Recombinant Salmonella schwarzengrund UPF0060 membrane protein ynfA (ynfA)

What is the structural characterization of Salmonella schwarzengrund UPF0060 membrane protein ynfA?

Salmonella schwarzengrund (strain CVM19633) UPF0060 membrane protein ynfA is a recombinant protein consisting of 108 amino acids (aa 1-108) . As a membrane protein, it possesses specific structural features that enable its integration into bacterial cell membranes. While detailed three-dimensional structural characterization studies specifically for ynfA remain limited in the literature, researchers typically approach membrane protein characterization through multiple complementary techniques:

• X-ray crystallography after stabilization with detergents

• Cryo-electron microscopy for visualization of native conformation

• Circular dichroism spectroscopy to analyze secondary structure components

• Nuclear magnetic resonance (NMR) spectroscopy for dynamic structural information

The amino acid sequence of ynfA protein suggests it contains transmembrane domains typical of UPF0060 family proteins, which are conserved across multiple Salmonella strains and potentially play roles in membrane integrity or transport functions.

Which expression systems are most effective for producing recombinant Salmonella ynfA protein?

Multiple expression systems can be utilized for recombinant Salmonella ynfA protein production, with specific advantages depending on research objectives:

The selection of an appropriate expression system depends on the specific research questions . For basic characterization studies, E. coli-based expression is often sufficient, while more complex functional analyses may benefit from eukaryotic expression systems that provide proper folding environments for membrane proteins. When designing expression constructs, inclusion of affinity tags (His6, GST, etc.) facilitates subsequent purification while codon optimization enhances expression efficiency in the chosen host system.

What purification strategies are most effective for isolating recombinant Salmonella ynfA protein?

Purification of recombinant Salmonella membrane proteins like ynfA requires specialized protocols that maintain protein structure and function:

• Membrane protein extraction: Use gentle detergents (n-dodecyl-β-D-maltoside, CHAPS, or Triton X-100) to solubilize the protein from membranes without denaturation

• Affinity chromatography: Utilize tag-based purification (His-tag, GST) with optimized binding and elution conditions

• Size exclusion chromatography: Remove aggregates and separate oligomeric states

• Ion exchange chromatography: Further refine purity based on charge properties

Quality assessment via SDS-PAGE typically reveals ynfA protein as a distinct band at approximately 12-15 kDa (for the 108 amino acid fragment) . Western blotting with anti-His antibodies (for His-tagged constructs) confirms identity, while mass spectrometry provides definitive molecular weight verification. The purified protein should be stored in buffer containing appropriate detergent concentrations to maintain solubility and prevent aggregation.

How can researchers verify the immunogenicity of recombinant Salmonella ynfA protein?

Verification of immunogenicity involves multiple assays:

• Antibody production assessment: Immunize laboratory animals (mice, rabbits, or chickens) with purified ynfA protein and adjuvant, followed by serum collection and antibody titer determination via ELISA

• Specificity testing: Confirm antibody specificity through Western blotting against both recombinant protein and native Salmonella lysates

• Epitope mapping: Identify specific immunogenic regions using peptide arrays or deletion mutants

• Cross-reactivity analysis: Test reactivity against related proteins from other Salmonella strains or species

ELISA-based methods can be optimized using ynfA protein at concentrations of approximately 156 ng per well, based on protocols developed for similar Salmonella outer membrane proteins . Antibody development typically follows a 28-42 day protocol with primary immunization and 1-2 booster doses to achieve high-titer antisera. The resulting antibodies can serve both as research reagents and as indicators of potential vaccine efficacy.

How can recombinant Salmonella ynfA protein be incorporated into attenuated Salmonella vaccine vectors?

