Recombinant Edwardsiella ictaluri ATP synthase subunit alpha (atpA), partial

What is Edwardsiella ictaluri and why is its ATP synthase significant in research?

Edwardsiella ictaluri is a Gram-negative facultative anaerobe that causes enteric septicemia in catfish, representing a major economic concern in aquaculture. This pathogen can survive inside catfish phagocytes, utilizing complex mechanisms including the Type VI Secretion System (T6SS) for intracellular survival . ATP synthase, particularly its alpha subunit (atpA), is essential for energy metabolism in bacterial pathogens and potentially contributes to pathogen survival under stress conditions in host environments. Understanding these components provides critical insights into bacterial persistence mechanisms and may identify potential therapeutic targets.

How does ATP synthase function in bacterial energy metabolism?

ATP synthase is a multi-subunit enzyme complex that couples electrochemical potential across the bacterial membrane to ATP synthesis. The F1 sector contains the catalytic core where the alpha subunit (atpA) works together with the beta subunit to form nucleotide binding sites necessary for ATP synthesis. In bacteria like E. ictaluri, ATP synthase is critical for both oxidative phosphorylation and maintaining proton homeostasis. While less information is available specifically about E. ictaluri atpA, studies on related bacterial ATP synthases indicate the alpha subunit contains nucleotide-binding domains and contributes to the conformational changes required for catalysis.

What structural characteristics distinguish atpA from other ATP synthase subunits?

The ATP synthase alpha subunit typically exhibits a three-domain architecture consisting of an N-terminal beta-barrel domain, a central nucleotide-binding domain, and a C-terminal domain involved in subunit interactions. Based on homology with related bacterial ATP synthases, E. ictaluri atpA likely contains highly conserved residues involved in nucleotide binding, including lysine and threonine in the Walker A motif, and glutamate in the Walker B motif. Unlike atpB (ATP synthase subunit a), which is a membrane-embedded subunit of the F0 sector , atpA is part of the water-soluble F1 sector that extends into the cytoplasm.

What expression systems are optimal for producing recombinant E. ictaluri atpA?

For optimal expression of recombinant E. ictaluri atpA, researchers should consider multiple expression parameters:

• Expression host: E. coli BL21(DE3) strains are typically suitable for ATP synthase component expression, similar to the approach used for other bacterial ATP synthase components .

• Vector design: Vectors with T7 promoters and appropriate fusion tags (His-tag is common) facilitate expression and subsequent purification. The placement of the tag (N-terminal vs. C-terminal) should be empirically determined, as observed with the atpB subunit where N-terminal His-tagging proved effective .

• Expression conditions: Optimization through temperature variation (16-30°C), IPTG concentration adjustment (0.1-1.0 mM), and induction time testing (4-24 hours) is critical for maximizing yield while maintaining proper folding.

What purification strategies yield the highest purity for recombinant atpA protein?

Based on methodologies applied to similar recombinant ATP synthase components, a multi-step purification approach is recommended:

• Initial capture using Immobilized Metal Affinity Chromatography (IMAC) with Ni-NTA resin for His-tagged protein, similar to the approach documented for atpB .

• Secondary purification using either ion exchange chromatography or size exclusion chromatography to remove co-purifying contaminants.

• Quality assessment via SDS-PAGE to confirm purity above 90%, comparable to the standards applied for commercial recombinant ATP synthase components .

The purification buffer composition should be optimized to maintain protein stability, typically including:

• 50 mM Tris-HCl or phosphate buffer (pH 7.5-8.0)

• 100-300 mM NaCl to maintain solubility

• Potential stabilizing additives (5-10% glycerol, 1 mM DTT)

• Protease inhibitors during initial lysis steps

What storage and handling protocols maximize stability of purified atpA?

For optimal stability of purified recombinant E. ictaluri atpA, researchers should implement the following evidence-based protocols:

• Storage temperature: Store at -20°C/-80°C for extended storage, with -80°C preferred for long-term preservation .

• Cryoprotection: Add glycerol to a final concentration of 5-50% (typically 50%) before freezing to prevent ice crystal formation and protein denaturation .

• Aliquoting: Divide purified protein into small working aliquots to avoid repeated freeze-thaw cycles, which significantly reduce activity .

• Working stock handling: Short-term working aliquots can be stored at 4°C for up to one week .

• Lyophilization consideration: Lyophilized forms typically have longer shelf life (12 months) compared to liquid forms (6 months) .

What methods can verify the structural integrity and activity of recombinant E. ictaluri atpA?

A comprehensive approach to verifying structural integrity and functionality includes:

• Structural integrity assessment:

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

  • Differential scanning fluorimetry to determine thermal stability

  • Size exclusion chromatography to assess oligomeric state

  • Limited proteolysis to probe proper folding

• Functional characterization:

  • Nucleotide binding assays using fluorescent ATP analogs

  • ATPase activity measurements (when reconstituted with other subunits)

  • Interaction studies with other ATP synthase components

• Identity confirmation:

  • Western blotting with anti-His antibodies and/or specific anti-atpA antibodies

  • Mass spectrometry for accurate molecular weight determination and peptide mapping

  • N-terminal sequencing to confirm the correct start of the protein

How can researchers study interactions between atpA and other ATP synthase subunits?

