Recombinant Haemophilus parasuis serovar 5 ATP synthase subunit c (atpE)

What is the structure and amino acid sequence of Haemophilus parasuis serovar 5 ATP synthase subunit c (atpE)?

The full-length recombinant Haemophilus parasuis serovar 5 ATP synthase subunit c (atpE) is an 84-amino acid protein. The complete amino acid sequence is:

METVITATIIGASILLAFAALGTAIGFAILGGKFLESSARQPELASSLQTKMFIVAGLLDAIAMIAVGISLLFIFANPFIDLLK

Structurally, atpE functions as part of the F0 sector of ATP synthase, participating in proton translocation across the bacterial membrane. The protein contains hydrophobic regions that facilitate its membrane insertion and proper functioning in the ATP synthase complex. When expressed recombinantly with an N-terminal His-tag, the protein maintains its structural integrity while allowing for simplified purification protocols .

How does atpE function within the ATP synthase complex of H. parasuis?

AtpE functions as subunit c within the F0 sector of ATP synthase, playing a crucial role in proton translocation across the bacterial membrane. This subunit forms a ring structure in the membrane that rotates as protons pass through, converting the proton gradient energy into mechanical energy that drives ATP synthesis.

Within the ATP synthase complex, different subunits perform complementary functions:

AtpE contributes to energy generation in H. parasuis, which is essential for bacterial survival and pathogenicity. The protein's highly conserved nature across different serovars makes it an attractive target for broad-spectrum vaccine development.

What expression systems are most effective for producing recombinant H. parasuis atpE?

E. coli expression systems have proven most effective for recombinant production of H. parasuis atpE. When expressing the full-length protein (1-84 amino acids), an N-terminal His-tag fusion significantly improves solubility and facilitates purification via affinity chromatography .

Key methodological considerations include:

• Codon optimization for E. coli expression

• Selection of appropriate promoter systems (T7 promoter systems work efficiently)

• Induction conditions optimization (IPTG concentration, temperature, duration)

• Cell lysis methods that preserve protein integrity

• Purification protocols that yield >90% purity as determined by SDS-PAGE

It's important to note that membrane proteins like atpE can present expression challenges. While F0 subunits like atpE are generally more amenable to recombinant expression than soluble F1 subunits, optimizing buffer conditions and detergent selection during purification is critical for maintaining structural integrity.

How does recombinant atpE contribute to protective immunity against H. parasuis infection?

Recombinant atpE has demonstrated significant immunoprotective potential against H. parasuis infection. As a secreted protein, atpE stimulates both humoral and cell-mediated immune responses in animal models. Specifically, immunization with recombinant atpE elicits:

• Robust antibody production with high specific IgG titers

• Enhanced cytokine secretion, particularly IL-2 and IFN-γ, indicating Th1-type immune response activation

• Increased CD4+ and CD8+ T cell proliferation, with CD4+ T cells showing more pronounced expansion

In challenge studies using mouse models, animals immunized with recombinant outer membrane proteins (OMPs) including atpE showed significant protection against lethal H. parasuis challenge. While single-antigen immunization provides partial protection, combining atpE with other immunogenic proteins enhances protective efficacy significantly .

The bactericidal activity of whole blood from immunized animals shows that anti-atpE antibodies contribute to bacterial clearance, with combination antigen formulations demonstrating superior bactericidal capacity compared to single-antigen approaches .

What methodologies are most effective for evaluating the immunogenicity of recombinant atpE?

Comprehensive evaluation of recombinant atpE immunogenicity requires multiple complementary approaches:

• Antibody response assessment:

  • Indirect ELISA for measuring antigen-specific serum IgG titers

  • Western blotting for confirming antibody specificity

  • Bactericidal activity assays using whole blood from immunized animals

• Cell-mediated immunity evaluation:

  • Flow cytometry analysis of CD4+ and CD8+ T cell proliferation

  • Cytokine profiling (particularly IL-2, IFN-γ) using ELISA or multiplex assays

  • Lymphocyte proliferation assays upon antigen re-stimulation

• Protection efficacy studies:

  • Challenge experiments with virulent H. parasuis strains

  • Survival rate monitoring

  • Bacterial load quantification in tissues

  • Histopathological examination of target organs

Research has demonstrated that mice immunized with recombinant proteins showed significantly higher antigen-specific antibody responses compared to control groups. The triple-antigen formulation containing atpE elicited the strongest immune response, with antibody titers reaching >1:10,000 dilutions in ELISA assays .

