Recombinant Haemophilus somnus ATP synthase subunit delta (atpH)

What is the ATP synthase operon structure in Haemophilus somnus, and how does it compare to other bacterial species?

The ATP synthase in Haemophilus somnus (now known as Histophilus somni) is encoded by the atpHAGDC operon, which contains the five genes coding for the F1 sector of the ATP synthase. This structure is similar to that found in other bacteria such as Rhodobacter capsulatus, where the atpHAGDC operon has been well-characterized . While genome analysis of H. somni strain 129Pt has revealed its complete genetic structure, comparative genomics studies with other Haemophilus species like H. influenzae Rd and H. ducreyi 35000HP have shown significant variations in genetic organization, with hundreds of unique coding sequences (CDSs) in each species . The ATP synthase components are generally conserved across bacterial species, as they perform essential functions in cellular energy metabolism.

How is the atpH gene in Haemophilus somnus regulated at the transcriptional level?

Transcriptional regulation of the atpH gene in H. somnus likely follows patterns similar to those observed in related bacteria. In bacterial systems like Rhodobacter capsulatus, the promoter region for ATP synthase operons has been defined through primer extension analysis . The regulation of ATP synthase genes is typically responsive to cellular energy needs and environmental conditions. In H. somni, which can adapt to different growth environments within its bovine host, the expression of ATP synthase genes including atpH is likely regulated as part of its metabolic adaptation strategies. The organism possesses an incomplete reductive TCA cycle, suggesting specialized metabolic regulation that would influence ATP synthase expression .

What is the functional significance of the delta subunit in bacterial ATP synthases?

The delta subunit (atpH) plays a critical role in the structure and function of bacterial F1F0-ATP synthase. It forms part of the central stalk that connects the F1 catalytic domain to the F0 membrane domain, contributing to the rotational catalytic mechanism. According to the rotational catalytic model supported by X-ray crystallography studies of F1 from bovine heart mitochondria, the interaction of single-copy subunits like delta confers different affinities for ATP, ADP, and phosphate to the catalytic sites in each αβ pair in a cyclical manner . The delta subunit is essential for proper assembly and function of the ATP synthase complex, and its absence would significantly impair energy production in bacterial cells.

What are the optimal expression systems and conditions for producing recombinant H. somnus atpH protein?

The optimal expression of recombinant H. somnus atpH protein can be achieved using Escherichia coli expression systems, with E. coli C41 strain showing particularly high overexpression potential when utilizing autoinduction processes . Based on experience with other H. somni recombinant proteins, the following methodology is recommended:

• Clone the atpH gene into a suitable expression vector such as pET41a with a GST or His tag for purification

• Transform into E. coli C41 (DE3) strain, which is engineered to handle potentially toxic membrane proteins

• Employ an autoinduction system rather than IPTG induction for higher yields

• Optimize growth conditions: culture at 30°C after induction to balance expression yield and protein solubility

• Extract and purify using affinity chromatography (e.g., glutathione for GST-tagged proteins or Ni-NTA for His-tagged proteins)

Expression levels should be monitored by SDS-PAGE and Western blotting, with expected molecular weight verification based on sequence analysis. Note that amino acid sequence analysis of recombinant H. somni proteins may reveal minor differences compared to reference sequences, as observed with other H. somni recombinant proteins .

How can researchers troubleshoot common issues with atpH protein folding and stability during recombinant expression?

When working with recombinant H. somnus atpH protein, researchers frequently encounter folding and stability challenges. Here are methodological approaches to address these issues:

• Inclusion body formation: If the protein forms inclusion bodies, employ solubilization strategies:

  • Reduce expression temperature to 16-20°C

  • Lower inducer concentration

  • Co-express with molecular chaperones like GroEL/GroES system

  • Consider fusion partners that enhance solubility (e.g., MBP, SUMO)

• Protein degradation: To minimize proteolytic degradation:

  • Use protease-deficient host strains (e.g., BL21)

  • Include protease inhibitors during purification

  • Minimize handling time and maintain cold conditions throughout purification

  • Optimize buffer composition (pH, salt concentration, glycerol content)

• Structure verification: Confirm proper folding using:

  • Circular dichroism spectroscopy to assess secondary structure

  • Limited proteolysis to evaluate domain stability

  • Thermal shift assays to optimize buffer conditions for maximum stability

The ATP synthase subunits typically require proper association with other complex components for full stability. Creating fusion constructs or co-expression with interacting partners may enhance stability of the isolated delta subunit.

