Recombinant Salmonella choleraesuis UPF0060 membrane protein YnfA (ynfA)

What is YnfA and how is it classified within membrane protein families?

YnfA from Salmonella choleraesuis is a small integral membrane protein belonging to the Small Multidrug Resistance (SMR) superfamily of transporters. Based on structural analyses, YnfA is approximately 11.9 kDa in size and consists of 108 amino acids organized into four α-transmembrane helices, which is a characteristic signature of SMR family members . The protein's gene is located at positions 1616397 to 1616723 in the S. choleraesuis genome, where it was initially annotated as an uncharacterized hypothetical protein . SMR family proteins like YnfA typically function as efflux transporters that can contribute to antimicrobial resistance by extruding various compounds from bacterial cells.

The amino acid sequence of YnfA (ATH67966.1) reveals its highly hydrophobic nature, which is consistent with its membrane localization and function. The protein structure includes four membrane-spanning domains with specific conserved residues that are likely essential for its transport mechanism and substrate recognition. Computational prediction tools such as TMHMM and TMpred have confirmed this four-helix topology, which is fundamental to understanding its functional mechanisms .

In comparative genomic analyses, YnfA homologs appear in various Gram-negative bacteria, suggesting evolutionary conservation of this protein's function across different bacterial species. Despite structural similarities among SMR transporters, subtle variations in amino acid composition likely influence substrate specificity and transport efficiency.

How does YnfA compare functionally to other well-characterized SMR transporters?

EmrE from Escherichia coli is considered the paradigm SMR efflux pump, having been extensively studied and often used as a foundation for investigating other members of this family . Like EmrE, YnfA functions as an integral inner membrane protein involved in small molecule transport. EmrE has been established as a 12 kDa protein responsible for the efflux of various antimicrobials including erythromycin, tetracycline, methyl viologen, ethidium bromide, acriflavine, and sulfadiazine . Based on homology and functional studies of SMR transporters, YnfA likely exhibits similar substrate profiles, though with potential variations in specificity and transport efficiency.

Functional analysis of YnfA homologs in bacteria like Shigella flexneri has demonstrated their role in conferring resistance to multiple antimicrobial compounds . When comparing YnfA to EmrE, both share the fundamental dual-topology architecture that facilitates their drug efflux mechanism. X-ray structural studies of EmrE have revealed important insights into this mechanism, which likely applies to YnfA as well, with the protein undergoing conformational changes during substrate binding and transport cycles .

The evolutionary conservation of critical functional residues between YnfA and other SMR transporters suggests mechanistic similarities, though the precise substrate range for YnfA may differ based on the specific microenvironment and physiological role in S. choleraesuis. Understanding these similarities and differences provides a foundation for targeted experimental approaches when studying YnfA's specific functional characteristics.

What expression systems are most effective for producing recombinant YnfA protein?

When expressing recombinant YnfA protein for research purposes, several expression systems can be employed, each with distinct advantages depending on research objectives. For initial characterization studies, E. coli-based expression systems offer practical advantages due to rapid growth, established genetic tools, and compatibility with membrane protein expression. Systems utilizing pET vectors with T7 promoters provide controlled, high-level expression when induced with IPTG, which is crucial for potentially toxic membrane proteins like YnfA.

For more challenging expression scenarios, researchers should consider specialized E. coli strains such as C41(DE3) or C43(DE3), which are specifically designed for membrane protein expression and can mitigate toxicity issues. Additionally, codon optimization of the ynfA gene for the expression host is recommended to enhance translation efficiency. The addition of fusion tags such as His6, FLAG, or MBP can facilitate detection and purification, though careful consideration should be given to tag placement (N- or C-terminal) to avoid interfering with membrane insertion or function.

Expression parameters requiring empirical optimization include induction temperature (typically lowered to 16-25°C for membrane proteins), inducer concentration, and expression duration. For functional studies, it may be advantageous to use homologous expression in Salmonella systems, which provide a native-like membrane environment for proper folding and function. Researchers investigating YnfA should initially test multiple expression constructs in parallel to identify optimal conditions for their specific experimental requirements.

What purification strategies work best for YnfA as a membrane protein?