How does atpE expression change during H. parasuis biofilm formation?

ATP synthase components, including atpE, show differential expression during biofilm formation compared to planktonic growth. Transcriptomic analysis of H. parasuis biofilms reveals:

• Altered energy metabolism gene expression patterns during biofilm development

• Differential regulation of ATP synthase components as biofilms mature

• Correlation between biofilm maturation stages and energy production pathway shifts

H. parasuis biofilms develop rapidly during the first 48 hours and stabilize by 60 hours. During this development, proteins and DNA form a significant proportion of the extracellular matrix. Genes involved in ATP metabolism show expression changes that correlate with biofilm developmental stages .

While transcriptomic studies have identified several differentially expressed genes (DEGs) involved in bacterial colonization and adhesion (artM, artQ, ssrS, pflA, and HutX), further functional validation is needed to precisely characterize atpE's role in biofilm formation. Current research indicates that metabolic pathway genes, including those involved in ATP synthesis, show significant enrichment in functional gene analysis of biofilm-forming H. parasuis .

What are the optimal storage and handling conditions for maintaining recombinant atpE stability?

Maintaining stability of recombinant atpE requires specific storage and handling protocols:

• Initial storage:

  • Store lyophilized powder at -20°C/-80°C upon receipt

  • Aliquoting is necessary for multiple use to avoid repeated freeze-thaw cycles

• Reconstitution protocol:

  • Briefly centrifuge vial before opening

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to 5-50% final concentration (50% recommended)

  • Aliquot for long-term storage at -20°C/-80°C

• Working conditions:

  • Store working aliquots at 4°C for up to one week

  • Avoid repeated freeze-thaw cycles which compromise protein integrity

  • Use Tris/PBS-based buffer with 6% trehalose (pH 8.0) for optimal stability

Research indicates that recombinant atpE protein maintains >90% purity when properly stored, as determined by SDS-PAGE analysis. For immunological studies, protein quality significantly impacts results, making adherence to these storage protocols essential for experimental reproducibility .

How can recombinant atpE be incorporated into vaccine development against Glässer's disease?

Developing effective vaccines using recombinant atpE involves several strategic approaches:

• Subunit vaccine formulation:

  • Purified recombinant atpE can be formulated with appropriate adjuvants

  • Combination with other immunogenic OMPs enhances cross-protection

  • Optimal antigen dosage determination through dose-response studies

• Multi-antigen vaccine strategies:

  • Combining atpE with other subunits (e.g., a, α) enhances cross-protection

  • Triple-antigen formulations show superior protection compared to single-antigen approaches

  • Recombinant protein combinations elicit broader immune responses against diverse H. parasuis serovars

• Delivery system considerations:

  • Adjuvant selection significantly impacts immune response quality

  • Nanoparticle or liposome encapsulation may enhance antigen presentation

  • Prime-boost strategies with varied formulations can enhance immunity breadth

Experimental data from mouse models demonstrates that multi-antigen formulations containing atpE provide superior protection against lethal challenge with H. parasuis serovar 5. While individual antigens confer partial protection, combination formulations significantly reduce bacterial colonization in tissues and prevent pathological changes following challenge .

What techniques can be used to study atpE interactions with host immune system components?