For investigating protein-protein interactions involving E. ictaluri atpA:

• In vitro reconstitution studies:

  • Co-expression of multiple ATP synthase subunits

  • Sequential addition of purified subunits to monitor complex formation

  • Analysis of assembled complexes by blue native PAGE or analytical ultracentrifugation

• Interaction mapping techniques:

  • Pull-down assays using differentially tagged subunits

  • Surface plasmon resonance to determine binding kinetics

  • Chemical cross-linking followed by mass spectrometry to identify interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry to map binding surfaces

• Functional reconstitution:

  • Liposome reconstitution to assess proton-pumping activity

  • ATP synthesis/hydrolysis measurements of reconstituted complexes

What approaches help resolve inconsistent results when working with recombinant atpA?

When facing experimental inconsistencies, researchers should systematically:

• Assess protein quality:

  • Verify protein identity and integrity through mass spectrometry

  • Check for degradation using western blotting

  • Evaluate batch-to-batch variation with activity assays

  • Examine potential aggregation using dynamic light scattering

• Optimize experimental conditions:

  • Test multiple buffer compositions (varying pH, salt concentration)

  • Evaluate the effect of different additives (reducing agents, metal ions)

  • Control temperature and incubation times precisely

  • Use freshly prepared reagents

• Implement standardization protocols:

  • Document detailed procedures for all experimental steps

  • Use internal controls consistently

  • Establish quantitative acceptance criteria for each assay

  • Implement statistical analysis for proper interpretation of variability

How can E. ictaluri atpA research contribute to vaccine development against fish pathogens?

ATP synthase components represent potential vaccine candidates due to their essential nature and surface accessibility in some bacteria. A methodical approach for vaccine development includes:

• Immunogenicity assessment:

  • Evaluation of recombinant atpA in generating specific antibody responses in fish

  • Testing of different administration routes (injection, immersion, oral delivery)

  • Measurement of both humoral and cell-mediated immune responses

• Protective efficacy studies:

  • Challenge trials with vaccinated fish exposed to virulent E. ictaluri

  • Quantification of survival rates and bacterial loads

  • Histopathological examination to assess infection progression

• Delivery system development:

  • Integration into balanced-lethal plasmid systems, similar to the asdA complementation approach described for E. ictaluri

  • Incorporation into nanoparticle or microencapsulation formulations for oral delivery

  • Combination with adjuvants to enhance immune responses

What role might atpA play in E. ictaluri virulence and pathogenesis?

While direct evidence for atpA's role in E. ictaluri virulence is limited, several research approaches can elucidate its potential contributions:

• Gene knockout/knockdown studies:

  • Creation of atpA conditional mutants (complete knockout may be lethal)

  • Assessment of growth under various environmental conditions

  • Evaluation of intracellular survival in fish macrophages, building on methodologies used for studying other E. ictaluri virulence factors

• Comparative expression analysis:

  • Transcriptomics to compare atpA expression during infection versus in vitro growth

  • Proteomic analysis under conditions mimicking the host environment

  • Correlation with expression of known virulence factors

• Host-pathogen interaction studies:

  • Localization of ATP synthase components during infection

  • Evaluation of host immune recognition of atpA

  • Assessment of potential moonlighting functions beyond energy metabolism

How can structural biology approaches enhance understanding of E. ictaluri atpA?

Structural biology provides crucial insights into function and potential targeting approaches:

• Structure determination methods:

  • X-ray crystallography of purified atpA (alone or in complex with nucleotides)

  • Cryo-electron microscopy of reconstituted ATP synthase complexes

  • NMR spectroscopy of domains or fragments to capture dynamic properties

• Computational approaches:

  • Homology modeling based on solved structures from related bacteria

  • Molecular dynamics simulations to study conformational changes

  • Virtual screening for potential inhibitors targeting atpA-specific features

• Structure-guided functional studies:

  • Site-directed mutagenesis of predicted catalytic residues

  • Domain swapping experiments with homologs from other species

  • Design of chimeric proteins to map species-specific functions

How does E. ictaluri atpA compare with homologous proteins in other bacterial species?

Comparative analysis provides evolutionary and functional insights:

What can proteomic approaches reveal about E. ictaluri atpA regulation during infection?

Proteomic studies offer insights into expression patterns and post-translational modifications:

• Comparative proteomics approaches:

  • 2D gel electrophoresis coupled with mass spectrometry, similar to methods used to study other E. ictaluri proteins under stress conditions

  • Quantitative techniques (iTRAQ, SILAC, label-free quantification) to measure atpA abundance during different infection stages

  • Enrichment techniques to study low-abundance interacting partners

• Post-translational modification analysis:

  • Phosphoproteomics to identify regulatory phosphorylation sites

  • Redox proteomics to assess oxidative modifications under stress conditions

  • Identification of other potential modifications that regulate activity

• In vivo studies:

  • Isolation of bacterial proteins from infected fish tissues

  • Analysis of bacterial protein expression in different fish organs

  • Correlation with host immune response markers