Recombinant Actinobacillus pleuropneumoniae serotype 3 ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in A. pleuropneumoniae and why is it significant for research?

ATP synthase subunit c (atpE) is a critical component of the F0F1-ATP synthase complex in Actinobacillus pleuropneumoniae, a highly contagious respiratory pathogen in swine. The protein forms part of the membrane-embedded F0 portion of ATP synthase, which is essential for cellular energy production. Its significance stems from being a highly conserved membrane protein that may serve as a potential antigenic target for vaccine development. A. pleuropneumoniae causes porcine contagious pleuropneumonia, characterized by severe fibrinous necrotizing hemorrhagic pleuropneumonia and represents a significant economic threat to the swine industry worldwide . As a membrane-associated protein, atpE may contribute to the bacterial outer membrane protein (OMP) fraction, which has been identified as an important component in effective multicomponent vaccines against this pathogen .

How does atpE conservation compare across different serotypes of A. pleuropneumoniae?

ATP synthase subunit c demonstrates high conservation across the 15 known serotypes of A. pleuropneumoniae due to its essential role in energy metabolism. This conservation makes it a potentially valuable target for cross-serotype protection strategies. Research examining genomic sequences across diverse A. pleuropneumoniae isolates has revealed that certain proteins maintain high sequence homology across serotypes, particularly those involved in essential metabolic processes . This conservation contrasts with more variable surface antigens that often define serotype differences. When developing vaccines against A. pleuropneumoniae, identifying conserved antigens is crucial as current subunit vaccines containing ApxI, ApxII, and ApxIII toxins provide only partial protection against heterologous serotypes . A systematic immunoproteomic analysis of outer membrane proteins and extracellular proteins from different serotypes would be required to definitively establish the conservation status of atpE across all 15 serotypes.

What techniques can be used to express and purify recombinant atpE for research purposes?

Several expression systems can be employed for the production of recombinant A. pleuropneumoniae atpE:

For membrane proteins like atpE, expression with a fusion tag (His6, GST, or MBP) facilitates purification and can improve solubility. Following expression, purification typically involves detergent solubilization of membrane fractions followed by affinity chromatography. For structural studies, detergent exchange and additional purification steps including size exclusion chromatography would be necessary. This methodological approach aligns with techniques used to express other A. pleuropneumoniae proteins, such as those employed in previous studies where six major virulence factor genes were successfully expressed for vaccine development .

How does recombinant atpE compare to established immunogenic proteins in A. pleuropneumoniae?

The immunogenic potential of recombinant atpE should be evaluated in comparison to well-established A. pleuropneumoniae antigens:

Current research indicates that multicomponent vaccines including rApxI, rApxII, rApxIII, and rOMP provide effective cross-protection against both homologous and heterologous APP challenge . The immunoproteomic analysis of A. pleuropneumoniae has identified 30 immunogenic proteins from outer membrane and extracellular fractions . While atpE is not specifically mentioned among these identified immunogens, its membrane localization makes it a potential candidate for further investigation. To properly position atpE among established immunogens, comparative studies measuring antibody titers, survival rates, and lung lesion reduction following challenge would be necessary.

What immunization protocols best evaluate the protective efficacy of recombinant atpE?

An optimal immunization protocol to evaluate recombinant atpE would follow established approaches used for other A. pleuropneumoniae antigens:

• Animal Model Selection: BALB/c mice are commonly used for initial evaluations, followed by studies in the natural host (pigs) .

• Dose and Adjuvant Optimization:

  • Typically 50-100 μg of purified recombinant protein per dose

  • Compare multiple adjuvants: Freund's adjuvant (research only), aluminum hydroxide, oil-in-water emulsions, or commercial veterinary adjuvants

• Immunization Schedule:

  • Primary immunization followed by two booster immunizations at 2-week intervals

  • Challenge with virulent A. pleuropneumoniae strains 2-4 weeks after final immunization

• Evaluation Parameters:

  • Antibody titers (ELISA)

  • IFN-γ and IL-4 production by stimulated lymphocytes (ELISPOT)

  • Survival rates and clinical scores

  • Lung lesion scores

  • Bacterial recovery from tissues

  • Indirect immunofluorescence detection of A. pleuropneumoniae in tissues

This approach aligns with methods used to evaluate other multicomponent recombinant vaccines against A. pleuropneumoniae, where efficacy was assessed based on antibody titers, survival rates, lung lesions, and indirect immunofluorescence detection following challenge with virulent strains .