The incorporation of ynfA protein into attenuated Salmonella vaccine vectors requires careful genetic engineering approaches:

• Vector selection: Choose an appropriate attenuated Salmonella strain (such as S. Choleraesuis rSC0016) with demonstrated safety and immunogenicity profiles

• Plasmid construction: Design expression plasmids with optimized promoters (e.g., nirB, pagC) for in vivo induction and balanced expression

• Transformation: Introduce the plasmid into the attenuated Salmonella strain using electroporation or chemical transformation

• Stability testing: Verify plasmid stability through multiple passages without antibiotic selection

• Expression verification: Confirm in vivo expression through Western blotting of bacterial lysates recovered from animal models

The regulated delayed attenuation and balanced lethal systems represent advanced approaches that enhance vector efficacy. For example, construction of vectors similar to those used for other Salmonella antigens can be applied, where the gene encoding ynfA is cloned into an Asd+ complemented plasmid (such as pYA3493) using appropriate restriction enzymes like EcoRI and SalI . The resulting construct can be verified by PCR amplification and restriction enzyme digestion before transformation into the attenuated Salmonella strain.

What immune responses are typically elicited by recombinant Salmonella vector vaccines expressing membrane proteins?

Recombinant Salmonella vector vaccines expressing membrane proteins like ynfA typically elicit multi-faceted immune responses:

The oral administration of recombinant Salmonella vectors induces strong mucosal immunity by effectively delivering antigens to gut-associated lymphoid tissues, which is crucial for protection against pathogens that infect via mucosal surfaces . Measurements of specific cytokines (IFN-γ for Th1 responses, IL-4 for Th2 responses) typically reveal a mixed Th1/Th2 response with Th1 predominance, as evidenced by higher numbers of IFN-γ secreting cells compared to IL-4 secreting cells in ELISPOT assays . This balanced immune response is beneficial for comprehensive protection.

What are the critical parameters for assessing protective efficacy of ynfA-expressing Salmonella vaccines in animal models?

Assessment of protective efficacy requires comprehensive evaluation across multiple parameters:

• Challenge model design:

  • Selection of appropriate challenge strain and dose

  • Determination of challenge route (oral, respiratory, etc.)

  • Establishment of challenge timeline (typically 2-4 weeks post-vaccination)

• Protection parameters:

  • Survival rates and time-to-death analysis

  • Bacterial colonization levels in target tissues

  • Weight loss/gain monitoring

  • Clinical symptom scoring

• Immunological correlates:

  • Antibody titers (mucosal and systemic)

  • Lymphocyte proliferation indices (SI values)

  • Cytokine profiles (IFN-γ/IL-4 ratios)

  • T-cell subpopulation analysis

• Histopathological evaluation:

  • Tissue damage scoring

  • Inflammatory cell infiltration quantification

  • Immunohistochemistry for pathogen detection

Protective efficacy is typically measured through challenge studies where immunized animals show reduced clinical symptoms, improved weight maintenance, decreased pathogen loads, and minimized tissue damage compared to control groups . For example, in similar Salmonella vector studies, vaccinated mice demonstrated significantly reduced weight loss, diminished lung pathological damage, and decreased inflammatory cell infiltration following challenge with the target pathogen .

How can researchers optimize antigen presentation of ynfA protein in Salmonella vectors to enhance immunogenicity?

Optimization of antigen presentation involves multiple engineering strategies:

• Subcellular localization targeting:

  • Periplasmic expression: Use appropriate signal sequences (e.g., OmpA, PhoA)

  • Surface display: Fusion with outer membrane proteins or autotransporter domains

  • Cytoplasmic expression: Direct expression without signal sequences

  • Secretion: Incorporation of type III secretion signals

• Promoter selection and regulation:

  • In vivo-inducible promoters (e.g., nirB, pagC, ssaG)

  • Delayed antigen expression systems using arabinose-regulated promoters

  • Balanced expression to avoid metabolic burden

• Codon optimization:

  • Adjust codon usage to match the Salmonella host

  • Remove rare codons that might limit translation efficiency

  • Optimize mRNA secondary structure to enhance translation initiation

• Fusion strategies:

  • Adjuvant fusion: Link ynfA to immunostimulatory molecules (flagellin, IL-2)

  • Carrier fusion: Couple with highly immunogenic carrier proteins

  • Multimer construction: Create concatemers of multiple epitopes

The regulated delayed antigen expression systems represent particularly advanced approaches that balance bacterial fitness with robust antigen production. Expression levels can be verified using quantitative Western blotting, with optimal expression typically showing antigen bands representing 1-5% of total cellular protein. These optimization strategies significantly impact vaccine efficacy, with appropriately engineered constructs demonstrating superior immune responses compared to basic expression systems .

