Recombinant Salmonella arizonae UPF0060 membrane protein ynfA (ynfA)

What is the amino acid sequence and structural characteristics of Salmonella arizonae ynfA?

The full amino acid sequence of Salmonella arizonae ynfA is: mLKTTLLFFVTALCEIIGCFLPWLWIKRGASVWWLLPAVASLALFVWLLTLHPAASGRVYAAYGGVYVCTALLWLRVVDGVRLTVYDWCGALIALCGmLIIVVGWGRT . The protein is characterized as a small membrane protein with four transmembrane segments, which is typical of the SMR protein family. These proteins are usually 100 to 140 amino acids in length . Based on structural studies using the model transporter EmrE from E. coli, computational techniques have been employed to predict the three-dimensional structure of ynfA . The protein contains several conserved amino acid residues that are critical for its transport function, including glutamic acid at position 15 (E15), glycine at position 18 (G18), and tyrosine at position 60 (Y60), which have been identified through mutational studies .

What is the functional role of the ynfA protein in bacterial cells?

The ynfA protein functions as an efflux transporter that belongs to the SMR family and plays a significant role in antimicrobial resistance mechanisms. Research on the homologous ynfA protein in Shigella flexneri has demonstrated that this transporter is involved in extruding various antimicrobial compounds from the bacterial cell, particularly cationic compounds such as ethidium bromide, acriflavine, methyl viologen, and crystal violet . By actively pumping these compounds out of the cell, ynfA contributes to the bacteria's ability to survive in the presence of these antimicrobials. Disruption of the ynfA gene renders bacteria more susceptible to these compounds, confirming its role in conferring resistance . This function is consistent with other members of the SMR family, which are known to contribute to bacterial multidrug resistance mechanisms.

How does the mutation of specific amino acid residues affect the transport activity of ynfA?

Mutational studies on the ynfA protein have revealed that specific amino acid residues are crucial for its transport activity and resistance-conferring capabilities. Based on research conducted on Shigella flexneri, mutations of certain conserved residues significantly impaired the protein's function. Specifically, mutations E15A (glutamic acid to alanine at position 15), G18A (glycine to alanine at position 18), and Y60A (tyrosine to alanine at position 60) led to substantial reductions in both transport activity and antimicrobial resistance when tested with substrates like ethidium bromide and acriflavine . The Y63A mutation showed moderate to slight differences in the resistance profile and transport activity compared to the wild-type protein .

In contrast, mutations FF-LL, WLL-QVV, GGV-AAA, Y67A, and Y86A did not cause significant changes in transport or resistance capabilities, displaying wild-type-like functionality . These findings align with previous mutational studies conducted on EmrE and other SMR transporters, suggesting conserved functional mechanisms across this protein family. The results indicate that E15, G18, and Y60 are likely involved in substrate binding or the conformational changes necessary for transport activity.

What experimental methods are most effective for studying ynfA function?

Multiple complementary experimental approaches have proven effective for studying ynfA function:

• Gene Knockout and Complementation: Creating a ynfA knockout mutant followed by complementation with the wild-type gene helps establish the protein's role in antimicrobial resistance. This approach allows researchers to observe phenotypic changes when the gene is absent and confirm that these changes are specifically due to the absence of ynfA by restoring the wild-type phenotype through complementation .

• Minimum Inhibitory Concentration (MIC) Assays: MIC₉₀ assays using microtiter plate dilution methods help determine the resistance profile conferred by ynfA against various antimicrobial compounds. These assays reveal the lowest concentration at which 90% of bacterial growth is inhibited .

• Drug Sensitivity Spot Assays: Spotting 10-fold dilutions of bacterial cultures on agar plates containing antimicrobial compounds at specific concentrations provides a visual assessment of growth differences between wild-type, knockout, and complemented strains .

• Fluorescence-Based Transport Assays: These assays directly measure the transport activity of ynfA using fluorescent substrates like ethidium bromide and acriflavine. Both accumulation (measuring substrate uptake) and efflux (measuring substrate extrusion) assays can be performed to assess transport capabilities .

• Site-Directed Mutagenesis: Creating specific amino acid mutations helps identify residues crucial for protein function. This approach, combined with functional assays, reveals which parts of the protein are involved in substrate binding, transport, or structural integrity .

• Protein Expression Analysis: Western blotting using anti-His antibodies helps confirm proper expression of wild-type and mutant ynfA proteins in experimental strains, ensuring that observed functional differences are not due to expression problems .

How does ynfA compare structurally and functionally to other SMR family transporters?