How should atpE be incorporated into multicomponent recombinant subunit vaccines?

Integration of atpE into multicomponent vaccines requires several considerations:

• Compatibility Assessment: Evaluate potential inhibitory or synergistic effects between atpE and other vaccine components through in vitro and in vivo studies.

• Optimal Ratio Determination: Test different concentration ratios of atpE to established antigens (ApxI, ApxII, ApxIII, OMP) to identify formulations that maximize protective immunity.

• Delivery Platform Options:

• Stability Testing: Evaluate the physical and chemical stability of atpE when combined with other vaccine components under various storage conditions.

Research has shown that multicomponent vaccines containing rApxI, rApxII, rApxIII, and rOMP provide superior protection compared to formulations with additional components (rApxIV, rApfa) . This finding suggests that careful selection of antigens rather than simply increasing antigen number is critical for effective vaccine design. Any new antigen, including atpE, should be evaluated for its incremental contribution to protection before inclusion in an established formulation.

How can phase variation affect the expression of atpE and other potential vaccine antigens in A. pleuropneumoniae?

Phase variation in A. pleuropneumoniae presents a significant consideration for vaccine antigen selection:

A. pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that create phasevarions (phase-variable regulons), resulting in genome-wide methylation differences within bacterial populations . These epigenetic mechanisms lead to altered expression of multiple genes, including potential vaccine antigens. Phase variation occurs through:

• Simple Sequence Repeats (SSRs): Type III DNA methyltransferases in A. pleuropneumoniae contain GCACA(n) repeat tracts in their 5' region, allowing for phase variation through slipped-strand mispairing .

• Inverted Repeats: Type I DNA methyltransferases contain duplicated variable hsdS genes with inverted repeats, enabling gene shuffling through homologous recombination .

While atpE is not specifically identified as phase-variable in the research, understanding its expression stability is crucial for vaccine development. Analysis of gene expression under different growth conditions and methylation states would determine whether atpE expression is influenced by phasevarions. The characterization of phase-variable elements in A. pleuropneumoniae is essential for "the selection of stably expressed antigens" and the "development of a rationally designed subunit vaccine" . Therefore, any vaccine candidate, including atpE, should be assessed for expression stability across different bacterial growth phases and environmental conditions.

What biotechnological approaches can improve the immunogenicity of recombinant atpE?

Several strategies can enhance the immunogenicity of recombinant atpE:

• Structural Modifications:

  • Deletion of transmembrane domains to improve solubility while preserving key epitopes

  • Creation of chimeric constructs by fusing immunodominant epitopes from multiple antigens

  • Site-directed mutagenesis to enhance stability or expose cryptic epitopes

• Expression Optimization:

  • Codon optimization for the expression host

  • Addition of secretion signals for improved yield

  • Selection of fusion partners that enhance folding and solubility

• Formulation Enhancements:

• Epitope Optimization: Computational prediction and experimental validation of B-cell and T-cell epitopes, followed by focused presentation of these regions.

These approaches align with current vaccine development strategies that have shown success for other A. pleuropneumoniae antigens. The multicomponent recombinant subunit vaccines composed of rApxI, rApxII, rApxIII, and rOMP have demonstrated effective cross-protection against both homologous and heterologous APP challenge , suggesting that similar approaches could be applied to optimize atpE as a vaccine component.

How can gene knockout or modification studies assess the role of atpE in A. pleuropneumoniae virulence?