What approaches can be used to study interaction between recombinant atpH and other ATP synthase subunits?

Studying the interactions between recombinant atpH and other ATP synthase subunits requires sophisticated biophysical and biochemical approaches:

• Co-immunoprecipitation (Co-IP):

  • Express atpH with epitope tags (His, FLAG)

  • Co-express with other ATP synthase subunits

  • Use tag-specific antibodies to pull down atpH and identify interacting partners

• Surface Plasmon Resonance (SPR):

  • Immobilize purified atpH on sensor chips

  • Flow other purified ATP synthase subunits over the surface

  • Measure binding kinetics and affinities in real-time

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding

  • Determine stoichiometry, binding constants, enthalpy and entropy changes

• Cross-linking coupled with mass spectrometry:

  • Use chemical cross-linkers to capture transient interactions

  • Digest cross-linked complexes and analyze by LC-MS/MS

  • Identify interaction interfaces using specialized software

• Bacterial two-hybrid systems:

  • Adapt bacterial two-hybrid methods to study atpH interactions

  • Monitor protein-protein interactions in vivo

These approaches can reveal not only which subunits interact with atpH but also the specific amino acid residues involved in these interactions, providing valuable insight into the structural organization of the H. somnus ATP synthase complex.

How does H. somnus atpH compare structurally and functionally to homologous proteins in other bacterial pathogens?

The H. somnus atpH protein shares structural and functional similarities with homologous proteins in other bacterial pathogens, particularly within the Pasteurellaceae family. Comparative genomic analysis has revealed significant protein sequence conservation among ATP synthase components across bacterial species, reflecting their essential role in cellular energy metabolism .

When examining the amino acid sequences, H. somnus atpH likely has significant homology with related species. For instance, genome comparisons have shown that H. somnus 129Pt shares 1,242 protein sequences with H. influenzae Rd and 1,091 sequences with H. ducreyi 35000HP . This conservation extends to essential metabolic proteins like ATP synthase components.

• pH tolerance during infection of different host tissues

• Temperature adaptation for optimal function in bovine body temperature

• Regulatory differences in gene expression under host-specific stress conditions

The evolutionary conservation of atpH reflects the essential nature of ATP synthase across bacterial species, with variations potentially contributing to host adaptation strategies.

What insights can comparative genomics provide about the evolution of ATP synthase in the Pasteurellaceae family?

Comparative genomics analysis of ATP synthase genes within the Pasteurellaceae family reveals important evolutionary patterns:

The genome sequencing of H. somnus 129Pt has enabled detailed comparative analysis with related pathogens like H. influenzae Rd and H. ducreyi 35000HP . These analyses show that while core metabolic functions are conserved, there are significant differences in gene content and arrangement. For example, pairwise BLAST comparisons have identified 319 coding sequences unique to H. somnus 129Pt, 228 unique to H. influenzae Rd, and 411 unique to H. ducreyi 35000HP .

The ATP synthase operon structure and regulation likely reflect evolutionary adaptations to different host environments. All three organisms maintain incomplete, reductive TCA cycles, which is common among bacteria and represents an ancient metabolic strategy primarily functioning in carbon assimilation and biosynthetic precursor generation . Phylogenetic evidence suggests that the original state of the TCA cycle was a reductive biosynthetic pathway .

The conservation of ATP synthase genes across these species, despite differences in other metabolic pathways, emphasizes the essential nature of ATP synthesis even as these pathogens evolved to colonize different niches within their respective hosts.

How can researchers design experiments to assess the immunogenicity of recombinant H. somnus atpH protein in cattle?

