Recombinant Salmonella agona UPF0060 membrane protein ynfA (ynfA)

What is YnfA and to which protein family does it belong?

YnfA is a membrane protein belonging to the Small Multidrug Resistance (SMR) family of efflux pumps. It is classified as a UPF0060 membrane protein and has been identified in various bacterial species including Salmonella agona, Salmonella heidelberg, Escherichia coli, and Shigella flexneri . The protein functions as a proton-coupled transporter that contributes to bacterial multidrug resistance by actively extruding antimicrobial compounds from the bacterial cell . YnfA plays a crucial role in bacterial defense mechanisms against various antibiotics and toxic compounds.

What is the structural composition of YnfA protein?

YnfA has a compact structure composed of four alpha-transmembrane helices, similar to the well-characterized EmrE transporter. Computational modeling using I-TASSER and AlphaFold has confirmed this structural arrangement with high confidence scores (coverage of 0.95 and a Normalized Z-score of 2.15) . The protein functions as a dimer in the bacterial membrane, with both monomers displaying a dual-topology arrangement. This configuration allows for the alternation between inward-facing and outward-facing conformations, enabling the efflux mechanism that exports antimicrobial compounds while importing protons . The structural similarity between YnfA and EmrE suggests a conserved mechanism of action among SMR family members.

How is YnfA implicated in antimicrobial resistance?

YnfA contributes significantly to antimicrobial resistance through its function as an efflux pump. Quantitative studies have shown that YnfA is abundantly expressed in multi-drug-resistant (MDR) Escherichia coli isolates from urinary tract infections . The expression level of ynfA was found to be consistently high among 75-80% of MDR isolates, suggesting its important role in conferring resistance to multiple antibiotics . When the ynfA gene is disrupted in bacterial strains like Shigella, the mutants show increased susceptibility to antimicrobial compounds, confirming its direct involvement in drug resistance mechanisms . YnfA appears to have substrate specificity for compounds such as ethidium bromide and acriflavine, as demonstrated by transport activity assays.

What experimental approaches are most effective for studying YnfA function?

Investigating YnfA function requires a multi-faceted experimental approach combining genetic, biochemical, and computational methods. For genetic studies, gene knockout techniques should be employed to create ynfA-deficient strains, which can then be compared with wild-type bacteria for antimicrobial susceptibility using standard minimum inhibitory concentration (MIC) assays . For transport activity assessment, fluorescent substrate accumulation assays using compounds like ethidium bromide provide quantitative measurements of efflux capacity .

Site-directed mutagenesis of conserved amino acid residues is crucial for identifying functional domains. Based on studies with the homologous protein EmrE, researchers should target conserved glutamate residues that are likely involved in proton coupling and substrate binding . Complementation studies, where the wild-type gene is reintroduced into knockout strains, can confirm phenotype restoration.

For structural studies, a combination of computational modeling using I-TASSER and AlphaFold with experimental validation through techniques like X-ray crystallography or cryo-electron microscopy is recommended. Heterologous expression systems in E. coli with affinity tags facilitate protein purification for in vitro functional assays and structural determination .

How does YnfA compare functionally to other SMR family transporters?

YnfA shares significant functional similarities with other SMR family transporters, particularly EmrE, while maintaining distinct characteristics. Both YnfA and EmrE function as proton-coupled antiporters that exchange two protons for one drug molecule per transport cycle . The structural comparison reveals that YnfA possesses the same four alpha-transmembrane helical topology as EmrE, with high threading alignment scores (coverage of 0.95 and a Normalized Z-score of 2.15) .

Unlike some larger multidrug resistance transporters that require accessory proteins, YnfA likely functions as a simple homodimer, similar to EmrE. This structural simplicity makes YnfA an excellent model system for studying fundamental transport mechanisms in the SMR family .

What is the precise mechanism of YnfA-mediated drug efflux at the molecular level?

The molecular mechanism of YnfA-mediated drug efflux appears to follow the alternating access model established for EmrE and other SMR transporters. YnfA functions through a dual-topology arrangement where the protein alternates between inward-facing and outward-facing conformations . This conformational switching is driven by substrate binding and proton translocation.

The transport cycle begins with the binding of a substrate molecule (typically a cationic hydrophobic compound) to the inward-facing conformation of YnfA at the cytoplasmic side. This binding triggers a conformational change to the outward-facing state, allowing the substrate to be released into the periplasmic space or external environment. Simultaneously, protons from the periplasm bind to conserved glutamate residues in the transmembrane domains, which then drives the transporter back to its inward-facing conformation, completing the cycle .

