Recombinant Salmonella schwarzengrund ATP synthase subunit alpha (atpA), partial

What is ATP synthase subunit alpha (atpA) in Salmonella schwarzengrund?

ATP synthase subunit alpha is an essential component of the F1F0-ATP synthase complex in Salmonella species, including S. schwarzengrund. This enzyme catalyzes the synthesis of ATP from ADP and inorganic phosphate during oxidative phosphorylation, with an EC classification of 3.6.3.14. In bacterial systems, ATP synthase consists of two main sectors: the membrane-embedded F0 sector and the catalytic F1 sector, with the alpha subunit being a key component of the F1 sector . The atpA gene encodes this alpha subunit, which contains nucleotide binding domains and plays a critical role in energy transduction during ATP synthesis. In S. schwarzengrund, this protein shares structural and functional similarities with ATP synthase subunits in other Salmonella serovars, though with sequence variations that reflect evolutionary adaptations specific to this serovar .

How does S. schwarzengrund differ from other Salmonella serovars in terms of genomic characteristics?

S. schwarzengrund demonstrates distinct genomic features that differentiate it from other Salmonella serovars. Comparative genomic analysis of S. schwarzengrund strain S16 (the first sequenced genome in the Republic of Korea) with other S. schwarzengrund genomes revealed a pangenome of 7112 genes, a core-genome of 3374 genes, an accessory-genome of 2906 genes, and a unique-genome of 835 genes . This strain contains 81 unique genes, including various hypothetical proteins and transcriptional regulators that are not found in other serovars .

S. schwarzengrund typically belongs to sequence type ST96, which has been associated with strains isolated from poultry and environmental sources . Genomic analysis confirms the presence of 153 virulence genes in S. schwarzengrund, including the Saf operon and cdtB gene, which may contribute to its pathogenicity . Unlike many other Salmonella serovars, S. schwarzengrund has shown increasing prevalence in human infections across Asia, the United States, Denmark, and Brazil, with multiple isolates exhibiting multidrug resistance .

What are the recommended storage and handling conditions for recombinant Salmonella proteins?

Based on established protocols for similar recombinant Salmonella proteins, the following storage and handling conditions are recommended:

• Storage temperature: -20°C for regular storage, or -80°C for extended preservation

• Reconstitution: Centrifuge the vial briefly before opening to bring contents to the bottom. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Glycerol addition: Add 5-50% glycerol (final concentration) and aliquot for long-term storage at -20°C/-80°C. A 50% final glycerol concentration is typically recommended

• Freeze-thaw cycles: Repeated freezing and thawing is not recommended. Working aliquots can be stored at 4°C for up to one week

• Shelf life: Liquid form typically has a shelf life of 6 months at -20°C/-80°C, while lyophilized form can maintain stability for 12 months at -20°C/-80°C

These conditions help maintain protein integrity and activity during storage and experimental use.

How can I verify the purity and identity of recombinant Salmonella atpA protein?

Verification of recombinant Salmonella atpA protein purity and identity involves several complementary analytical techniques:

• SDS-PAGE analysis: To confirm protein size and assess purity. A well-purified recombinant protein should show >85% purity by SDS-PAGE

• Western blotting: Using anti-atpA or anti-tag antibodies to confirm protein identity

• Mass spectrometry (MS):

  • MALDI-TOF MS analysis for molecular weight confirmation

  • LC-MS/MS for peptide sequence verification against known atpA sequences

• N-terminal sequencing: To verify the start of the protein sequence matches the expected atpA sequence

• Functional assays: ATP hydrolysis activity measurements to confirm biological activity

• Tag verification: If the recombinant protein contains affinity tags, specific assays can detect their presence (e.g., anti-His antibodies for His-tagged proteins)

For experimental applications, it's recommended to perform at least three independent verification methods to ensure both identity and quality of the recombinant protein.

What methodologies are most effective for heterologous expression of Salmonella schwarzengrund ATP synthase components?

