Recombinant Salmonella heidelberg UPF0060 membrane protein ynfA (ynfA)

How does the ynfA protein from Salmonella heidelberg compare to homologs in other Salmonella serotypes?

The ynfA protein shows high conservation across different Salmonella serotypes, similar to other membrane proteins in this pathogen. When comparing the amino acid sequences between Salmonella heidelberg and Salmonella paratyphi A, there is only a single amino acid difference at position 26, where lysine (K) in S. heidelberg is replaced by isoleucine (I) in S. paratyphi A (LWLKR vs. LWIKR) . This high degree of conservation suggests functional importance and potential cross-reactivity of antibodies against different Salmonella serotypes, similar to what has been observed with the FlgK protein, which shows >97% conservation across different Salmonella serovars .

What is the predicted structure and topology of the ynfA membrane protein?

Based on the amino acid sequence and computational analysis, ynfA is a typical integral membrane protein with multiple transmembrane domains. The protein contains hydrophobic stretches consistent with membrane-spanning regions, as indicated by its GRAVY (Grand Average of Hydropathicity) score. The presence of sequences like "LCEIIGCFLPWLWLKR" and "AAASLALFVWLLTLHP" suggests transmembrane helices that anchor the protein within the bacterial membrane . Similar to other bacterial membrane proteins, it likely has portions exposed to both the cytoplasmic and periplasmic sides of the membrane, though detailed structural studies would be needed to confirm its exact topology.

What are the potential functional roles of ynfA in Salmonella pathogenesis and virulence?

While the specific function of ynfA has not been fully characterized in the provided search results, its conservation across Salmonella serotypes suggests biological significance. By analogy with other membrane proteins in Salmonella (such as FlgK, which is involved in flagellar function), ynfA may play roles in:

• Membrane integrity and permeability

• Bacterial adhesion to host cells

• Environmental stress responses

• Transport of molecules across the bacterial membrane

• Potential involvement in antibiotic resistance mechanisms

Research approaches similar to those used for the FlgK protein could be applied to explore ynfA's role in virulence, using techniques such as gene knockout studies, adhesion assays, and infection models . The protein's high conservation across serovars (as demonstrated by the near-identical sequences between S. heidelberg and S. paratyphi A) suggests it may perform an essential function .

How can epitope mapping techniques be applied to study the immunogenic properties of ynfA?

Based on methodologies applied to other Salmonella proteins like FlgK, researchers can employ both in silico prediction and in vivo experimental approaches for epitope mapping of ynfA:

In silico prediction methods:

• Use of multiple B-cell epitope prediction software (e.g., Bepipred, Kolaskar and Tongaonkar antigenicity software)

• Analysis with VaxiJen v2.0 for physicochemical property-based epitope prediction

• Assessment of antigenicity, allergenicity, and toxicity using tools such as AllerTOP and ToxinPred

In vivo experimental approaches:

• Immunization of animal models (e.g., chickens) with recombinant ynfA protein

• Mass spectrometry-based immunoproteomics with immunized sera

• Experimental validation of predicted epitopes

This dual approach (computational and experimental) can identify consensus epitope sequences with potential for vaccine development, as demonstrated in the FlgK study where three overlapping consensus epitope sequences were identified .

What are the predicted cross-reactivity patterns of antibodies raised against ynfA across different Salmonella serotypes?

Given the high sequence conservation between ynfA proteins of S. heidelberg and S. paratyphi A (differing by only one amino acid at position 26), antibodies raised against ynfA from one serotype are likely to show strong cross-reactivity with ynfA from other serotypes . This prediction is supported by observations with the FlgK protein, where >97% conservation across serotypes led to cross-reactive antibodies.

The significance for research and diagnostics includes:

• Potential for broad-spectrum diagnostic assays targeting multiple Salmonella serotypes

• Development of vaccines with cross-protection capabilities

• Use as a universal biomarker for Salmonella detection

Experimental validation through Western blotting or ELISA using recombinant ynfA proteins from different serotypes would be necessary to confirm these cross-reactivity patterns .

What are the optimal expression and purification conditions for recombinant ynfA protein?

Based on the commercial protein specifications and general recombinant membrane protein methodologies:

Expression system:

• E. coli is the recommended expression host for recombinant ynfA

• N-terminal His-tag facilitates purification while minimizing impact on protein structure

• Expression under T7 promoter with IPTG induction is typical for these constructs

Purification protocol:

• Cell lysis under membrane-protein compatible conditions (detergent-based)

• Immobilized metal affinity chromatography (IMAC) using the His-tag

• Optional secondary purification step (ion exchange or size exclusion chromatography)

• Buffer exchange to a stabilizing formulation

Storage and handling:

• The purified protein should be stored as a lyophilized powder

• For reconstitution, use deionized sterile water to achieve 0.1-1.0 mg/mL concentration

• Add glycerol to 5-50% final concentration for long-term storage

• Store at -20°C/-80°C and avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

How can researchers assess the structural integrity and functionality of purified recombinant ynfA?

