Recombinant Salmonella gallinarum UPF0060 membrane protein ynfA (ynfA)

What is Salmonella gallinarum UPF0060 membrane protein ynfA and what is its structure?

Salmonella gallinarum UPF0060 membrane protein ynfA is a membrane-associated protein found in Salmonella gallinarum strain 287/91 / NCTC 13346 with UniProt accession number B5RAG1. The protein consists of 108 amino acid residues with the sequence: mLKTTLLFFVTALCEIIGCFLPWLWLKRGASVWWLLPAAASLALFVWLLTLHPAASGRVYAAYGGVYVCTALLWLRVVDGVRLTVYDWCGALIALCGmLIIVVGWGRT . The hydrophobic regions in this sequence suggest multiple transmembrane domains, consistent with its classification as a membrane protein. When working with this protein, researchers should consider its hydrophobic nature when designing expression and purification protocols.

What is known about the function of ynfA in Salmonella gallinarum pathogenesis?

While the specific function of ynfA in Salmonella gallinarum pathogenesis is not fully characterized, membrane proteins often play crucial roles in bacterial pathogenicity through mechanisms including adhesion, invasion, and immune evasion. Current research methodologies to investigate its function include gene knockout studies, protein-protein interaction assays, and experimental infection models. Studies examining the interaction between Salmonella Gallinarum and avian immune cells have shown significant differences in pathogen association with monocytes and heterophils compared to other Salmonella serovars, which may involve membrane proteins like ynfA . Researchers investigating this protein should design comparative studies between wild-type and ynfA-deficient strains to elucidate its specific contribution to virulence.

How does Salmonella gallinarum infection affect the avian immune system?

Experimental infection of commercial layers with Salmonella Gallinarum induces significant changes in white blood cell counts and serum protein profiles. Studies have shown that infection leads to leukocytosis characterized by increased lymphocyte and heterophil counts, with a particularly notable increase in heterophils causing an inversion of the heterophil:lymphocyte ratio . This response appears to be dose-dependent, with higher bacterial concentrations eliciting stronger immune responses. Additionally, infection triggers changes in acute-phase proteins, including increased ceruloplasmin, haptoglobin, and hemopexin levels, along with decreased transferrin concentrations . When designing experiments to study ynfA's role in this process, researchers should include time-course analyses to capture the dynamic nature of these immune responses.

How do different chicken lines respond to Salmonella Gallinarum infection, and how might this affect studies of ynfA?

Research has demonstrated significant differences in immune responses to Salmonella Gallinarum between chicken lines with different laying performance capabilities. High-producing chicken lines (e.g., WLA) showed a decrease in both heterophil numbers and Salmonella counts over time following infection, suggesting more effective bacterial clearance . In contrast, low-producing lines (e.g., R11) maintained more stable bacterial loads in the blood . These chicken line-dependent variations must be considered when designing experiments to study ynfA function in vivo.

The methodological approach should include:

• Selection of appropriate chicken lines for the research question

• Control groups with matched genetic backgrounds

• Sufficient sample sizes to account for individual variation

• Time-course analysis to capture dynamic immune responses

• Measurement of both bacterial load and immune parameters

How does Salmonella Gallinarum interact with different avian immune cell populations compared to other Salmonella serovars?

Flow cytometric analyses have revealed that Salmonella primarily interacts with monocytes, followed by heterophils and thrombocytes in avian blood. Comparative studies between Salmonella Enteritidis (SE) and Salmonella Gallinarum (SG) have shown higher proportions of monocytes associated with SE than with SG . This differential interaction suggests host adaptation mechanisms that may involve membrane proteins like ynfA.

The survival rates of different Salmonella serovars also vary depending on the chicken line. Both Salmonella Enteritidis and Salmonella Gallinarum showed better survival in blood from low-producing chickens (R11) compared to high-producing chickens (WLA) . This suggests that the strength of the immune defense depends on both the Salmonella serovar and the chicken line. When investigating ynfA's role in these interactions, researchers should use flow cytometry with fluorescently labeled bacteria to quantify association with specific immune cell populations.

What role might ynfA play in the host-adaptation of Salmonella Gallinarum compared to broader-host-range serovars?

Salmonella Gallinarum is host-restricted to avian species, unlike the broader-host-range Salmonella Enteritidis. The molecular basis for this host adaptation remains incompletely understood but likely involves multiple factors including membrane proteins like ynfA. Experimental data shows that Salmonella Gallinarum interacts differently with avian immune cells compared to Salmonella Enteritidis , potentially contributing to their different disease presentations in chickens.