Gene knockout or modification studies of atpE would provide valuable insights into its role in A. pleuropneumoniae biology:

• Knockout Strategy Selection:

  • Complete deletion may be lethal given the essential nature of ATP synthase

  • Conditional knockdown systems (inducible promoters)

  • Partial gene deletions or site-directed mutagenesis to alter function without eliminating it

• Transformation Methods:

  • MIV (membrane integrity/DNA uptake specificity/viscosity) transformation has been successfully used for genetic manipulation of A. pleuropneumoniae

  • Construct design should include chloramphenicol acetyltransferase gene (cat) as a selectable marker

  • Incorporation of uptake signal sequences (USS) improves transformation efficiency

• Phenotypic Characterization:

  • Growth kinetics under various conditions

  • ATP production and proton motive force measurements

  • Biofilm formation capacity

  • Resistance to environmental stresses

  • Adhesion to host cells

  • Virulence in cellular and animal models

• Complementation Studies:

  • Expression of wild-type atpE in mutant strains to confirm phenotypic restoration

  • Expression of altered versions to identify functional domains

The methodology for creating knockout mutants in A. pleuropneumoniae has been established in previous research, where genes were successfully replaced with selectable markers through homologous recombination . Transformants can be confirmed by PCR and sequencing to verify the correct insertion or replacement. This approach would provide valuable insights into the biological role of atpE and its potential as a vaccine target.

How does atpE expression change under different growth conditions and in different phases of infection?

Understanding the expression dynamics of atpE requires comprehensive analysis across various conditions:

• In vitro Expression Analysis:

  • Quantitative RT-PCR to measure transcript levels

  • Western blotting to quantify protein expression

  • Conditions to test: aerobic vs. anaerobic growth, nutrient limitation, biofilm vs. planktonic growth, different growth phases, presence of host factors

• In vivo Expression Studies:

  • Transcriptomics of bacteria recovered from infected tissues

  • In vivo expression technology (IVET) to identify promoter activity during infection

  • Immunohistochemistry to detect protein expression in infected tissues

• Regulation Mechanisms:

A. pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control phasevarions, resulting in genome-wide methylation differences and altered gene expression patterns . These systems may influence atpE expression under different conditions. Understanding how methylation patterns affect atpE expression would be crucial for vaccine development, as it would help ensure consistent antigen expression in vivo.

What structural features of atpE contribute to its immunogenicity and function?

A comprehensive structure-function analysis of atpE would examine:

• Structural Characterization:

  • Transmembrane topology prediction using bioinformatic tools

  • Protein modeling based on homologous structures

  • Advanced structural determination through X-ray crystallography or cryo-electron microscopy if feasible

• Epitope Mapping:

  • In silico prediction of B-cell and T-cell epitopes

  • Experimental validation through peptide arrays or phage display

  • Conformational epitope mapping using hydrogen-deuterium exchange mass spectrometry

• Key Functional Regions:

• Structure-Based Design: Information from structural studies could guide rational design of improved vaccine antigens through:

  • Stabilization of immunodominant epitopes

  • Display of multiple epitopes on scaffold proteins

  • Design of chimeric proteins incorporating protective epitopes from multiple antigens

This approach would build upon methodologies used for other A. pleuropneumoniae antigens. The identification of immunogenic proteins from outer membrane and extracellular fractions has provided valuable information for vaccine development , and similar approaches could be applied to thoroughly characterize atpE.

What is the current research landscape for novel vaccine approaches against A. pleuropneumoniae?