Computational structural models of YnfA suggest that, like EmrE, it exchanges two protons for one drug molecule per transport cycle . The proton-drug antiport mechanism utilizes the proton motive force across the bacterial membrane to energize the export of toxic compounds against their concentration gradient. This energy coupling mechanism is critical for the efficient functioning of YnfA as an antimicrobial resistance determinant.

How can recombinant YnfA protein be effectively expressed and purified for functional studies?

Recombinant expression and purification of YnfA require specialized approaches due to its hydrophobic nature as a membrane protein. For optimal expression, E. coli BL21(DE3) or C43(DE3) strains, which are specifically designed for membrane protein expression, should be used as host cells. The ynfA gene should be cloned into expression vectors containing strong inducible promoters (like T7) and appropriate affinity tags (His-tag or Strep-tag) for purification .

Expression should be performed at lower temperatures (16-20°C) to minimize inclusion body formation and use mild inducers (0.1-0.5 mM IPTG) for slower expression rates. For membrane extraction, detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) are recommended as they effectively solubilize membrane proteins while maintaining structural integrity.

Purification should employ a two-step approach:

• Initial purification using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin for His-tagged constructs

• Secondary purification using size exclusion chromatography to obtain homogeneous protein preparations

For functional reconstitution, the purified protein should be incorporated into proteoliposomes using E. coli polar lipid extracts at a protein:lipid ratio of approximately 1:100. Transport activity can then be assessed using fluorescent substrates or radiolabeled compounds to measure efflux rates.

What experimental systems are most suitable for studying YnfA-mediated antimicrobial resistance?

Several experimental systems can be employed to study YnfA-mediated antimicrobial resistance, each with distinct advantages for investigating specific aspects of YnfA function:

• Genetic knockout models: Creating ynfA deletion mutants in Salmonella agona, E. coli, or Shigella flexneri provides a direct approach to assess the contribution of YnfA to antimicrobial resistance. These knockout strains can be compared to wild-type bacteria in antimicrobial susceptibility assays to quantify the impact of YnfA on resistance to various compounds .

• Complementation systems: Reintroducing wild-type or mutant ynfA genes into knockout strains allows for structure-function analyses and identification of critical amino acid residues. This approach has been successfully used to study the effect of mutating conserved residues on transport activity and antimicrobial resistance .

• Heterologous expression systems: Expressing ynfA in hypersensitive E. coli strains lacking major efflux systems provides a clean background for assessing YnfA function without interference from other transporters.

• Real-time transport assays: Fluorescent substrate accumulation assays using compounds like ethidium bromide provide dynamic measurements of YnfA-mediated efflux activity. These assays can be performed with intact cells or with reconstituted proteoliposomes .

• Clinical isolate studies: Analyzing the expression levels of ynfA in clinical isolates of multidrug-resistant bacteria helps correlate its expression with resistance phenotypes, as demonstrated in studies with urinary E. coli isolates where ynfA expression was consistently high in 75-80% of MDR strains .

How can site-directed mutagenesis be utilized to identify critical residues in YnfA function?

Site-directed mutagenesis is a powerful approach for identifying functionally crucial amino acid residues in YnfA. Based on structural similarities with the well-characterized EmrE transporter, researchers should target conserved residues likely involved in substrate binding, proton translocation, or structural integrity .

The mutagenesis strategy should focus on:

• Conserved charged residues: Glutamate residues (particularly E14) that are likely involved in proton coupling should be mutated to neutral amino acids (e.g., E14Q) to assess their role in the transport mechanism.

• Aromatic residues in transmembrane domains: Tyrosine and tryptophan residues that may participate in substrate binding through π-stacking interactions should be mutated to alanine or phenylalanine to evaluate their contribution.

• Residues at monomer interfaces: Amino acids at the dimer interface should be targeted to understand oligomerization requirements.

• Loop regions: Residues in connecting loops between transmembrane segments that might be involved in conformational changes should be modified.

Each mutant should be characterized through:

• Expression level analysis using western blotting

• Membrane localization assessment using fractionation techniques

• Functional assays including MIC determinations for various antimicrobials

• Transport activity measurements using fluorescent substrates like ethidium bromide

• Protein stability assessments through thermal shift assays

The combined results from these analyses will reveal which residues are essential for different aspects of YnfA function, providing insights into its molecular mechanism of action .

How can YnfA research contribute to developing novel antimicrobial strategies?

YnfA research offers several promising avenues for developing new antimicrobial strategies to combat multidrug-resistant bacteria. As a significant contributor to antimicrobial resistance in pathogens like Salmonella and E. coli, YnfA represents an attractive target for inhibitor development . By understanding the molecular mechanism of YnfA-mediated efflux, researchers can design small molecule inhibitors that specifically block its transport activity, thereby restoring bacterial susceptibility to existing antibiotics.