To investigate ynfA's potential role in host adaptation, researchers should:

• Compare ynfA sequences across Salmonella serovars with different host ranges

• Create chimeric proteins or targeted mutations to identify regions critical for host-specific interactions

• Use ex vivo models like the whole blood infection assay to assess how ynfA variants affect pathogen-immune cell interactions

• Evaluate ynfA expression levels during different stages of infection and in different host environments

What are the optimal conditions for expressing and purifying recombinant Salmonella gallinarum ynfA protein?

Expressing membrane proteins like ynfA presents significant challenges due to their hydrophobic nature. Based on available information about the protein, researchers should consider these methodological approaches:

• Expression system selection:

  • E. coli systems with specialized strains (C41/C43) designed for membrane protein expression

  • Yeast expression systems (P. pastoris) for eukaryotic processing if needed

  • Cell-free expression systems to avoid toxicity issues

• Fusion tag considerations:

  • Multiple tag options are available, with selection determined during the production process

  • Consider solubility-enhancing tags (MBP, SUMO) to improve yield

  • Include a cleavable tag if native protein is required for downstream applications

• Purification strategy:

  • Two-phase extraction using detergents for initial solubilization

  • IMAC purification using histidine or other affinity tags

  • Size exclusion chromatography for final polishing

  • Store in Tris-based buffer with 50% glycerol for stability

• Storage conditions:

  • Store at -20°C for regular use

  • For extended storage, maintain at -80°C

  • Avoid repeated freeze-thaw cycles

  • Prepare working aliquots for storage at 4°C for up to one week

How can researchers design effective ex vivo models to study ynfA interactions with avian immune cells?

Based on successful experimental approaches with Salmonella Gallinarum, researchers can design ex vivo models to study ynfA-immune cell interactions using these methodological guidelines:

• Whole blood infection model:

  • Collect blood samples from chickens of defined genetic background and age

  • Use anticoagulants suitable for maintaining immune cell function

  • Prepare standardized bacterial inocula (specific CFU concentrations)

  • Maintain physiological temperature and conditions during co-incubation

  • Include appropriate negative controls (uninfected blood) and positive controls (known immunostimulants)

• Flow cytometric analysis protocol:

  • Use GFP-expressing Salmonella strains for tracking bacterial associations

  • Include appropriate antibodies for identifying avian immune cell populations

  • Set proper compensation controls for multi-color analysis

  • Analyze both percentage of cells associated with bacteria and the intensity of association

• Survival assays:

  • Perform time-course sampling (e.g., every 30 minutes for 4 hours)

  • Use appropriate dilution series and selective media for bacterial enumeration

  • Calculate survival rates relative to initial inoculum concentration

What considerations are important when designing ELISA assays using recombinant ynfA?

When developing ELISA assays using recombinant Salmonella gallinarum UPF0060 membrane protein ynfA, researchers should consider:

• Antigen preparation:

  • Use highly purified recombinant protein (50 μg or another appropriate quantity)

  • Ensure proper folding of the protein, particularly for conformational epitopes

  • Determine optimal coating concentration through titration experiments

  • Consider the impact of tags on antibody recognition

• Assay optimization:

  • Test multiple blocking agents to minimize background

  • Optimize primary and secondary antibody dilutions

  • Determine appropriate incubation times and temperatures

  • Validate specificity against related proteins from other Salmonella serovars

• Controls and standards:

  • Include positive and negative control samples

  • Prepare a standard curve using purified ynfA protein

  • Include controls for non-specific binding

How should researchers analyze and interpret changes in leukocyte populations following Salmonella Gallinarum infection?

When analyzing leukocyte responses to Salmonella Gallinarum infection, which may involve ynfA interactions, researchers should:

• Quantitative analysis approach:

  • Monitor absolute counts of different leukocyte populations (heterophils, lymphocytes, monocytes, thrombocytes)

  • Calculate heterophil:lymphocyte ratios as an indicator of stress and infection severity

  • Perform time-course analysis to capture dynamic changes

  • Compare infected vs. non-infected controls at each time point

• Statistical considerations:

  • Use appropriate statistical tests for time-course data (repeated measures ANOVA)

  • Account for individual variation within experimental groups

  • Consider both statistical and biological significance

• Interpretation framework:

  • Early infection (24h post-infection): Look for initial changes in leukocyte distribution

  • Mid-infection (3-5 days): Evaluate leukocytosis and shifts in cell population ratios

  • Late infection (7+ days): Assess resolution patterns or chronic inflammation markers

The typical pattern observed in experimental infections includes leukocytosis characterized by increased heterophil and lymphocyte counts, with a proportionally higher increase in heterophils leading to an inversion of the heterophil:lymphocyte ratio . This pattern may vary depending on bacterial dose, chicken line, and age.

How can researchers effectively analyze the interaction between ynfA and specific immune cell populations?