The research landscape for A. pleuropneumoniae vaccines continues to evolve:

• Current Status of Vaccine Development:

  • Traditional inactivated whole-cell vaccines provide partial protection with concerns about side effects

  • Live attenuated vaccines show promise but raise safety concerns

  • Subunit vaccines containing Apx toxins and outer membrane proteins demonstrate improved safety profiles but variable cross-protection

• Emerging Approaches:

• Integration of atpE Research: As a highly conserved membrane protein, atpE represents a potential target for next-generation vaccines, particularly if it can be shown to:

  • Elicit cross-protective antibodies

  • Demonstrate stable expression across growth conditions

  • Contribute to bacterial fitness or virulence

Current research indicates that multicomponent vaccines containing carefully selected antigens offer the best protection. The combination of rApxI, rApxII, rApxIII, and rOMP has been shown to provide effective cross-protection against both homologous and heterologous APP challenge . Additionally, the characterization of phase-variable DNA methyltransferases in A. pleuropneumoniae has highlighted the importance of selecting stably expressed antigens for rational vaccine design . These findings suggest that the integration of atpE into vaccine development efforts would need to consider both its protective potential and expression stability.

What are the most effective strategies for troubleshooting expression and purification issues with recombinant atpE?

Common challenges and solutions for recombinant atpE production:

• Expression Troubleshooting:

• Solubilization and Purification Strategies:

  • For membrane proteins, screening multiple detergents is essential (DDM, LDAO, Triton X-100)

  • Use fusion partners that enhance solubility (MBP, SUMO, thioredoxin)

  • Develop detergent-free alternatives using amphipols or nanodiscs for structural studies

  • Implement on-column refolding protocols for proteins isolated from inclusion bodies

• Quality Control Assessments:

  • Size exclusion chromatography to verify monodispersity

  • Circular dichroism to confirm secondary structure formation

  • Thermal shift assays to assess stability

  • Functional assays to verify biological activity

These approaches align with established protocols for producing other A. pleuropneumoniae proteins for vaccine research . The successful expression and purification of multiple recombinant proteins from A. pleuropneumoniae, including ApxI, ApxII, ApxIII, and OMP, demonstrates that effective production systems can be developed for bacterial membrane and secreted proteins .

How can bioinformatic approaches enhance atpE research and vaccine development?

Bioinformatic tools offer valuable insights for atpE research:

• Sequence Analysis:

  • Multiple sequence alignment to identify conserved regions across serotypes

  • Phylogenetic analysis to understand evolutionary relationships

  • Homology modeling based on ATP synthase structures from related organisms

• Epitope Prediction and Analysis:

• Structural Analysis:

  • Protein structure prediction using AlphaFold or RoseTTAFold

  • Molecular dynamics simulations to study flexibility and interactions

  • Protein-protein docking to model interactions with antibodies or other ATP synthase components

• Systems Biology Integration:

  • Analysis of gene expression data across conditions

  • Integration with metabolic models to predict effects of atpE modulation

  • Network analysis to understand relationships with virulence factors

These approaches complement experimental methods and can guide hypothesis generation. The identification of phase-variable DNA methyltransferases in A. pleuropneumoniae through bioinformatic analysis of REBASE data demonstrates how computational approaches can provide valuable insights that inform experimental design and vaccine development strategies.

What controls and validation steps are essential when evaluating atpE as a vaccine candidate?

Rigorous controls and validation steps ensure reliable results:

• Immunological Assay Controls:

• Cross-Reactivity Assessment:

  • Test recognition of homologous proteins from commensals and other pathogens

  • Evaluate potential autoimmunity through reactivity with host proteins

  • Measure antibody specificity across different A. pleuropneumoniae serotypes

• Functional Validation:

  • Opsonophagocytic assays to confirm antibody functionality

  • Complement-mediated killing assays

  • Neutralization tests if atpE contributes to specific virulence mechanisms

  • Adhesion inhibition assays if atpE plays a role in host cell interactions

• Reproducibility Considerations:

  • Biological replicates using different protein preparations

  • Technical replicates to assess assay variability

  • Independent laboratory validation of key findings

  • Use of different animal models and/or genetic backgrounds

These validation approaches are essential for meaningful evaluation of vaccine candidates. Previous studies evaluating multicomponent recombinant vaccines against A. pleuropneumoniae used rigorous controls and multiple assessment parameters, including antibody titers, survival rates, lung lesion evaluation, and indirect immunofluorescence detection . Similar comprehensive validation would be necessary to properly evaluate atpE as a potential vaccine antigen.