Recombinant Campylobacter fetus subsp. fetus ATP synthase subunit alpha (atpA), partial

Basic Research Questions

• What is the structure and function of ATP synthase subunit alpha in Campylobacter fetus?

  The ATP synthase subunit alpha (atpA) is part of the F1 complex of the ATP synthase enzyme. This enzyme synthesizes ATP using an electrical and chemical gradient to bind ADP to inorganic phosphate, forming a new phosphate-phosphate bond and releasing ATP as the final product . The enzyme consists of two main components: the F0 complex embedded in the membrane and the F1 complex that spins like a motor to catalyze ATP synthesis . The alpha subunit plays a crucial role in the catalytic activity of the enzyme. Methodologically, researchers can study the structure-function relationship through protein crystallography, cryo-electron microscopy, and enzymatic activity assays under varying conditions.

• How is the atpA gene utilized in molecular typing and identification of Campylobacter species?

  The atpA gene serves as one of the key loci in Multilocus Sequence Typing (MLST) systems for Campylobacter species . MLST analyzes sequence variations in multiple housekeeping genes to differentiate strains and identify clonal lineages. For Campylobacter, atpA sequences have been categorized into distinct phylogenetic groups (as shown in the table below), with specific alleles predominantly associated with certain species .

  Locus	Group	Alleles	% Nucleotide identity to other C. jejuni alleles
  atpA	II	17, 28, 36, 37, 38, 41, 42, 67, 68, 73, 74, 75, 76, 79	85-88 (85-88 between groups I and II)
  	III	56	85-88

  The methodological approach involves PCR amplification of the atpA gene followed by sequencing and comparison to reference databases to assign allele numbers and sequence types.

• What are the optimal storage and handling conditions for recombinant C. fetus ATP synthase subunit alpha?

  Based on similar recombinant proteins, the optimal storage conditions for recombinant C. fetus ATP synthase subunit alpha typically include:

  • Storage temperature: -20°C for short-term storage and -20°C to -80°C for extended storage

  • Avoiding repeated freeze-thaw cycles, with working aliquots kept at 4°C for up to one week

  • Addition of glycerol (typically 5-50% final concentration) for long-term storage

  • Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  Proper handling ensures protein stability and activity for experimental applications.

Advanced Research Questions

• How does pH affect the structure and function of C. fetus ATP synthase, and what are the implications for pathogenesis?

  Recent research has begun examining ATP synthase under acidic conditions, which is physiologically relevant as mitochondria often become acidic in cells affected by diseases such as cancer and cardiac ischemia . A study led by Stuti Sharma at Stony Brook University revealed new insights into ATP synthase function by examining it at an acidic state just below neutral on the pH scale .

  Methodologically, researchers can investigate pH effects through:

  • Structural studies using cryo-electron microscopy at varying pH levels

  • Enzymatic activity assays across pH gradients

  • Molecular dynamics simulations to predict conformational changes

  • Correlation of pH-dependent activity with pathogen survival in host environments

  Understanding these pH-dependent changes is particularly relevant for C. fetus pathogenesis, as the bacterium must adapt to varying pH environments within hosts.

• What genetic variations exist in atpA genes across Campylobacter species, and how do they impact evolutionary relationships?

  Multilocus sequence typing has revealed significant genetic diversity in atpA genes across Campylobacter species. The table below demonstrates the diversity of atpA alleles in different Campylobacter isolates:

  Name	Type	atpA	Source	Year	Location
  C. coli RM1908	I	36	Swine	Unknown	USA (Texas)
  C. coli RM1908	II	36	Swine	Unknown	USA (Texas)
  C. lari RM2816	I	13	Seawater	1989	UK
  C. lari RM2816	II	1	Seawater	1989	UK
  C. upsaliensis RM3949	I	1	Canine	1998	USA (California)
  C. upsaliensis RM3949	II	1	Canine	1998	USA (California)
  C. upsaliensis RM4048	I	7	Human	1997	South Africa
  C. upsaliensis RM4048	II	5	Human	1997	South Africa

  Phylogenetic analyses have identified three distinct groups of atpA alleles, with approximately 85-88% nucleotide identity between groups . This genetic diversity impacts phylogenetic relationships and may reflect adaptation to different ecological niches.

• How does horizontal gene transfer of atpA affect the evolution and adaptation of Campylobacter fetus subspecies?

  Evidence of putative lateral transfer among thermotolerant Campylobacter species has been documented, including the transfer of atpA alleles . For example, the study identified Campylobacter coli strains containing both C. jejuni and C. coli MLST loci .

