein belonging to the Small Multidrug Resistance (SMR) family of efflux pumps. Found in Escherichia fergusonii (strain ATCC 35469 / DSM 13698 / CDC 0568-73), this 108-amino acid transmembrane protein functions as an efflux transporter involved in antimicrobial resistance mechanisms. The protein is cataloged in the UniProt database under accession number B7LRA1. Quantitative studies have demonstrated that ynfA is consistently expressed at high levels in multi-drug-resistant (MDR) Escherichia coli clinical isolates, particularly those from urinary tract infections, suggesting its significant role in antibiotic resistance .

What is the predicted structure of ynfA protein?

The three-dimensional structure of ynfA has been predicted using advanced computational techniques. The I-TASSER (Iterative Threading ASSEmbly Refinement) tool utilized the already resolved crystal structure of EmrE transporter (PDB ID: 3b61) from E. coli as a template to predict ynfA's structure . This prediction revealed that ynfA consists of four alpha-transmembrane helices with a threading alignment coverage of 0.95 and a Normalized Z-score of 2.15, indicating high confidence in the structural model .

The predicted structure was further validated using the AlphaFold protein structure database, which generated consistent results for both ynfA and EmrE, confirming the four alpha-transmembrane helical configuration . This structural information is invaluable for understanding ynfA's transport mechanism and for designing inhibitors targeting this efflux pump.

How should recombinant ynfA protein be stored for experimental use?

Proper storage of recombinant ynfA is critical for maintaining its structural integrity and functional properties. The recommended storage conditions are as follows:

• Standard storage: -20°C

• Extended storage: -20°C or -80°C

• Working aliquots: 4°C for up to one week

Repeated freezing and thawing cycles should be avoided as they can compromise protein stability and function . The expected shelf life is approximately 6 months for the liquid form and 12 months for the lyophilized form when stored at -20°C/-80°C . These storage parameters are essential for ensuring reproducible experimental results, particularly in functional assays investigating antimicrobial resistance mechanisms.

What expression systems are used for producing recombinant ynfA?

Recombinant ynfA is typically produced using in vitro E. coli expression systems optimized for membrane protein production . The expression construct typically includes the full-length protein (amino acids 1-108) with an N-terminal 10xHis-tag to facilitate purification through affinity chromatography . When expressing membrane proteins like ynfA, researchers must consider several critical factors:

• Expression strain selection: Strains optimized for membrane protein expression

• Induction conditions: Temperature, inducer concentration, and duration

• Membrane extraction: Detergent selection for solubilization

• Purification strategy: Balancing yield with protein activity

These considerations are crucial for obtaining functional protein for downstream structural and functional analyses.

What is the role of ynfA in antimicrobial resistance?

ynfA plays a significant role in antimicrobial resistance as a member of the SMR family efflux pump system. Quantitative research using real-time PCR has demonstrated that ynfA expression is consistently high in 75-80% of multi-drug-resistant E. coli isolates from urinary tract infections . This prevalence suggests that ynfA contributes substantially to the antibiotic resistance phenotype in these clinical isolates.

The mechanism of ynfA-mediated resistance involves active efflux of antimicrobial compounds from the bacterial cell, thereby reducing intracellular drug concentrations below their effective thresholds . This process is likely energetically coupled to proton translocation across the membrane, similar to other SMR family transporters. Recent findings indicate that ynfA's contribution to resistance extends beyond E. coli to other Gram-negative pathogens such as Shigella flexneri, where it has been identified as a functional efflux transporter involved in antimicrobial resistance .

This widespread distribution and functional conservation make ynfA an important target for developing efflux pump inhibitors to combat antimicrobial resistance in Gram-negative pathogens.

How does the structure of ynfA compare to other SMR family proteins like EmrE?

Structural comparison between ynfA and the well-characterized EmrE transporter reveals significant similarities that inform our understanding of ynfA's function:

The I-TASSER prediction used EmrE (PDB ID: 3b61) as a template, resulting in a high coverage score (0.95) and a good Normalized Z-score (2.15), indicating reliable structural similarity . Both ynfA and EmrE share the characteristic four alpha-transmembrane helical architecture of SMR family transporters . This structural homology suggests that ynfA likely employs a similar antiport mechanism, where substrate transport is coupled to proton translocation in the opposite direction.

What experimental approaches are used to characterize ynfA function?

Multiple complementary experimental approaches are employed to comprehensively characterize ynfA function:

• Gene Expression Analysis: Quantitative real-time PCR (qRT-PCR) is used to measure ynfA expression levels in clinical isolates and laboratory strains, correlating expression with antimicrobial resistance profiles .

• Structural Prediction and Analysis: Computational methods such as I-TASSER and AlphaFold are employed to predict protein structure and identify potential functional domains .

• Mutagenesis Studies: Site-directed mutagenesis targeting specific amino acid residues helps identify crucial residues for substrate binding and transport function, based on structural predictions and comparison with homologs like EmrE .

• Antimicrobial Susceptibility Testing: Methods such as agar dilution and broth microdilution are used to determine minimum inhibitory concentrations (MICs) of various antibiotics in wild-type strains versus ynfA knockout or overexpression strains .

• Conjugation Experiments: These are employed to study the transferability of resistance mechanisms, particularly relevant when ynfA is encoded on mobile genetic elements .

• Transport Assays: Fluorescent substrate accumulation/efflux assays can measure the transport activity of ynfA using fluorescent antimicrobial compounds or dyes as substrates.

These approaches collectively provide comprehensive insights into ynfA function, substrate specificity, and potential for inhibition as a strategy to combat antimicrobial resistance.

How can mutations in ynfA affect its efflux pump activity?

Mutations in ynfA can significantly impact its efflux pump activity through several mechanisms:

Understanding these mutation effects provides insights into the structure-function relationship of ynfA and can guide the development of inhibitors targeting specific functional domains of the protein to overcome antimicrobial resistance.

What statistical considerations are important when designing experiments involving ynfA?

When designing experiments involving ynfA, several statistical considerations are crucial for obtaining robust and reproducible results:

These statistical considerations ensure that research findings related to ynfA function are reliable, reproducible, and scientifically sound.

Characteristics of Recombinant ynfA Protein

This table summarizes the key characteristics of recombinant ynfA protein, providing essential information for researchers working with this protein in laboratory settings .

Expression Levels of Efflux Pump Genes in MDR E. coli Clinical Isolates

This table highlights the significant prevalence of ynfA expression in multi-drug-resistant E. coli clinical isolates compared to other efflux pump genes, emphasizing its importance in antimicrobial resistance mechanisms .

Experimental Approaches for ynfA Functional Characterization

This table summarizes the complementary experimental approaches used to characterize ynfA function, providing a methodological framework for researchers investigating this important efflux pump .