Purification of YnfA presents typical membrane protein challenges that require specialized approaches. The purification protocol should begin with optimized cell lysis using techniques that effectively disrupt bacterial membranes while maintaining protein stability, such as sonication or high-pressure homogenization in buffer systems containing glycerol (10-20%) as a stabilizing agent. Following lysis, membrane fraction isolation via differential centrifugation is essential to separate membrane-bound YnfA from cytoplasmic proteins.

The critical step involves membrane protein solubilization using appropriate detergents. For SMR family proteins, detergents such as n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucopyranoside (OG), or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) have proven effective. Selection of detergent concentration is crucial—typically starting at 1-2% for solubilization and reducing to 0.05-0.1% for subsequent purification steps to maintain protein stability.

For affinity purification, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective for His-tagged YnfA. Multiple wash steps with increasing imidazole concentrations (10-40 mM) help eliminate non-specific binding before elution with higher imidazole concentrations (250-500 mM). Size exclusion chromatography serves as a final purification step to separate monomeric/oligomeric states and remove aggregates. Throughout the purification process, maintaining consistent detergent concentration above its critical micelle concentration (CMC) is essential to prevent protein aggregation.

The purified YnfA protein should be characterized by SDS-PAGE, Western blotting, and mass spectrometry to confirm identity and purity. For functional studies, reconstitution into proteoliposomes may be necessary to assess transport activity in a membrane-like environment.

How can researchers functionally characterize YnfA's transport activity?

Functional characterization of YnfA's transport activity requires multiple complementary approaches. Transport assays using fluorescent substrates such as ethidium bromide and acriflavine provide direct evidence of YnfA's efflux capacity . These assays typically monitor substrate accumulation in cells or membrane vesicles expressing YnfA compared to controls lacking the transporter. A typical protocol involves incubating cells with the fluorescent substrate, followed by washing to remove extracellular substrate, and then measuring fluorescence changes over time using spectrofluorometry. Active efflux by YnfA would result in decreased intracellular fluorescence compared to control samples.

Substrate specificity can be determined through competition assays where various potential substrates compete with a reporter substrate (such as ethidium bromide) for transport. This approach helps identify the range of compounds that YnfA can transport. For more detailed kinetic analyses, researchers can employ radioisotope-labeled substrates to precisely measure transport rates under varying conditions, enabling determination of kinetic parameters such as Km and Vmax.

Electrophysiological techniques, including patch-clamp methods applied to reconstituted systems or whole cells, can provide insights into the electrogenic nature of transport and ion coupling mechanisms. Additionally, pH sensitivity assays help determine if transport is coupled to proton gradients, which is common for SMR transporters. For structure-function studies, site-directed mutagenesis targeting conserved residues followed by functional assays reveals critical amino acids involved in substrate binding and transport.

The following table summarizes common functional assays for YnfA characterization:

What evidence supports YnfA's function as an antimicrobial efflux pump?

Evidence supporting YnfA's function as an antimicrobial efflux pump comes primarily from studies on YnfA homologs in related bacterial species. Research with Shigella flexneri has demonstrated that YnfA acts as an efflux transporter belonging to the SMR family . When the ynfA gene was knocked out in S. flexneri, the resulting mutant strain showed increased susceptibility to certain antimicrobial compounds compared to the wild-type strain, providing direct evidence of YnfA's role in conferring resistance . Additionally, transport assays using fluorescent substrates such as ethidium bromide and acriflavine showed reduced efflux capacity in the ynfA knockout mutant, further confirming its function as an active transporter .

The functional characterization of YnfA places it within the broader context of bacterial efflux systems that contribute significantly to antimicrobial resistance. Efflux pumps such as YnfA represent one of the principal mechanisms employed by bacteria to gain resistance against various antimicrobial compounds . The structural features of YnfA, particularly its four transmembrane helices characteristic of SMR family proteins, support its function as a membrane transporter capable of extruding toxic compounds from the bacterial cell.