Heterologous expression of S. schwarzengrund ATP synthase components requires optimization of several parameters based on research with related Salmonella proteins:

Expression Systems Comparison:

Optimization Strategies:

• Codon optimization: Essential for membrane proteins like ATP synthase components

• Fusion partners: MBP, SUMO, or thioredoxin can improve solubility

• Temperature modulation: Lower temperatures (16-25°C) often improve proper folding

• Induction parameters: For IPTG-inducible systems, concentrations of 0.1-0.5 mM are typically optimal

Studies with recombinant Salmonella proteins have shown that yeast expression systems often provide the best balance between yield and proper folding for ATP synthase components . When expressing membrane-associated portions, detergent screening (DDM, LDAO, Fos-choline) is critical for maintaining native conformation.

How do recombination events influence the evolution of virulence and antimicrobial resistance in S. schwarzengrund?

Recombination plays a significant role in S. schwarzengrund evolution, particularly regarding virulence and antimicrobial resistance. Studies on Salmonella enterica population structure have revealed that recombination contributes substantially to genetic diversity and adaptation .

Analysis of various Salmonella lineages shows that the relative frequency and effect of recombination versus mutation (ρ/θ ratio) varies considerably across different populations, with some lineages demonstrating values as high as 2.95 . This indicates that recombination introduces approximately three times more substitutions than mutation in certain Salmonella lineages.

Regarding antimicrobial resistance in S. schwarzengrund specifically:

• Genotypic analysis confirms resistance to multiple antibiotics including amikacin, ciprofloxacin, sulfamethoxazole, streptomycin, and tetracycline

• Unlike other S. schwarzengrund genomes, strain S16 shows a distinctive mutation in gyrB

• The acquisition of resistance genes like aphA1 (found predominantly in kanamycin-resistant Salmonella isolates) occurs through horizontal gene transfer and recombination events

The prevalence of S. schwarzengrund has increased annually in certain regions, becoming the predominant serovar in some areas such as Kagoshima Prefecture in Japan . This rise correlates with increased kanamycin resistance, attributed specifically to the S. schwarzengrund population expansion . Genomic analysis indicates that recombination events facilitate the spread of resistance determinants across different Salmonella populations, with the aphA1 gene identified as the main antimicrobial resistance gene in several isolates .

What are the most effective experimental approaches for studying interactions between recombinant atpA and other components of the ATP synthase complex?

Studying interactions between recombinant atpA and other ATP synthase components requires sophisticated biophysical and biochemical techniques:

Biophysical Techniques for Protein-Protein Interaction Studies:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified recombinant atpA on sensor chips

  • Flow other ATP synthase components as analytes

  • Measure real-time binding kinetics (kon, koff)

  • Determine binding affinity (KD)

• Isothermal Titration Calorimetry (ITC):

  • Provides thermodynamic parameters (ΔH, ΔS, ΔG)

  • Quantifies binding stoichiometry

  • No protein labeling required

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps interaction interfaces between atpA and partner proteins

  • Identifies conformational changes upon binding

  • Provides regional resolution of interactions

• Cryo-Electron Microscopy:

  • Visualizes complete ATP synthase complex structure

  • Determines position and orientation of atpA

  • Identifies conformational states during catalytic cycle

Biochemical and Molecular Approaches:

• Co-immunoprecipitation with Recombinant Components:

  • Express atpA with affinity tags (His, FLAG, etc.)

  • Pull-down experiments with other subunits

  • Western blot analysis of binding partners

• Cross-linking Mass Spectrometry:

  • Use chemical cross-linkers (BS3, DSS, formaldehyde)

  • Identify cross-linked peptides by MS/MS

  • Map distance constraints between interacting regions

• Bacterial Two-Hybrid Systems:

  • Modified for membrane protein interactions

  • Quantifiable reporter gene activation

  • In vivo validation of interactions

For functional studies, reconstitution of the ATP synthase complex in liposomes followed by ATP synthesis/hydrolysis assays can verify whether recombinant atpA properly integrates into the functional complex. Additionally, site-directed mutagenesis of conserved residues can identify critical interaction points between atpA and other subunits.