  Methodological approaches to study horizontal gene transfer include:

  • Comparative genomic analyses across Campylobacter species

  • Phylogenetic incongruence testing to identify discordant gene histories

  • Analysis of GC content and codon usage bias

  • Experimental verification through transformation assays

  These horizontal transfer events may contribute to bacterial adaptation to new environments and hosts, potentially influencing virulence and host specificity.

Experimental Design and Methodology

• What are the optimal expression conditions for producing recombinant C. fetus ATP synthase subunit alpha in E. coli systems?

  Based on information from similar recombinant proteins, the optimal expression system typically includes:

  • Expression host: E. coli is commonly used for recombinant protein production

  • Purification strategy: Affinity chromatography followed by additional purification steps

  • Quality assessment: SDS-PAGE analysis to confirm >85% purity

  • Tag considerations: Selection of appropriate fusion tags to enhance solubility and facilitate purification

  Researchers must systematically optimize expression temperature, induction conditions, and buffer compositions to maximize yield and maintain protein function. Experimental designs should include controls to verify protein activity after purification.

• How can researchers design experiments to investigate the role of ATP synthase in antimicrobial resistance in C. fetus?

  ATP synthase is a known drug target for various infectious diseases . To investigate its role in antimicrobial resistance, researchers could design experiments including:

  • Comparative genomics of atpA sequences from susceptible and resistant C. fetus isolates

  • Site-directed mutagenesis to introduce or revert mutations associated with resistance

  • Enzyme inhibition studies using known ATP synthase inhibitors like bedaquiline

  • Measurement of ATP production in resistant strains under antimicrobial pressure

  • Development of combination therapies targeting both ATP synthase and conventional antibiotic targets

  These approaches would provide valuable insights into potential resistance mechanisms and guide the development of new therapeutic strategies.

• What methodological approaches are most effective for studying ATP synthase function under oxygen-limited conditions relevant to C. fetus pathogenesis?

  Since mitochondria often turn acidic in cells affected by oxygen-deficient or hypoxic conditions like cancer and cardiac ischemia , studying ATP synthase under these conditions is physiologically relevant. Methodological approaches include:

  • Anaerobic chamber cultivation of C. fetus

  • Oxygen-controlled bioreactors for bacterial growth

  • Measurement of ATP production under varying oxygen concentrations

  • RNA-seq analysis to assess transcriptional responses to oxygen limitation

  • Metabolomic studies to map energy pathway utilization under hypoxic conditions

  These approaches would help understand how C. fetus adapts its energy metabolism during host colonization and infection, potentially revealing new targets for intervention.

Data Analysis and Interpretation

• How should researchers interpret conflicting phylogenetic signals in atpA sequences from different C. fetus isolates?

  Conflicting phylogenetic signals may arise from horizontal gene transfer events, as observed in some Campylobacter species with atpA alleles . To address this challenge:

  • Employ multiple phylogenetic inference methods (Maximum Likelihood, Bayesian)

  • Use recombination detection programs to identify potential recombination breakpoints

  • Analyze individual domains of the protein separately

  • Develop network-based phylogenetic representations rather than strictly bifurcating trees

  • Compare phylogenies from multiple genes to identify incongruent patterns

  This comprehensive approach allows researchers to distinguish between vertical inheritance and horizontal transfer, providing a more accurate understanding of evolutionary relationships among C. fetus isolates.

• What statistical approaches are most appropriate for analyzing sequence variation in atpA genes across environmental and clinical C. fetus isolates?

  To analyze sequence variation effectively, researchers should consider:

  • Calculation of nucleotide diversity (π) and haplotype diversity

  • Tajima's D test to detect selection pressures

  • FST analyses to quantify genetic differentiation between populations

  • AMOVA (Analysis of Molecular Variance) to partition genetic variation

  • Regression analyses to correlate genetic distances with ecological or clinical variables

  These statistical approaches would help identify patterns of genetic variation associated with host adaptation, geographic distribution, or clinical outcomes.

• How can researchers assess the potential of C. fetus ATP synthase as a vaccine target through epitope prediction and immunoinformatics?

  To evaluate the potential of C. fetus ATP synthase as a vaccine target:

  • Perform in silico epitope prediction using algorithms like IEDB, BepiPred, and NetMHC

  • Assess epitope conservation across C. fetus strains

  • Evaluate potential cross-reactivity with host ATP synthase

  • Use structural modeling to map epitopes to accessible regions of the protein

  • Design validation experiments including peptide-based ELISA and T-cell activation assays

  This integrated computational and experimental approach would identify promising antigenic regions while minimizing the risk of autoimmunity, guiding rational vaccine design.