Comparative analyses with well-characterized SMR transporters like EmrE provide additional evidence for YnfA's role in antimicrobial resistance. EmrE has been extensively studied and shown to confer resistance to various compounds including erythromycin, tetracycline, ethidium bromide, and acriflavine through active efflux . Given the structural and functional similarities between YnfA and EmrE, it is reasonable to conclude that YnfA likely possesses similar capabilities, though potentially with a different substrate specificity profile.

What specific antimicrobial compounds are potentially transported by YnfA?

Based on research with YnfA homologs and related SMR transporters, YnfA likely transports a range of antimicrobial compounds. Studies with similar SMR transporters, particularly EmrE, have established that these proteins can transport various cationic and hydrophobic compounds. In particular, experiments with YnfA homologs have demonstrated transport activity against fluorescent substrates such as ethidium bromide and acriflavine, which are commonly used as indicators of efflux activity .

The substrate specificity of YnfA likely extends to various classes of antimicrobials, including certain antibiotics, quaternary ammonium compounds, and aromatic cations. The following table summarizes potential substrates of YnfA based on research with homologous SMR transporters:

The substrate range of YnfA is likely influenced by several factors, including the physicochemical properties of the compounds (such as charge, hydrophobicity, and size) and their ability to interact with specific amino acid residues within the transporter's binding pocket. The relatively small size of YnfA (108 amino acids) suggests that it may have limitations in accommodating larger antimicrobial compounds, consistent with the general characteristics of SMR transporters.

It's important to note that the exact substrate profile of YnfA from S. choleraesuis would require direct experimental validation through transport assays and resistance studies with the purified protein or in vivo systems expressing YnfA. Such studies would help establish YnfA's specific contribution to antimicrobial resistance in this pathogen.

Which amino acid residues are critical for YnfA function based on structural predictions?

The functional activity of YnfA likely depends on specific conserved residues within its transmembrane domains. Based on comparative analyses with well-characterized SMR transporters like EmrE, several amino acid residues are predicted to be crucial for YnfA function. In EmrE, glutamate-14 (E14) has been identified as essential for substrate binding and proton coupling during transport . The corresponding glutamate residue in YnfA's sequence would likely play a similar critical role in its transport mechanism.

Other conserved residues in SMR transporters typically include aromatic amino acids (tryptophan, tyrosine, phenylalanine) that contribute to the substrate binding pocket through π-π interactions with aromatic substrates. Positively charged residues (lysine, arginine) near the membrane-cytoplasm interface often participate in substrate recognition and translocation. Based on the amino acid sequence of YnfA (MLKTTLLFFVTALCEIIGCFLPWLWLKRGASVWWLLPAAASLALFVWLTLHPAASGRVYAAYGGYVCTA LLWLRVVDGVRLTVYDWCGALIALCGMLIIVVGWGRT) , residues such as tryptophan (W) at positions like 25, 26, 35, and 36 may be particularly important for substrate interactions.

The predicted four-transmembrane helix structure of YnfA suggests that these helices form a central pore or cavity for substrate binding and translocation across the membrane. Residues lining this cavity would be directly involved in substrate recognition and transport. Additionally, residues at the interfaces between transmembrane helices likely contribute to conformational changes during the transport cycle, which are essential for alternating access of the substrate binding site to either side of the membrane.

Site-directed mutagenesis targeting these predicted critical residues would provide experimental validation of their importance for YnfA function. Substitution with alanine or other amino acids would be expected to alter transport activity, substrate specificity, or resistance profiles if the residues are indeed functionally important.

How can advanced structural biology techniques advance our understanding of YnfA?

Advanced structural biology techniques offer promising avenues to elucidate YnfA's structure-function relationships at the molecular level. Cryo-electron microscopy (cryo-EM) has revolutionized membrane protein structural biology and would be particularly valuable for determining YnfA's three-dimensional structure. Unlike X-ray crystallography, which has traditionally been challenging for small membrane proteins, cryo-EM can resolve structures in more native-like environments without the need for crystal formation. For YnfA, this approach could reveal critical details about substrate binding sites, conformational states during transport cycles, and oligomerization patterns.