Several complementary approaches can verify the quality of purified recombinant ynfA:

Structural integrity assessment:

• SDS-PAGE analysis for purity (>90% purity is standard for research applications)

• Western blotting with anti-His antibodies to confirm full-length expression

• Circular dichroism (CD) spectroscopy to assess secondary structure elements

• Limited proteolysis to evaluate proper folding

• Size-exclusion chromatography to detect aggregation

Functional assays:

• Liposome incorporation assays to confirm membrane integration capability

• Assessment of specific binding partners (if known)

• Antibody recognition tests using antisera from Salmonella-infected hosts

• Thermal stability assays to determine protein stability

For membrane proteins like ynfA, maintaining the native structure can be challenging, and verification that the recombinant form adopts a conformation similar to the native protein is crucial for meaningful experimental results .

What are the challenges in designing immunization studies using recombinant ynfA protein?

Researchers should consider several challenges when designing immunization studies with recombinant ynfA:

Formulation challenges:

• Membrane proteins may require specific adjuvants to maintain structure and enhance immunogenicity

• Freund's incomplete adjuvant has been successfully used with other Salmonella proteins

• Protein concentration typically ranges from 50-100 μg per dose (100 μg was used for FlgK studies)

Study design considerations:

• Primary immunization followed by a booster (typically 2-3 weeks apart)

• Sample collection timing (optimal at 2 weeks post-booster)

• Control groups must include adjuvant-only and unimmunized subjects

• Appropriate animal models (chickens are relevant for Salmonella research)

Assessment methods:

• ELISA to measure antibody titers

• Mass spectrometry-based immune-capture proteomics for epitope mapping

• Challenge studies to assess protective efficacy

• Cross-reactivity testing against multiple Salmonella serotypes

The immunization protocol should be approved by the appropriate Institutional Animal Care and Use Committee, with animals housed according to standard guidelines with ad libitum access to feed and water .

How can bioinformatic approaches enhance understanding of ynfA protein properties?

Bioinformatic analysis can provide valuable insights into ynfA properties prior to experimental work:

Similar to approaches used for FlgK, multiple tools should be employed for consensus prediction. For example, epitope prediction would benefit from at least 5 different software tools to identify overlapping consensus epitope sequences .

How does ynfA compare to other membrane proteins in Salmonella as potential vaccine or diagnostic targets?

While ynfA remains less studied than other Salmonella proteins like FlgK, comparative analysis suggests several considerations:

What novel approaches could advance understanding of ynfA function in Salmonella virulence?

Several cutting-edge approaches could illuminate ynfA's role in Salmonella pathogenesis:

Genetic approaches:

• CRISPR-Cas9 gene editing to create precise ynfA knockout and point mutation strains

• Conditional expression systems to study essentiality in different growth conditions

• Transposon sequencing (Tn-seq) to identify genetic interactions

Structural biology:

• Cryo-electron microscopy of ynfA in membrane environments

• X-ray crystallography of detergent-solubilized protein

• NMR studies of specific domains or the full protein in membrane mimetics

Functional characterization:

• Bacterial two-hybrid systems to identify protein-protein interactions

• Lipidomics to assess impact on membrane composition

• Single-cell tracking of GFP-tagged ynfA during infection processes

• Comparative transcriptomics between wild-type and ynfA mutants

Immunological studies:

• Application of epitope mapping approaches similar to those used for FlgK

• Assessment of ynfA-specific T-cell responses

• Evaluation as a potential DIVA (Differentiating Infected from Vaccinated Animals) marker

How might mRNA vaccine technology be applied to ynfA-based Salmonella vaccine development?

Building on the success of mRNA vaccines for COVID-19, similar approaches could be applied to ynfA-based Salmonella vaccines:

mRNA vaccine design considerations:

• Codon optimization for enhanced expression in host cells

• Selection of optimal 5' and 3' UTRs for stability and translation efficiency

• Incorporation of modified nucleosides to reduce innate immune sensing

• Lipid nanoparticle formulation optimized for target tissue delivery

Potential advantages over protein-based vaccines:

• Avoidance of challenges associated with membrane protein purification

• In vivo expression may maintain native conformation better than recombinant proteins

• Potential for co-delivery of multiple Salmonella antigens in a single construct

• Rapid adaptation to emerging strains or serotypes

Research pathway:

• In vitro transcription and testing of ynfA mRNA constructs

• Cellular assays to confirm expression and localization

• Small animal immunogenicity studies

• Challenge studies in relevant animal models (e.g., poultry)

This approach would align with the trend noted in the literature toward applying RNA vaccine technology to bacterial pathogens following successful COVID-19 mRNA vaccine development .