Designing experiments to assess the immunogenicity of recombinant H. somnus atpH protein in cattle should follow a systematic approach similar to that used for other H. somni recombinant proteins:

• Protein preparation:

  • Express and purify recombinant atpH protein using optimized protocols

  • Confirm protein purity using SDS-PAGE and Western blotting

  • Verify structural integrity using circular dichroism or other biophysical methods

• Immunization protocol:

  • Design a double immunization schedule with appropriate dosage (e.g., 20 μg per animal, as used for H. somni OMP40)

  • Include proper adjuvants suitable for veterinary applications

  • Establish control groups receiving adjuvant only

• Immune response monitoring:

  • Collect serum samples at regular intervals

  • Quantify antibody responses using ELISA for different antibody isotypes (IgG1, IgG2, IgM)

  • Assess antibody specificity through Western blotting against native H. somnus proteins

  • Evaluate cross-reactivity with other bacterial species

• Functional assays:

  • Perform bactericidal/growth inhibition assays with immune sera

  • Assess opsonophagocytic activity

  • Conduct delayed-type hypersensitivity tests

• Challenge studies:

  • Expose immunized and control animals to controlled H. somnus challenge

  • Monitor clinical parameters, bacterial loads, and disease progression

This approach would provide comprehensive data on both humoral and cellular immune responses to the recombinant atpH protein, similar to studies conducted with H. somni OMP40 that demonstrated significant increases in IgG1 and IgG2 antibodies after immunization .

What experimental approaches can determine if atpH could serve as a target for novel antimicrobial development?

To evaluate the potential of H. somnus atpH as a target for novel antimicrobial development, researchers should implement a multi-faceted experimental strategy:

• Target validation:

  • Attempt to create atpH deletion mutants in H. somnus to assess essentiality

  • If direct deletion is challenging (as seen with ATP synthase genes in other bacteria) , employ conditional expression systems

  • Use gene transfer agent transduction combined with conjugation methods to construct strains with mutations in essential genes

• High-throughput screening:

  • Develop biochemical assays measuring ATP synthase activity

  • Screen chemical libraries for compounds that specifically inhibit the delta subunit function

  • Validate hits using secondary assays for specificity

• Structure-based drug design:

  • Determine the crystal structure of H. somnus atpH

  • Identify potential binding pockets using computational modeling

  • Design inhibitors that specifically target these structures

• Cytotoxicity and selectivity assessment:

  • Test candidate inhibitors against mammalian ATP synthases to evaluate selectivity

  • Assess toxicity in mammalian cell cultures

  • Determine minimum inhibitory concentrations against H. somnus and other pathogens

• In vivo efficacy studies:

  • Evaluate pharmacokinetics and pharmacodynamics in animal models

  • Assess efficacy in relevant infection models

  • Monitor for resistance development

This comprehensive approach would determine whether atpH is both essential (and thus a viable target) and sufficiently different from host proteins to allow selective targeting by antimicrobial compounds.

How might recombinant atpH be incorporated into multi-component vaccines against bovine respiratory disease complex?

Incorporating recombinant H. somnus atpH into multi-component vaccines against bovine respiratory disease complex (BRDC) represents a promising strategy that could be implemented through the following approaches:

• Antigen combination rationale:

  • Combine atpH with other immunoprotective antigens from H. somni (such as IbpA DR2/Fic or OMP40)

  • Include antigens from other BRDC pathogens (Mannheimia haemolytica, Pasteurella multocida, bovine viral diarrhea virus)

  • Evaluate potential synergistic or antagonistic effects between antigens

• Formulation optimization:

  • Test different adjuvant systems to enhance immune responses

  • Develop delivery systems (liposomes, nanoparticles) that present antigens effectively

  • Determine optimal antigen ratios and concentrations

• Cross-protection assessment:

  • Evaluate if anti-atpH antibodies exhibit cross-reactivity with ATP synthase from other BRDC pathogens

  • Test for cross-protection in challenge studies with heterologous pathogens

  • Similar to H. somni OMP40, which showed cross-reactivity with antigens from other gram-negative pathogens

• Immune response characterization:

  • Profile the type of immune response (Th1/Th2 balance)

  • Monitor IgG subclass distribution (IgG1, IgG2) as indicators of immune bias

  • Assess mucosal immunity through IgA measurements

• Field trial design:

  • Implement appropriate vaccination schedules

  • Monitor not only protection but also production parameters

  • Assess long-term immunity and need for booster vaccinations

This approach leverages findings from studies of other H. somni antigens like OMP40, which has demonstrated significant immunogenicity in calves and cross-reactivity with antigens from other Pasteurellaceae and Enterobacteriaceae family members , suggesting potential for broader protection against multiple respiratory pathogens.

What are the most reliable methods for confirming the identity and purity of recombinant H. somnus atpH protein?