X-ray crystallography, despite its challenges, remains valuable if diffraction-quality crystals can be obtained. This might require extensive screening of crystallization conditions, use of specialized detergents, and potentially the application of lipidic cubic phase (LCP) crystallization methods that provide a more membrane-like environment. Crystallographic studies could provide atomic-resolution details of YnfA's structure, particularly if the protein can be captured in different conformational states with and without bound substrates.

Nuclear magnetic resonance (NMR) spectroscopy offers another powerful approach, particularly solution NMR and solid-state NMR, which are well-suited for small membrane proteins like YnfA. These techniques can provide information not only about static structure but also about dynamics and conformational changes during transport. For YnfA, NMR could help identify residues involved in substrate binding and characterize the flexibility of different protein regions during the transport cycle.

Complementary to experimental methods, computational approaches such as molecular dynamics (MD) simulations can model YnfA's behavior in membrane environments and predict interactions with various substrates. Homology modeling based on the structures of related transporters like EmrE can provide initial structural models for further refinement and analysis. These computational methods are particularly valuable when integrated with experimental data from mutagenesis studies or spectroscopic measurements.

The following table summarizes the advanced structural biology techniques applicable to YnfA research:

How might recombinant Salmonella Choleraesuis expressing YnfA be utilized in vaccine research?

Recombinant attenuated Salmonella strains have emerged as powerful live vaccine vectors capable of eliciting strong mucosal immune responses against various pathogens . In this context, recombinant S. Choleraesuis expressing YnfA could potentially be developed as a vaccine component, particularly if YnfA contains immunogenic epitopes relevant to protective immunity. The attenuated S. Choleraesuis vector rSC0016, which incorporates regulated delayed attenuation and delayed antigen expression systems, provides a promising platform for such applications .

When considering YnfA as a vaccine component, researchers must evaluate several factors. First, while YnfA is primarily a membrane protein involved in transport functions, certain exposed epitopes might serve as targets for antibody recognition or cellular immune responses. Second, membrane proteins can sometimes elicit strong immune responses due to their multimeric structures and stable presentation of epitopes. The design of a recombinant construct would need to optimize YnfA expression levels and cellular localization to maximize immunogenicity.

Studies with recombinant attenuated Salmonella vaccines have demonstrated their ability to induce strong mucosal immunity, cell-mediated immunity, and humoral immunity, generating a mixed Th1/Th2-type response . Similar immunological profiles might be expected with recombinant S. Choleraesuis expressing YnfA. The oral route of administration for such vaccines is particularly advantageous for stimulating mucosal immunity at sites of potential pathogen entry.

The development process would involve constructing expression plasmids containing the ynfA gene under appropriate promoters, transforming these into attenuated S. Choleraesuis strains, and evaluating expression levels and stability. Immunization studies in animal models would assess protective efficacy, immune response profiles, and safety. Similar approaches have been successful with other Salmonella-based vaccines expressing protective antigens such as P42 and P97 from Mycoplasma hyopneumoniae .

What experimental design considerations are crucial when evaluating recombinant YnfA immunogenicity?

When evaluating the immunogenicity of recombinant YnfA in the context of vaccine development, researchers must implement a comprehensive experimental design that addresses multiple immunological parameters. The study design should include appropriate control groups (e.g., vector-only control, irrelevant antigen control) and sufficient sample sizes for statistical power. Animal models should be carefully selected based on their ability to recapitulate relevant aspects of the target disease and immune response.

Immune response assessment should be multi-faceted, examining mucosal, cellular, and humoral immunity. For mucosal immunity, researchers should collect and analyze secretory IgA levels in mucosal secretions from relevant sites (e.g., intestinal, respiratory). Cellular immunity evaluation should include T-cell proliferation assays upon antigen stimulation, cytokine profiling (particularly IL-4 and IFN-γ levels), and assessment of memory T-cell populations . Humoral immunity should be assessed through measurement of YnfA-specific antibody titers in serum, including analysis of different antibody isotypes to characterize the Th1/Th2 balance.