The ynfA protein belongs to the SMR family of transporters, which includes well-characterized members such as EmrE, SugE, YdgE/F, and QacC. EmrE from E. coli, first identified in 1992, serves as the archetypal model for the SMR family and has been extensively studied structurally and functionally . Like other SMR family members, ynfA is a small inner membrane protein (approximately 100-140 amino acids) with four transmembrane segments .

Functionally, ynfA demonstrates similar substrate preferences to other SMR transporters, particularly showing affinity for cationic compounds such as ethidium bromide, acriflavine, methyl viologen, and crystal violet . This substrate profile aligns with the known preference of SMR transporters for cationic lipophilic compounds.

The functional significance of specific amino acid residues in ynfA, as determined through mutational studies, corresponds to findings from similar studies on EmrE. For instance, the critical importance of E15 in ynfA parallels the essential role of the corresponding glutamic acid residue in EmrE for transport activity . This suggests conservation of key functional mechanisms across the SMR family despite variations in primary sequence.

What are the optimal conditions for storing and handling recombinant ynfA protein?

For optimal storage and handling of recombinant ynfA protein, the following conditions are recommended:

• Storage Temperature: Store the protein at -20°C for short-term storage, and at -20°C or -80°C for extended storage periods .

• Storage Buffer: A Tris-based buffer with 50% glycerol, optimized specifically for this protein, is recommended for storage .

• Freeze-Thaw Cycles: Repeated freezing and thawing should be avoided as this can lead to protein degradation and loss of activity. Working aliquots can be stored at 4°C for up to one week .

• Handling Considerations: When working with recombinant ynfA, it's important to maintain the integrity of its membrane-associated properties. As a hydrophobic membrane protein, ynfA may aggregate or lose activity if subjected to conditions that disrupt its native conformation.

• Quantity Considerations: Standard research quantities begin at 50 μg, though other quantities may be available upon request for specific experimental needs .

Following these storage and handling guidelines will help ensure the protein maintains its structural integrity and functional activity for experimental use.

What are the most effective methods for assessing ynfA-mediated antimicrobial resistance?

Several complementary methods have proven effective for assessing ynfA-mediated antimicrobial resistance:

• MIC₉₀ Determination Using Microtiter Plate Dilution:

  • This method involves preparing serial 2-fold dilutions of antimicrobial compounds in 96-well plates.

  • Bacterial cultures (wild-type, ynfA knockout, and complemented strains) are added to each well.

  • After incubation (typically 16 hours at 37°C), optical density (OD₆₀₀) is measured.

  • The MIC₉₀ is determined as the lowest concentration that inhibits 90% of bacterial growth .

• Drug Sensitivity Spot Assay:

  • Prepare 10-fold serial dilutions of bacterial cultures.

  • Spot 5 μL of each dilution onto agar plates containing antimicrobial compounds at specific concentrations.

  • Include a control plate without antimicrobials to confirm equal growth potential.

  • Incubate overnight and compare growth patterns between strains .

• Transport Activity Assessment Using Fluorescent Substrates:

  • For accumulation assays: Expose bacterial cells to fluorescent substrates like ethidium bromide or acriflavine, and measure the increase in fluorescence over time as the substrate accumulates.

  • For efflux assays: Pre-load cells with the fluorescent substrate, then measure the decrease in fluorescence as the substrate is extruded .

Table 1. MIC₉₀ values for various antimicrobial compounds in Shigella strains with different ynfA status

*Exact MIC₉₀ values for ethidium bromide and acriflavine were not provided in the source material, but relative resistance patterns were reported .

These methods collectively provide a comprehensive assessment of how ynfA contributes to antimicrobial resistance.

What control experiments should be included when studying ynfA knockouts?

When studying ynfA knockouts, several critical control experiments should be included to ensure valid and interpretable results:

• Growth Curve Analysis in the Absence of Antimicrobials:

  • This control is essential to confirm that any growth differences observed in the presence of antimicrobials are due to altered resistance rather than inherent growth defects.

  • Compare growth patterns of wild-type, knockout, and complemented strains in standard media without antimicrobials.

  • In previous studies, no significant differences were observed in the growth patterns of Shigella strains with different ynfA status in the absence of antimicrobial compounds .

• Complementation Controls:

  • Include a strain where the knockout has been complemented with the wild-type ynfA gene.

  • This confirms that phenotypic changes in the knockout are specifically due to the absence of ynfA rather than polar effects or unintended genetic alterations.