Investigating atpE interactions with host immune components requires sophisticated methodological approaches:

• Cellular interaction studies:

  • Dendritic cell activation assays measuring MHC-II upregulation and cytokine production

  • Macrophage phagocytosis assays with fluorescently-labeled bacteria after opsonization

  • Neutrophil activation and respiratory burst measurement following atpE exposure

• Epitope mapping techniques:

  • Peptide microarray analysis to identify immunodominant epitopes

  • T cell epitope prediction followed by experimental validation

  • B cell epitope characterization through truncation mutant analysis

• Functional immunology assays:

  • Opsonophagocytic killing assays to evaluate antibody functionality

  • Complement activation studies measuring C3 deposition

  • Cytokine profiling following immune cell stimulation with atpE

Research demonstrates that immunization with recombinant atpE significantly increases CD4+ and CD8+ T cell proliferation, with CD4+ T cells showing more robust responses than CD8+ T cells. This suggests that atpE primarily activates helper T cell responses, which facilitate antibody production by B cells .

How does genomic variation in atpE across H. parasuis strains impact vaccine development?

Genomic analysis of atpE across H. parasuis strains reveals important considerations for vaccine development:

• Sequence conservation analysis:

  • The atpE gene shows high conservation across different H. parasuis serovars

  • Complete genomic sequencing of H. parasuis SH0165 (a virulent serovar 5 strain) identified atpE within the 2,269,156 base pair circular chromosome

  • Comparative genomics reveals atpE as part of the core genome with limited variation

• Structural implications:

  • Limited amino acid substitutions typically occur in non-critical functional domains

  • Epitope conservation analysis helps identify universally recognized regions

  • Structural modeling predicts consistent protein folding despite minor sequence variations

• Cross-protection potential:

  • High sequence conservation suggests broad cross-protection potential

  • Experimental validation across serovars confirms immunological cross-reactivity

  • Combination with other conserved antigens enhances protection breadth

The high conservation of atpE across H. parasuis strains makes it particularly valuable for developing broadly protective vaccines. While some surface-exposed proteins show significant variation across serovars, ATP synthase components like atpE maintain consistent structure and function, providing targets for cross-protective immunity .

What methodological approaches would advance our understanding of atpE's role in H. parasuis pathogenesis?

Several methodological approaches could significantly enhance our understanding of atpE's role in pathogenesis:

• Genetic manipulation studies:

  • Conditional knockdown of atpE to assess viability and virulence impacts

  • Site-directed mutagenesis to identify critical functional residues

  • Complementation studies to confirm phenotypic effects

• Host-pathogen interaction models:

  • Ex vivo infection models using porcine respiratory epithelial cells

  • Transcriptomic analysis of host cells following atpE exposure

  • In vivo tracking of atpE expression during different infection stages

• Structural biology approaches:

  • Cryo-electron microscopy of the complete ATP synthase complex

  • Hydrogen-deuterium exchange mass spectrometry to identify surface-exposed regions

  • Protein-protein interaction studies to identify host binding partners

Current research on H. parasuis biofilm formation has identified differential gene expression patterns, including metabolic pathways and ATP-binding cassette (ABC) transporters. Future studies should specifically examine atpE expression dynamics during infection progression and correlate these with virulence phenotypes .

How might atpE be combined with other antigens to create more effective vaccine formulations?

Optimizing multi-antigen vaccine formulations containing atpE requires systematic investigation:

• Antigen combination screening:

  • Systematic evaluation of different antigen combinations

  • Assessment of potential antigenic competition or enhancement

  • Optimization of relative antigen ratios for maximal immunogenicity

• Adjuvant optimization:

  • Evaluation of different adjuvant systems for specific immune profile induction

  • Compatibility testing between adjuvants and multiple protein antigens

  • Stability studies of complex formulations under various storage conditions

• Delivery platform innovation:

  • Development of recombinant vectored vaccines expressing atpE

  • Self-assembling nanoparticle displays of multiple antigens

  • Controlled-release formulations for enhanced immune stimulation

Experimental data demonstrates that triple-antigen formulations containing outer membrane proteins induce significantly stronger immune responses than single-antigen approaches. For example, mice immunized with triple-rOMP formulations showed the highest antigen-specific responses and demonstrated superior protection against lethal challenge compared to individual antigen immunization .