Recombinant Shigella flexneri serotype 5b UPF0060 membrane protein YnfA (ynfA)

What is YnfA and what is its significance in Shigella flexneri?

YnfA is a membrane protein belonging to the Small Multidrug Resistance (SMR) family of transporters found in Shigella flexneri. It functions as an efflux transporter involved in promoting antimicrobial resistance. The protein consists of 108 amino acids with the sequence: MIKTTLLFFATALCEIIGCFLPWLWLKRNASIWLLLPAGISLALFVWLLTLHPAASGRVY AAYGGVYVCTALMWLRVVDGVKLSLYDWTGALIALCGmLIIVAGWGRT .

S. flexneri is one of the leading bacterial causes of dysentery worldwide, particularly in low-income countries, making the study of its resistance mechanisms critically important . YnfA has been identified as an uncharacterized hypothetical protein that plays a functional role in antimicrobial resistance, potentially serving as a new drug/inhibitor target to combat antimicrobial resistance in this pathogen .

How does YnfA contribute to antimicrobial resistance in Shigella?

YnfA contributes to antimicrobial resistance through its function as an efflux transporter that extrudes various antimicrobial compounds from the bacterial cell. Research has demonstrated that:

Functional studies have confirmed that YnfA plays a direct role in the efflux of specific compounds, contributing to S. flexneri's ability to survive in the presence of these antimicrobials.

What structural features of YnfA are important for its function?

The structural analysis of YnfA has revealed several important features:

The 3D structure of YnfA has been predicted using computational approaches including I-TASSER and validated through the AlphaFold protein structure database. These predictions used the already resolved crystal structure of the EmrE transporter (PDB ID: 3b61) as a template, suggesting functional similarities between these two proteins .

Mutational studies targeting conserved amino acid residues have shown alterations in the resistance profile and efflux activity of the mutant YnfA transporter, highlighting the importance of these residues for proper function .

How does YnfA compare with other efflux transporters in bacterial pathogens?

• YnfA belongs to the SMR superfamily of transporters that are characterized by their small size and role in multidrug resistance

• Unlike larger multidrug resistance transporters that require energy from ATP hydrolysis, SMR transporters like YnfA typically use proton motive force for substrate transport

• The specificity of substrates that YnfA can efflux appears to differ from other transporters, suggesting a specialized role in S. flexneri's resistance mechanisms

Comparative analysis with other efflux systems in Gram-negative bacteria has shown that YnfA contributes to a complex network of resistance mechanisms, with potential synergistic effects when multiple efflux systems are expressed simultaneously.

What is the relationship between YnfA and host immune responses during Shigella infection?

While direct evidence linking YnfA to host immune evasion is limited in the search results, we can make some connections based on the broader context of Shigella pathogenesis:

S. flexneri employs various effector proteins, such as IpaH1.4 and IpaH2.5, which can antagonize host immune responses by preventing LPS ubiquitylation . Though YnfA is not directly implicated in this process, its role in antimicrobial resistance may complement these immune evasion strategies by:

• Enhancing bacterial survival in the presence of host-derived antimicrobial peptides

• Contributing to persistent colonization, which is a characteristic pattern observed in S. flexneri infections

• Potentially interfering with the efficacy of innate immune responses that rely on antimicrobial compounds

Research into the interplay between YnfA-mediated resistance and host immune responses represents an important frontier for understanding Shigella pathogenesis.

What are the optimal conditions for expressing recombinant YnfA protein?

Based on information from commercial sources and standard practices for membrane protein expression, the following conditions are recommended for recombinant YnfA expression:

For laboratory-scale expression, the E. coli system is most commonly used with the following considerations:

• Use of a weak promoter to prevent toxic overexpression

• Growth at lower temperatures (16-25°C) to facilitate proper folding

• Addition of specific detergents during extraction to maintain protein stability

• Use of fusion tags (such as His-tag) to facilitate purification

When expressing membrane proteins like YnfA, it's crucial to optimize detergent concentration and buffer conditions to maintain the native conformation of the protein.

What methods are effective for studying YnfA's role in antimicrobial resistance?

Several complementary approaches have proven effective for studying YnfA's role in antimicrobial resistance:

• Gene knockout studies: Creating a ynfA knockout (KO) mutant and comparing its properties with wild-type S. flexneri strains

• MIC assays: Determining minimum inhibitory concentrations of various antimicrobial compounds using plate dilution methods

• Drug sensitivity assays: Evaluating growth and resistance patterns

• Transport assays: Using fluorescent substrates like ethidium bromide and acriflavine to directly measure efflux capacities

• Site-directed mutagenesis: Modifying conserved amino acid residues to identify crucial functional elements

These methods have successfully demonstrated that disruption of YnfA renders S. flexneri more susceptible to certain antimicrobial compounds and significantly affects transport activity.