What methods can be employed to evaluate cross-protective potential of ynfA-based vaccines against heterologous Salmonella strains?

Evaluation of cross-protection requires systematic analysis:

• Sequence analysis and epitope prediction:

  • Alignment of ynfA sequences across Salmonella strains

  • Identification of conserved and variable regions

  • B-cell and T-cell epitope prediction using algorithms (IEDB, BepiPred)

  • Construction of consensus sequences covering multiple strains

• In vitro cross-reactivity assessment:

  • ELISA and Western blot with ynfA proteins from multiple strains

  • Epitope mapping using peptide arrays

  • Competitive binding assays with sera from immunized animals

• Heterologous challenge studies:

  • Sequential challenge with different Salmonella serotypes

  • Measurement of protection parameters across strains

  • Correlation of protection with immune response breadth

• Passive transfer experiments:

  • Transfer of immune sera between animal models

  • Assessment of protection conferred against heterologous strains

  • Identification of protective antibody subclasses and epitopes

The potential for cross-protection depends largely on the conservation of immunodominant epitopes across Salmonella strains. Sequence analysis typically reveals higher conservation in functional domains, which can be targeted specifically for broader protection. Challenge studies with heterologous strains provide the most definitive evidence of cross-protection, with successful vaccines demonstrating at least 50-70% protection against related strains compared to the homologous strain.

What are the optimal ELISA protocols for detecting antibodies against Salmonella ynfA protein?

Optimized ELISA protocols for ynfA-specific antibody detection require careful parameter adjustment:

• Antigen coating optimization:

  • Recombinant ynfA protein concentration: 156 ng per well (based on similar Salmonella membrane proteins)

  • Coating buffer selection: Carbonate-bicarbonate buffer (pH 9.6)

  • Coating temperature and duration: 4°C overnight or 37°C for 2 hours

• Blocking and washing conditions:

  • Blocking buffer: 3-5% BSA or 5% skim milk in PBS

  • Blocking duration: 1-2 hours at room temperature

  • Wash buffer: PBS-T (0.05% Tween-20)

  • Wash cycles: 3-5 washes between steps

• Sample processing:

  • Serum dilutions: Two-fold serial dilutions starting at 1:100

  • Incubation conditions: 1-2 hours at 37°C or room temperature

  • Secondary antibody selection: Species-appropriate conjugates (typically HRP or AP)

• Signal development and analysis:

  • Substrate selection: TMB, ABTS, or pNPP depending on conjugate

  • Development time: 10-30 minutes (monitored for optimal signal-to-noise ratio)

  • Cutoff determination: Mean of negative controls plus 3 standard deviations

For field applications, standardized ELISA conditions can achieve sensitivity and specificity exceeding 90% when properly optimized . Comparison of different antigen preparations (whole cell, Omp extract, and recombinant protein) shows that recombinant proteins provide superior specificity while maintaining comparable sensitivity to crude preparations .

How can researchers differentiate immune responses to ynfA from responses to other Salmonella antigens?

Differential analysis of immune responses requires selective approaches:

• Competitive inhibition assays:

  • Pre-incubation of sera with purified ynfA or other Salmonella proteins

  • Measurement of differential inhibition patterns

  • Calculation of percent inhibition to quantify specificity

• Epitope-specific assays:

  • Synthesis of unique ynfA peptide fragments

  • ELISA or ELISPOT using peptide-specific antigens

  • B-cell and T-cell epitope mapping

• Absorption studies:

  • Sequential absorption of sera with various Salmonella antigen preparations

  • Analysis of reactivity before and after absorption

  • Identification of ynfA-specific antibody populations

• Recombinant antigen panels:

  • Parallel testing with multiple defined Salmonella antigens

  • Development of antibody profile signatures

  • Statistical analysis to identify ynfA-specific responses

Cross-reactivity analysis with antisera against other bacteria (E. coli, Klebsiella, etc.) confirms specificity of the assay, with properly designed tests showing no significant reactivity with heterologous antisera . The differential diagnosis can be further refined by calculating the ratio of reactivity to ynfA versus other Salmonella antigens, with ynfA-specific responses typically showing >3:1 ratios compared to non-specific antigens.