The most reliable methods for confirming the identity and purity of recombinant H. somnus atpH protein include a combination of analytical techniques:

• Mass spectrometry analysis:

  • Peptide mass fingerprinting after tryptic digestion

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for sequence confirmation

  • Intact protein mass determination to verify the entire protein sequence

• Immunological verification:

  • Western blotting using antibodies against the recombinant protein or tag

  • ELISA with specific anti-atpH antibodies

• Purity assessment:

  • SDS-PAGE with densitometry analysis (aim for >95% purity)

  • Size-exclusion chromatography to detect aggregates or contaminants

  • Capillary electrophoresis for high-resolution purity analysis

• Sequence verification:

  • N-terminal sequencing to confirm proper translation initiation

  • BLAST analysis to compare with reference sequences

  • Detection of amino acid variations from reference sequences (as observed with other H. somni recombinant proteins where differences at certain positions were found)

• Functional validation:

  • Activity assays appropriate for the protein

  • Structural characterization (circular dichroism, intrinsic fluorescence)

These methods collectively provide comprehensive confirmation of recombinant protein identity, purity, and integrity, essential for downstream application in research or vaccine development.

How can researchers optimize conditions for long-term storage and stability of recombinant atpH protein?

Optimizing conditions for long-term storage and stability of recombinant H. somnus atpH protein requires systematic testing of various conditions and additives:

• Buffer optimization:

  • Test pH range (typically 7.0-8.0)

  • Evaluate different buffer systems (phosphate, Tris, HEPES)

  • Optimize ionic strength (typically 100-300 mM NaCl)

  • Add stabilizing agents (5-10% glycerol, 1-2 mM DTT)

• Storage temperature assessment:

  • Compare protein stability at 4°C, -20°C, -80°C, and in liquid nitrogen

  • Evaluate freeze-thaw effects through multiple cycles

  • Consider flash-freezing in small aliquots to avoid repeated freeze-thaw

• Lyophilization protocols:

  • Test lyophilization with different cryoprotectants (trehalose, sucrose)

  • Optimize reconstitution procedures

  • Compare activity before and after lyophilization

• Stability enhancers:

  • Evaluate protein stabilizers (amino acids like arginine, glycine)

  • Test metal chelators (EDTA) if metal-catalyzed oxidation is a concern

  • Consider carrier proteins for dilute solutions

• Monitoring methods:

  • Implement accelerated stability testing at elevated temperatures

  • Use analytical methods (SEC-HPLC, dynamic light scattering) to detect aggregation

  • Perform periodic activity assays to confirm functional integrity

• Storage container considerations:

  • Use low-protein-binding tubes

  • Minimize headspace to reduce oxidation

  • Protect from light if photosensitive

Each lot of purified protein should undergo stability testing to establish a validated shelf-life under optimal storage conditions, ensuring consistent performance in downstream applications.

What are the most sensitive methods for detecting potential contamination of recombinant atpH with host cell proteins or endotoxins?

Ensuring the purity of recombinant H. somnus atpH with respect to host cell proteins and endotoxins is critical for research reliability and safety. The following methods represent the most sensitive approaches for contamination detection:

• Host Cell Protein (HCP) detection:

  • ELISA using antibodies raised against E. coli whole cell lysate (detection limit: 1-10 ng/mL)

  • Mass spectrometry-based proteomics (detection limit: sub-ng/mL)

    • Data-dependent acquisition (DDA)

    • Multiple reaction monitoring (MRM)

  • Western blotting with anti-E. coli antibodies

  • 2D-PAGE with silver staining for visual detection of contaminants

• Endotoxin testing:

  • Limulus Amebocyte Lysate (LAL) assay variants:

    • Gel-clot method (detection limit: 0.03-0.1 EU/mL)

    • Chromogenic method (detection limit: 0.005-0.01 EU/mL)

    • Turbidimetric method (detection limit: 0.01 EU/mL)

  • Recombinant Factor C assay (detection limit: 0.001 EU/mL)

  • Monocyte Activation Test using human blood cells

• DNA contamination assessment:

  • qPCR targeting host cell DNA sequences

  • PicoGreen assay for double-stranded DNA

  • Threshold Alert System to monitor process consistency

• Removal strategies for identified contaminants:

  • Endotoxin removal using polymyxin B affinity chromatography

  • Additional ion exchange chromatography steps

  • Ultrafiltration with appropriate molecular weight cut-offs

  • Specific HCP precipitation methods