How can researchers overcome challenges in membrane protein characterization when studying YnfA?

Membrane proteins like YnfA present specific challenges for characterization. Researchers can employ the following strategies to overcome these obstacles:

• Use of mass photometry: This relatively recent bioanalytical technique provides high-resolution information on sample heterogeneity within minutes, helping to assess protein purity and oligomeric state

• NanoDSF (Differential Scanning Fluorimetry): A technology that requires minimal material and can analyze the thermal stability of proteins containing aromatic amino acids that change their local chemical environment upon denaturation

• Computational approaches: Tools like I-TASSER and AlphaFold have successfully predicted the 3D structure of YnfA based on homology with the EmrE transporter, providing structural insights without the need for crystallization

• Biochemical assays in native-like environments: Using nanodiscs or liposomes to reconstitute YnfA in a membrane-like environment for functional studies

• Fusion with stabilizing proteins: Creating chimeric constructs with well-folding proteins to enhance stability during purification and characterization

These approaches can help overcome the inherent difficulties in working with membrane proteins, which constitute approximately 20-30% of gene coding proteins but remain challenging to study due to their hydrophobic nature and instability when removed from the membrane environment .

How can YnfA be targeted for development of novel antimicrobial strategies?

YnfA represents a promising target for novel antimicrobial strategies due to its role in resistance. Potential approaches include:

• Efflux pump inhibitors (EPIs): Developing compounds that specifically block YnfA's transport function could restore sensitivity to existing antibiotics

• Combination therapies: Using EPIs targeting YnfA alongside conventional antibiotics to enhance efficacy

• Structure-based drug design: Utilizing the predicted 3D structure of YnfA to design molecules that bind to and inhibit its function

• Peptidomimetics: Developing synthetic peptides that mimic substrates but block the transport channel

The research explicitly identifies YnfA as "a new drug/inhibitor target that could be exploited in the future to fight antimicrobial resistance in bacterial pathogens and aid in combatting shigellosis caused by drug-resistant strains of Shigella" .

What is the evolutionary significance of YnfA in Shigella flexneri lineages?

While the search results don't directly address the evolutionary history of YnfA, we can infer its significance in the context of S. flexneri evolution:

S. flexneri has been subdivided into seven phylogenetic groups (PGs), each characterized by distinct virulence gene complements and geographic ranges . The species demonstrates a pattern of:

• Long-term colonization and persistence in specific geographic regions

• Local acquisition of antimicrobial resistance (AMR) determinants

• Limited evidence of intercontinental spread of AMR strains

YnfA, as an efflux transporter contributing to antimicrobial resistance, likely plays a role in this evolutionary pattern by:

• Enhancing bacterial survival during long-term colonization

• Contributing to the baseline intrinsic resistance that may have been present before acquisition of additional AMR determinants

• Potentially interacting with other resistance mechanisms that have evolved in geographically restricted sub-lineages

Comparative genomic studies across S. flexneri lineages could reveal variations in the YnfA sequence and expression that might correlate with differences in virulence and resistance profiles among strains.

What are the key considerations when designing experiments involving recombinant YnfA?

When designing experiments with recombinant YnfA, researchers should consider:

• Protein stability: YnfA, like other membrane proteins, requires appropriate detergents or membrane mimetics to maintain stability

• Expression system selection: Choosing between E. coli, yeast, baculovirus, or mammalian expression systems based on experimental needs and available resources

• Functional assays: Implementing appropriate transport assays using fluorescent substrates like ethidium bromide and acriflavine to directly measure efflux activity

• Controls: Including both positive controls (known SMR transporters like EmrE) and negative controls (catalytically inactive variants) in functional studies

• Storage conditions: Maintaining protein stability with appropriate buffer conditions, potentially including 50% glycerol and storage at -20°C or -80°C for extended periods

• Validation approaches: Using multiple complementary techniques to confirm findings, particularly when studying structure-function relationships

These considerations are essential for generating reliable and reproducible data when working with this challenging but important membrane protein.

How does understanding YnfA contribute to broader knowledge of bacterial resistance mechanisms?

The study of YnfA contributes to our broader understanding of bacterial resistance mechanisms in several ways:

• It highlights the importance of efflux transporters belonging to the SMR family in intrinsic resistance to antimicrobials

• It provides insights into how small membrane proteins can effectively contribute to drug resistance despite their limited size

• It offers a model system for studying the relationship between protein structure and transport function in the context of resistance

• It exemplifies how bacteria like S. flexneri employ multiple complementary mechanisms to survive antimicrobial pressure

• It underscores the importance of considering efflux-based resistance when developing new antimicrobial strategies

Understanding YnfA and similar transporters is crucial for developing comprehensive approaches to combat the growing global challenge of antimicrobial resistance, particularly in important pathogens like S. flexneri that cause significant disease burden worldwide .