Timing of immunological assessments is crucial, with measurements taken at multiple time points post-immunization to capture both primary and memory responses. Challenge studies, where appropriate, should evaluate protection against relevant pathogens, with endpoints including clinical symptoms, pathogen load, and histopathological evaluation of target tissues . The following table outlines key parameters to measure when evaluating recombinant YnfA immunogenicity:

Additionally, researchers should consider dose-response studies to determine optimal antigen levels and vaccination schedules. Route of administration comparisons (oral, intranasal, parenteral) may reveal differences in immune response profiles. Advanced immunological analyses, such as epitope mapping and B/T-cell receptor repertoire analysis, can provide deeper insights into the specific nature of YnfA-induced immunity.

How can YnfA serve as a target for novel antimicrobial development strategies?

The emerging role of efflux transporters like YnfA in antimicrobial resistance presents an opportunity for targeting these proteins as part of novel therapeutic strategies. Efflux pump inhibitors (EPIs) that specifically target YnfA could potentially restore bacterial susceptibility to antibiotics that would otherwise be extruded from the cell. The development of such inhibitors requires detailed understanding of YnfA's structure, substrate binding sites, and transport mechanism.

Structure-based drug design approaches, utilizing computational methods such as molecular docking and virtual screening, can identify potential inhibitor candidates that bind to critical regions of YnfA. These in silico methods can screen large compound libraries to select molecules with high predicted binding affinity and specificity for YnfA. Promising candidates from virtual screening would then undergo experimental validation through binding assays, transport inhibition studies, and evaluation of their ability to potentiate antibiotic activity against resistant strains.

Another innovative approach involves the development of "Trojan horse" antimicrobials – compounds that are recognized as substrates by YnfA but contain moieties that irreversibly bind to the transporter or deliver toxic payloads into the bacterial cell. This strategy exploits the transport function of YnfA to deliver antimicrobial agents rather than simply blocking its activity. Additionally, researchers could explore peptide-based inhibitors designed to mimic critical regions of YnfA involved in oligomerization or conformational changes, thereby disrupting its functional assembly.

The potential clinical application of YnfA inhibitors would likely involve their use in combination with existing antibiotics to overcome resistance mechanisms. This combinatorial approach could extend the useful lifespan of current antibiotics while reducing the required dosages, potentially decreasing side effects. The development pathway for such inhibitors would include in vitro efficacy studies, toxicity assessments, pharmacokinetic/pharmacodynamic analyses, and eventually clinical trials to evaluate safety and efficacy in humans.

What emerging technologies might revolutionize YnfA research in the next decade?

The research landscape for membrane proteins like YnfA is poised for transformation through several emerging technologies over the next decade. Single-particle cryo-electron microscopy (cryo-EM) continues to advance rapidly, with improvements in resolution now allowing visualization of smaller membrane proteins and their conformational states. For YnfA research, these advancements may soon enable direct visualization of substrate binding and transport mechanisms in near-native conditions, potentially revealing transient states that have been challenging to capture with traditional structural techniques.

CRISPR-Cas9 genome editing technologies offer unprecedented precision for creating YnfA variants and studying their function in native contexts. This approach allows for rapid generation of point mutations, deletions, or insertions in the ynfA gene within the bacterial genome, enabling detailed structure-function analyses. Combined with high-throughput phenotypic screening, CRISPR-based approaches could systematically map the functional landscape of YnfA residues and domains.

Artificial intelligence and machine learning algorithms are increasingly being applied to protein structure prediction, as demonstrated by breakthrough platforms like AlphaFold2. These computational tools could generate highly accurate structural models of YnfA and its variants, predict interactions with potential substrates or inhibitors, and guide experimental design. The integration of AI with molecular dynamics simulations may also enable modeling of complete transport cycles at timescales previously unattainable.

Microfluidic and organ-on-a-chip technologies present opportunities for studying YnfA function in more physiologically relevant environments. These platforms can recreate aspects of the host-pathogen interface, allowing researchers to investigate how YnfA contributes to bacterial survival and antibiotic resistance under conditions that more closely mimic in vivo settings. They also enable high-throughput screening of potential inhibitors with reduced sample requirements.

The following table summarizes emerging technologies with transformative potential for YnfA research:

These technological advances, when applied to YnfA research, have the potential to not only deepen our understanding of this specific membrane protein but also provide broader insights into bacterial transport mechanisms and antimicrobial resistance, ultimately contributing to the development of novel therapeutic strategies.