  • The complemented strain should display susceptibilities and MIC₉₀ values similar to the wild-type strain .

• Empty Vector Control:

  • Include a strain containing the empty expression vector used for complementation.

  • This controls for any effects the vector itself might have on antimicrobial resistance or transport activity.

  • In previous studies, this control was used as the baseline for comparing fold increases in resistance for various mutants .

• Protein Expression Verification:

  • Perform Western blotting to confirm appropriate expression of the ynfA protein in complemented strains and mutants.

  • This ensures that observed functional differences are not due to expression problems or protein instability .

• Multiple Antimicrobial Compounds Testing:

  • Test resistance against multiple compounds with different properties.

  • This helps establish the substrate range of ynfA and confirms that observed effects are consistent across different antimicrobials .

Including these controls helps ensure experimental rigor and allows for confident interpretation of results regarding ynfA function.

How can researchers distinguish between direct and indirect effects of ynfA knockout on antimicrobial resistance?

Distinguishing between direct and indirect effects of ynfA knockout requires a multi-faceted experimental approach:

• Complementation Studies: The most direct method to confirm that observed phenotypes are specifically due to the absence of ynfA is through complementation. When the wild-type ynfA gene is reintroduced into the knockout strain and the original phenotype is restored, this strongly indicates a direct effect. Research with Shigella flexneri demonstrated that complementation with the ynfA gene restored wild-type resistance levels to various antimicrobials, confirming direct involvement of ynfA .

• Transport Assays with Known Substrates: Direct measurement of transport activity using fluorescent substrates like ethidium bromide and acriflavine provides evidence for the direct involvement of ynfA in efflux. If the knockout strain shows significantly reduced efflux activity that is restored upon complementation, this suggests a direct role for ynfA in transporting these compounds .

• Specificity of Substrate Profile: Comparing the susceptibility profile of the ynfA knockout to knockouts of other transporters helps determine the specific contribution of ynfA. If the pattern of altered susceptibility is distinct from that seen with other transporter knockouts, this suggests direct and specific effects of ynfA .

• Protein-Substrate Interaction Studies: Direct binding assays or competition studies can demonstrate physical interaction between ynfA and putative substrates, providing evidence for direct effects.

• Cross-Resistance Analysis: Examining whether resistance to multiple compounds is affected similarly can help distinguish between direct efflux effects and indirect physiological adaptations.

By employing these approaches collectively, researchers can build a strong case for distinguishing between direct and indirect effects of ynfA knockout on antimicrobial resistance.

What does mutational analysis reveal about the structure-function relationship of ynfA?

Mutational analysis has provided significant insights into the structure-function relationship of ynfA. Research on Shigella flexneri ynfA has revealed that certain amino acid residues are critical for the protein's transport activity and resistance-conferring capabilities:

• Critical Functional Residues: Mutations E15A (glutamic acid to alanine at position 15), G18A (glycine to alanine at position 18), and Y60A (tyrosine to alanine at position 60) significantly impaired both transport activity and resistance to ethidium bromide and acriflavine. This suggests these residues are essential for substrate binding, transport, or maintaining the proper conformation of the protein .

• Moderately Important Residues: The Y63A mutation showed moderate effects on ynfA function, suggesting this residue contributes to but is not critical for activity .

• Non-Essential Residues: Mutations FF-LL, WLL-QVV, GGV-AAA, Y67A, and Y86A did not significantly affect ynfA function, indicating these residues are not essential for the core transport mechanism .

• Structural Implications: The critical importance of E15 in ynfA parallels the essential role of the corresponding glutamic acid residue in EmrE, suggesting conservation of key functional mechanisms across the SMR family. This glutamic acid residue is likely involved in proton coupling or substrate binding .

• Transmembrane Orientation: The pattern of functional and non-functional mutations provides insights into which protein regions face the transport channel versus those facing the lipid bilayer.

This mutational analysis supports a model where specific conserved residues form a substrate-binding pocket or transport pathway, while other regions maintain structural integrity without directly participating in transport.

How can researchers integrate data from different experimental approaches to build a comprehensive model of ynfA function?

Building a comprehensive model of ynfA function requires the integration of data from multiple experimental approaches:

• Combining Genetic and Biochemical Data: Correlate the results from genetic studies (knockouts, complementation) with biochemical assays (transport activity, MIC determinations) to establish fundamental functional properties. For instance, research on Shigella flexneri demonstrated that ynfA knockout resulted in both increased susceptibility to antimicrobials in MIC assays and reduced transport activity in fluorescence-based assays, providing complementary evidence for ynfA's role as an efflux pump .