What are the critical considerations for designing recombinant Salmonella strains as vaccine vectors for ynfA expression?

Design of effective vaccine vectors involves multiple engineering considerations:

• Attenuation strategy selection:

  • Metabolic drift mutations (aroA, aroC, purA)

  • Virulence gene deletion (phoP, phoQ, sopB)

  • Regulated delayed attenuation systems

  • Combination approaches for balanced attenuation

• Plasmid stability engineering:

  • Balanced-lethal systems (e.g., Asd+ plasmids in Δasd strains)

  • Antibiotic resistance-free selection systems

  • Origin of replication optimization

  • Partition systems to prevent plasmid loss

• Expression control optimization:

  • In vivo-inducible promoters

  • Codon optimization for Salmonella host

  • Translation efficiency enhancement

  • Secretion signal selection

• Safety enhancement features:

  • Programmed cell lysis systems

  • Biocontainment mechanisms

  • Reduced inflammatory potential (e.g., modified LPS)

  • Tissue tropism modification

Recent advances incorporate sophisticated regulated delayed attenuation and regulated delayed antigen synthesis systems that balance safety with immunogenicity . For example, the recombinant S. Choleraesuis attenuated vector rSC0016 incorporates a regulated delayed attenuation system and a delayed antigen expression system while also having the sopB gene knocked out to reduce intestinal inflammatory responses . These engineered vector systems have demonstrated superior immunogenicity compared to traditional attenuation approaches.

What techniques are most effective for monitoring in vivo expression of ynfA protein in Salmonella vaccine vectors?

Monitoring in vivo expression requires specialized techniques:

• Reporter gene fusions:

  • Transcriptional fusions with luciferase, GFP, or β-galactosidase

  • Measurement of reporter activity in recovered bacteria

  • Real-time imaging for luciferase reporters in animal models

• Tissue sampling and bacterial recovery:

  • Collection of tissues at different timepoints post-immunization

  • Selective plating to recover vaccine strains

  • Preservation of samples for protein and RNA analysis

• Direct antigen detection:

  • Immunohistochemistry of tissue sections

  • Flow cytometry of recovered bacteria

  • Western blotting of tissue lysates

  • Mass spectrometry-based proteomics

• Transcriptional analysis:

  • RT-PCR of bacterial RNA from recovered tissues

  • RNA-seq for comprehensive expression profiling

  • Single-cell transcriptomics for heterogeneity assessment

For quantitative assessment, recovery of bacteria from tissues followed by Western blotting with densitometric analysis provides reliable data on in vivo expression levels. Typically, successful vectors show detectable antigen expression for at least 7-10 days post-immunization, with peak expression occurring 3-5 days after administration . This expression kinetics ensures sufficient antigen presentation to the immune system while allowing for eventual clearance of the vaccine vector.

How does ynfA compare with other Salmonella membrane proteins as a vaccine antigen?

Comparative analysis reveals important distinctions:

What are the most promising future research directions for ynfA-based vaccines and diagnostics?

Several promising research avenues emerge:

• Multi-epitope vaccine development:

  • Identification of immunodominant epitopes within ynfA

  • Combination with epitopes from other conserved antigens

  • Design of chimeric proteins with enhanced immunogenicity

  • Development of particle-based delivery systems

• Advanced delivery platforms:

  • mRNA-based delivery of ynfA antigen

  • Virus-like particles displaying ynfA epitopes

  • Nanoparticle formulations for enhanced stability

  • Plant-based expression systems for oral delivery

• Diagnostic applications:

  • Multiplex assay development incorporating ynfA with other markers

  • Point-of-care rapid tests using recombinant ynfA

  • Machine learning algorithms for pattern recognition in antibody profiles

  • Development of ynfA-based biosensors

• Structural biology approaches:

  • Detailed structural characterization of ynfA

  • Structure-based epitope design

  • Protein engineering for enhanced stability

  • Rational antigen design based on structural insights

The integration of computational approaches with experimental validation represents a particularly promising direction. For example, epitope prediction algorithms combined with experimental verification can identify highly conserved, immunogenic regions of ynfA that could serve as the basis for next-generation subunit vaccines or diagnostic targets.