To analyze interactions between ynfA and immune cells, researchers should:

• Flow cytometry analysis approach:

  • Use fluorescently labeled recombinant ynfA or ynfA-expressing bacteria

  • Quantify both the percentage of cells interacting with ynfA and the intensity of interaction

  • Perform time-course analysis to capture dynamic interactions

  • Compare different immune cell populations (monocytes, heterophils, thrombocytes, lymphocytes)

• Data visualization:

  • Present both percentage data and mean fluorescence intensity

  • Use multi-parameter plots to identify cell subpopulations with differential ynfA interactions

  • Include time-course graphs to show dynamic changes

• Interpretation guidelines:

  • Consider differential interactions across cell types (typically monocytes > heterophils > thrombocytes)

  • Evaluate chicken line-dependent variations in interaction patterns

  • Compare ynfA interactions to those of other bacterial proteins

What approaches should be used to analyze acute-phase protein responses in relation to ynfA exposure?

Acute-phase proteins show characteristic changes during Salmonella Gallinarum infection. When analyzing these responses in relation to ynfA, researchers should:

• Protein analysis methodology:

  • Use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation

  • Quantify protein concentrations in g/dL for total protein and mg/dL for specific fractions

  • Monitor positive acute-phase proteins (ceruloplasmin, haptoglobin, hemopexin) and negative acute-phase proteins (transferrin, albumin)

• Data analysis approach:

  • Track protein kinetics over time, noting that different proteins may peak at different timepoints

  • Compare experimental groups with appropriate statistical tests

  • Analyze relationships between bacterial load and protein concentration changes

• Interpretation framework:

  • Consider the different kinetics of acute-phase proteins (ceruloplasmin typically showing early increases, followed by haptoglobin and hemopexin)

  • Evaluate differences between high and low bacterial dose groups

  • Correlate changes in acute-phase proteins with other immunological parameters

The table below summarizes typical acute-phase protein responses observed in experimental Salmonella Gallinarum infection:

These patterns provide a framework for interpreting the potential immunomodulatory effects of recombinant ynfA protein .

How might recombinant ynfA be utilized in vaccine development against Salmonella Gallinarum?

The potential application of recombinant ynfA in vaccine development would require:

• Immunogenicity assessment:

  • Evaluate antibody responses to purified recombinant ynfA in different chicken lines

  • Assess T-cell responses using appropriate assays

  • Determine cross-reactivity with other Salmonella serovars

• Vaccine formulation considerations:

  • Test different adjuvants to enhance immunogenicity

  • Compare subunit vaccine approaches vs. live-attenuated strategies incorporating ynfA

  • Evaluate mucosal vs. systemic delivery routes

• Efficacy testing framework:

  • Perform challenge studies with Salmonella Gallinarum after immunization

  • Analyze markers of protection including antibody titers, bacteremia levels, and clinical scores

  • Compare protection in different chicken lines (high vs. low producers)

Researchers should note that chicken line differences in immune responses to Salmonella infection may significantly impact vaccine efficacy, requiring testing in diverse genetic backgrounds.

What new research directions could advance our understanding of ynfA function?

Promising research directions include:

• Structural biology approaches:

  • Determine the three-dimensional structure of ynfA using X-ray crystallography or cryo-EM

  • Identify potential binding partners or ligands

  • Map functional domains through targeted mutagenesis

• Systems biology integration:

  • Perform transcriptomic analysis of host responses to purified ynfA

  • Compare host gene expression patterns between wild-type and ynfA-deficient Salmonella strains

  • Integrate proteomic and metabolomic data to build comprehensive models of ynfA function

• Comparative genomics:

  • Analyze sequence conservation and variation in ynfA across Salmonella serovars

  • Identify potential selection pressures acting on the ynfA gene

  • Correlate sequence variations with host range and virulence phenotypes

These approaches would provide a more comprehensive understanding of ynfA's role in Salmonella Gallinarum pathogenesis and host-pathogen interactions.

What are the common pitfalls in working with recombinant membrane proteins like ynfA?

Researchers working with recombinant ynfA should anticipate and address these common challenges:

• Expression challenges:

  • Low yield due to toxicity or inclusion body formation

  • Improper folding affecting functionality

  • Potential solutions include using lower induction temperatures, specialized host strains, and fusion tags

• Purification obstacles:

  • Aggregation during extraction and purification

  • Co-purification of host proteins or lipids

  • Detergent selection critical for maintaining protein structure and function

• Storage and stability issues:

  • Loss of activity during freeze-thaw cycles

  • Protein precipitation over time

  • Use of Tris-based buffer with 50% glycerol can improve stability

  • Prepare working aliquots to avoid repeated freeze-thaw cycles

• Functional assay limitations:

  • Difficulty distinguishing specific from non-specific effects

  • Challenges in maintaining native conformation in different assay conditions

  • Need for appropriate negative controls and complementation tests