Several approaches can be pursued:

• Direct YnfA inhibitors: Compounds that competitively bind to the substrate-binding pocket of YnfA without being transported can act as effective inhibitors. Structure-based drug design utilizing the computational models of YnfA can accelerate the discovery of such inhibitors .

• Proton-coupling disruptors: Molecules that interfere with the proton translocation mechanism of YnfA would disable its energy source, rendering it non-functional. This approach targets the fundamental energy coupling mechanism of SMR transporters.

• Oligomerization inhibitors: Since YnfA functions as a dimer, compounds that prevent dimer formation would inhibit its activity. Peptides or small molecules targeting the monomer-monomer interface represent a promising strategy.

• Expression modulators: Compounds that downregulate ynfA expression could reduce efflux capacity and enhance antimicrobial efficacy. This approach requires understanding the regulatory mechanisms controlling ynfA expression.

These YnfA-targeted approaches could be particularly valuable against multidrug-resistant Salmonella Agona, which has been implicated in food-borne outbreaks and shown to harbor numerous antimicrobial resistance genes . By specifically targeting efflux mechanisms, these strategies may overcome existing resistance mechanisms while minimizing selective pressure for new resistance determinants.

What insights can functional studies of YnfA provide about bacterial adaptation to environmental stresses?

Functional studies of YnfA can provide significant insights into how bacteria adapt to environmental stresses beyond antimicrobial resistance. As a membrane transporter involved in exporting toxic compounds, YnfA likely plays broader roles in bacterial stress responses and environmental adaptation .

Investigating YnfA function under various stress conditions could reveal:

• Response to host-derived antimicrobial compounds: YnfA may contribute to bacterial survival in host environments by exporting host-derived antimicrobial peptides or bile salts, particularly relevant for enteric pathogens like Salmonella.

• Adaptation to food preservation compounds: For food-borne pathogens like Salmonella Agona, YnfA might facilitate survival in food products containing preservatives or disinfectants, contributing to food safety concerns .

• Biofilm formation and persistence: Efflux pumps have been implicated in biofilm formation through the export of quorum sensing molecules. YnfA might participate in biofilm-associated processes, enhancing bacterial persistence in hostile environments.

• Heavy metal resistance: The research on multidrug-resistant Salmonella Agona indicates the presence of genes conferring resistance to six different heavy metals (gold, tellurium, arsenic, mercury, copper, and nickel/cobalt) often co-located with antibiotic resistance genes . YnfA might contribute to heavy metal resistance alongside antimicrobial resistance.

• Virulence regulation: Efflux systems can influence bacterial virulence by modulating the internal concentration of signaling molecules. Investigating potential links between YnfA activity and virulence factor expression could reveal new facets of pathogenesis regulation.

By understanding these broader functions of YnfA, researchers can gain insights into bacterial adaptation strategies and potentially identify novel approaches to control bacterial infections and transmission.

How conserved is YnfA across different bacterial species and what does this suggest about its evolutionary significance?

YnfA displays significant conservation across various bacterial species, particularly within Enterobacteriaceae, suggesting its evolutionary importance. Comparative genomic analyses reveal YnfA homologs in multiple bacterial genera including Salmonella, Escherichia, and Shigella . This conservation pattern indicates that YnfA likely emerged early in the evolution of these bacterial lineages and has been maintained due to its functional significance.

The structural and functional similarities between YnfA proteins from different species are particularly noteworthy. For instance, computational analyses show that YnfA from Shigella flexneri shares significant structural homology with EmrE from E. coli, with both proteins exhibiting the characteristic four transmembrane helix topology typical of SMR family transporters . This structural conservation suggests strong selective pressure to maintain YnfA's molecular architecture across species.

Interestingly, while the core structure of YnfA is conserved, subtle sequence variations exist between species, potentially reflecting adaptations to specific ecological niches or antimicrobial challenges. These variations might influence substrate specificity, transport efficiency, or regulatory control, allowing bacteria to fine-tune their efflux capabilities in response to environmental pressures.

The widespread distribution of YnfA in pathogenic and non-pathogenic bacteria suggests it serves fundamental physiological roles beyond antimicrobial resistance. Its conservation across diverse bacterial lineages indicates YnfA likely contributes to general stress responses and cellular homeostasis, making it an essential component of bacterial survival mechanisms.

What is the relationship between YnfA and mobile genetic elements in the spread of antimicrobial resistance?