• Integrating Structural Predictions with Mutational Data: Use computational structural predictions based on homology to well-characterized transporters like EmrE, combined with results from mutational studies, to develop a structural model of ynfA. This approach helped identify critical functional residues (E15, G18, Y60) that likely play key roles in transport activity .

• Cross-Species Comparative Analysis: Compare functional data from ynfA homologs across different bacterial species (Salmonella arizonae, Escherichia coli, Shigella flexneri) to identify conserved mechanisms and species-specific adaptations .

• Substrate Profile Analysis: Integrate data from resistance assays using different antimicrobial compounds to define the substrate specificity of ynfA. Research has shown that ynfA affects resistance to multiple cationic compounds including ethidium bromide, acriflavine, methyl viologen, and crystal violet .

• Developing a Mathematical Model: For advanced analysis, develop mathematical models that account for transport kinetics, resistance levels, and protein structural data to predict how changes in ynfA expression or structure might affect bacterial resistance under different conditions.

By synthesizing data from these diverse approaches, researchers can develop a comprehensive model that describes not only what ynfA does (function) but also how it accomplishes this function (mechanism) and why it has evolved these properties (adaptation).

How might ynfA be targeted in antimicrobial development strategies?

The role of ynfA as an efflux transporter that contributes to antimicrobial resistance makes it a potential target for novel therapeutic strategies:

• Efflux Pump Inhibitors (EPIs): Developing specific inhibitors that block ynfA transport activity could restore bacterial sensitivity to existing antimicrobials. The identified critical residues (E15, G18, Y60) could serve as targets for rational drug design of such inhibitors .

• Combination Therapy Approaches: Using ynfA inhibitors in combination with existing antimicrobials could enhance treatment efficacy against resistant strains. Research has shown that disrupting ynfA increases bacterial susceptibility to compounds like ethidium bromide, acriflavine, methyl viologen, and crystal violet .

• Broad-Spectrum Inhibitor Development: Since ynfA belongs to the SMR family with homologs across various Gram-negative bacteria, inhibitors targeting conserved functional features could potentially work against multiple pathogens.

• Structure-Guided Drug Design: Utilizing the structural models of ynfA based on homology to EmrE, combined with the identification of critical functional residues through mutational studies, could guide the development of small molecules that specifically interfere with ynfA function .

• Antisense Therapy: Developing antisense oligonucleotides or RNA interference strategies to reduce ynfA expression could provide an alternative approach to inhibit its function in bacterial cells.

The development of such strategies could be particularly valuable for treating infections caused by multidrug-resistant Gram-negative bacteria, where efflux pumps like ynfA contribute significantly to resistance mechanisms.

What are the most pressing unanswered questions regarding ynfA function and structure?

Despite recent advances in understanding ynfA, several critical questions remain unanswered:

• High-Resolution Structure: While computational models based on EmrE provide insights, a high-resolution structure of ynfA would significantly advance our understanding of its mechanism. Key questions include how ynfA's structure compares to other SMR family members and what structural features determine its substrate specificity.

• Precise Transport Mechanism: The exact mechanism by which ynfA transports substrates across the membrane remains unclear. Questions persist about proton coupling, conformational changes during transport, and the stoichiometry of transport.

• Physiological Role: Beyond conferring resistance to exogenous antimicrobials, what is the natural physiological role of ynfA in bacterial cells? Does it transport endogenous compounds or environmental substances encountered in natural habitats?

• Regulation of Expression: How is ynfA expression regulated in response to environmental conditions, stress, or the presence of antimicrobials? Understanding these regulatory mechanisms could provide additional targets for intervention.

• Species-Specific Adaptations: How do ynfA homologs differ functionally between bacterial species like Salmonella arizonae, Escherichia coli, and Shigella flexneri? Do these differences reflect adaptations to specific ecological niches or pathogenic lifestyles ?

• Interaction with Other Resistance Mechanisms: How does ynfA function interact with other resistance mechanisms such as target site modifications or additional efflux systems? Understanding these interactions is crucial for developing comprehensive antimicrobial strategies.

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, microbiology, and computational modeling.

How can researchers leverage comparative genomics to better understand ynfA evolution and function?

Comparative genomics offers powerful approaches to illuminate ynfA evolution and function:

By leveraging these comparative genomics approaches, researchers can develop a more comprehensive understanding of how ynfA has evolved and adapted across bacterial species, potentially revealing new insights into its function and opportunities for targeted intervention.