How can whole-genome sequencing and comparative genomics improve our understanding of S. schwarzengrund atpA variations?

Whole-genome sequencing (WGS) and comparative genomics provide powerful approaches to understanding atpA variations in S. schwarzengrund and their functional implications:

Advances in WGS technology have become economically viable alternatives to conventional typing methods for outbreak investigation and public health surveillance . For S. schwarzengrund specifically, WGS enables:

• Phylogenetic Analysis:

  • Placement of S. schwarzengrund strains within broader Salmonella evolutionary context

  • Average Nucleotide Identity (ANI) analysis shows S. schwarzengrund strain S16 shares 98.42% identity with S. enterica subsp. enterica NCTC 12416T

  • Sequence type determination (e.g., ST96 for strain S16) links isolates to potential sources

• Comparative Genomic Analysis of atpA:

  • Identification of conserved versus variable regions in atpA across different isolates

  • Detection of selection pressure signatures (dN/dS ratios) on specific domains

  • Correlation of sequence variations with geographical distribution or host specificity

• Pangenome Analysis:

  • Determination whether atpA belongs to core genome (present in all isolates) or accessory genome

  • Analysis of flanking genomic regions for evidence of horizontal gene transfer events

  • Identification of atpA allelic variants specific to antibiotic-resistant lineages

• Structure-Function Predictions:

  • Mapping sequence variations to protein structures to predict functional impacts

  • Molecular dynamics simulations of variant atpA proteins to assess stability and interaction changes

  • Integration with experimental data to validate in silico predictions

The methodological approach would involve:

• Collecting diverse S. schwarzengrund isolates from different geographical locations and sources

• Performing whole-genome sequencing (Illumina paired-end plus Nanopore/PacBio for complete assemblies)

• Genome annotation focusing on atpA and ATP synthase operon

• Multiple sequence alignment of atpA sequences

• Construction of phylogenetic trees and recombination analysis

• Correlation of atpA sequence types with phenotypic characteristics and source information

This approach has already yielded valuable insights in related studies, such as identifying virulence determinants and antibiotic resistance mechanisms in S. schwarzengrund .

What strategies can be employed to develop recombinant vaccines using S. schwarzengrund ATP synthase components?

Development of recombinant vaccines using S. schwarzengrund ATP synthase components can build upon established approaches for Salmonella vaccine development, with specific modifications:

Recombinant Antigen Expression Strategies:

• Heterologous Expression Systems:

  • Express S. schwarzengrund atpA in attenuated Salmonella vectors

  • Target conserved epitopes with cross-protection potential

  • Previous studies with S. Typhimurium expressing heterologous O-antigens from S. Choleraesuis demonstrated protection against both homologous and heterologous challenges

• Attenuation Strategies for Live Vectors:

  • Deletion of critical virulence genes (crp/cya) for safety

  • Regulated expression systems using arabinose-inducible promoters (e.g., araC PBAD)

  • Balanced attenuation to maintain immunogenicity while ensuring safety

• Multivalent Vaccine Design:

  • Expression of multiple ATP synthase components

  • Combination with other immunogenic Salmonella antigens

  • Bivalent vaccines expressing heterologous antigens have shown 50-83% protection against heterologous challenges

Immunological Considerations:

Evaluation Protocol:

• Expression and purification of recombinant S. schwarzengrund atpA

• Construction of attenuated vaccine vectors (Δasd mutants with complementing plasmids)

• Animal immunization studies (mice, then target species)

• Evaluation of humoral (IgG, IgA) and cellular (CD4+, CD8+) responses

• Challenge studies with virulent S. schwarzengrund

• Assessment of protection percentages and bacterial burden in tissues

Previous studies using similar approaches with heterologous O-antigen expression in S. Typhimurium have demonstrated significant protection against lethal homologous challenges (100%) and substantial heterologous protection (50-83%) . This provides a promising foundation for developing effective recombinant vaccines utilizing S. schwarzengrund ATP synthase components.