The relationship between YnfA and mobile genetic elements represents a critical aspect of antimicrobial resistance dissemination. While the chromosomal ynfA gene itself may not frequently be mobile, it often functions in conjunction with resistance determinants carried on mobile genetic elements .

In multidrug-resistant Salmonella Agona, extensive genomic analysis has revealed the presence of large plasmids carrying multiple antibiotic resistance genes . For instance, the plasmid pSE18-SA00377-1 (295,499 bp) belonging to the IncHI2 plasmid family harbors 16 antibiotic resistance genes organized in two distinct clusters, each associated with putative composite transposons . While ynfA may be chromosomally encoded, its efflux function likely complements the resistance mechanisms conferred by these plasmid-borne genes, creating a multi-layered defense system against antimicrobials.

Comparative analysis of such resistance plasmids has demonstrated that both the plasmid backbone and identical or similar antibiotic resistance gene clusters can be found across various bacterial genera from different geographical origins and isolation sources . This suggests extensive horizontal gene transfer and highlights how efflux systems like YnfA can work synergistically with acquired resistance determinants to enhance bacterial survival under antimicrobial pressure.

The functional interplay between chromosomal efflux pumps like YnfA and mobile resistance elements likely contributes to the rapid evolution and spread of multidrug resistance in clinical settings. Understanding these relationships is crucial for developing comprehensive strategies to combat antimicrobial resistance.

What are the most promising approaches for structural characterization of YnfA?

Future structural characterization of YnfA should employ advanced techniques that overcome the challenges associated with membrane protein analysis. While computational models based on homology with EmrE have provided valuable insights , experimental determination of YnfA's structure at atomic resolution would significantly advance our understanding of its mechanism.

The most promising approaches include:

• Cryo-electron microscopy (Cryo-EM): This technique has revolutionized membrane protein structural biology and could be applied to purified YnfA in nanodiscs or detergent micelles. Cryo-EM would be particularly valuable for capturing YnfA in different conformational states of the transport cycle.

• X-ray crystallography with lipidic cubic phase (LCP): LCP crystallization has proven successful for numerous membrane transporters and could potentially yield high-resolution structures of YnfA, especially if conformationally stabilized mutants are employed.

• Single-particle analysis combined with molecular dynamics simulations: This integrated approach can provide insights into the dynamic aspects of YnfA function, revealing how substrate binding induces conformational changes.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can map conformational changes and solvent accessibility of different regions of YnfA under various conditions, providing valuable functional insights.

• Site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy: These methods can determine distances between specific residues in different conformational states, helping to validate and refine existing structural models.

• Solid-state NMR spectroscopy: This technique can provide structural information about YnfA in a membrane-like environment, potentially revealing details about its interactions with lipids and substrates.

A multi-technique approach combining these methods would provide comprehensive structural insights into YnfA function and facilitate structure-based drug design targeting this important antimicrobial resistance determinant.

How might YnfA research inform broader understanding of bacterial membrane transport systems?

YnfA research has the potential to significantly advance our understanding of bacterial membrane transport systems more broadly. As a member of the SMR family, YnfA represents one of the simplest and most ancient classes of transporters, making it an excellent model system for studying fundamental transport mechanisms .

Several aspects of YnfA research could inform broader membrane transport concepts:

• Minimal functional units: With just four transmembrane helices, YnfA represents one of the smallest functional transport units in nature. Understanding how this minimal architecture achieves efficient substrate transport could reveal fundamental principles of membrane transport bioenergetics.

• Dual-topology arrangement: YnfA likely employs a dual-topology model where the protein alternates between inward and outward-facing conformations . Elucidating the molecular details of this conformational switching would provide insights applicable to more complex transporters that follow similar mechanisms.

• Oligomerization requirements: YnfA functions as a homodimer, and understanding the structural basis for this oligomerization could inform research on assembly mechanisms of larger multisubunit transporters.

• Proton-coupling mechanisms: YnfA's function as a proton-coupled antiporter makes it valuable for studying how proton gradients are harnessed to drive substrate transport, a fundamental mechanism in bioenergetics.

• Substrate recognition determinants: Identifying the molecular features that determine YnfA's substrate specificity would enhance our understanding of how transporters discriminate between different compounds, with implications for drug design.

• Evolutionary relationships: Comparative studies of YnfA across different bacterial species could reveal evolutionary pathways leading to the diversification of membrane transport systems, potentially identifying fundamental design principles conserved throughout bacterial evolution.

These insights from YnfA research would contribute to a more comprehensive understanding of membrane transport biology, with implications extending beyond antimicrobial resistance to fundamental aspects of cellular physiology and evolutionary biology.