Recombinant Bartonella henselae ATP synthase subunit delta (atpH)

What is the role of ATP synthase subunit delta (atpH) in Bartonella henselae metabolism?

The ATP synthase subunit delta (atpH) in B. henselae is an essential component of the F1F0-ATP synthase complex that plays a crucial role in energy production. This subunit helps connect the F1 catalytic domain to the membrane-embedded F0 domain, serving as part of the peripheral stalk that prevents unproductive rotation of the α3β3 hexamer during ATP synthesis. In B. henselae, ATP synthase is particularly important as this bacterium relies heavily on oxidative phosphorylation for energy generation, especially when residing within host cells. Research has shown that B. henselae does not derive carbon and energy from glucose catabolism, which is consistent with genome sequence data suggesting an incomplete glycolytic pathway. Instead, the bacterium depletes amino acids from the environment and accumulates ammonia, indicating amino acid catabolism through a tricarboxylic acid (TCA) cycle-dependent mechanism .

How does the amino acid sequence of B. henselae ATP synthase subunit delta compare to homologous proteins in other bacteria?

While the search results don't provide the specific amino acid sequence for B. henselae ATP synthase subunit delta (atpH), comparative analysis between B. henselae and other alphaproteobacteria typically shows significant conservation of ATP synthase subunits. Based on studies of related ATP synthase subunits, such as the ATP synthase subunit b 1 (atpF1), which has 188 amino acids and shares homology with ATP synthase subunits from other bacteria , we can infer that atpH likely shows similar evolutionary conservation. For context, the ATP synthase epsilon chain (atpC) from B. henselae is also commercially available as a recombinant protein , suggesting that multiple ATP synthase components from this pathogen have been characterized and produced for research purposes.

What expression systems are most effective for producing recombinant B. henselae ATP synthase subunits?

E. coli expression systems have proven most effective for the recombinant production of B. henselae ATP synthase subunits. According to the available data, recombinant ATP synthase subunit b 1 (atpF1) was successfully expressed in E. coli with an N-terminal His tag . Similarly, the ATP synthase epsilon chain (atpC) is commercially available as an E. coli-expressed recombinant protein . When expressing ATP synthase subunits, researchers should consider the following optimization parameters:

For purification, His-tag affinity chromatography followed by size exclusion chromatography yields preparations with >90% purity as determined by SDS-PAGE .

How can recombinant B. henselae ATP synthase subunit delta be used to study host-pathogen interactions?

Recombinant B. henselae ATP synthase subunit delta (atpH) serves as a valuable tool for investigating host-pathogen interactions through several methodological approaches:

• Immunological profiling: The purified protein can be used to raise specific antibodies for detecting native ATP synthase expression during different stages of infection. Western blot analysis has demonstrated that B. henselae-induced cell proliferation involves the mitochondrial intrinsic apoptotic pathway .

• Protein-protein interaction studies: Recombinant atpH can be employed in pull-down assays or Surface Plasmon Resonance (SPR) to identify host factors that interact with bacterial ATP synthase components during infection. This is particularly relevant as research has shown that different B. henselae strains (Houston-1, JK-40, U-4, and JK-47) exhibit varying abilities to adhere to and invade host cells .

• Vaccination studies: As a conserved bacterial protein, recombinant atpH can be tested as a potential vaccine candidate to evaluate protective immune responses against B. henselae infection.

• Metabolic modulation analysis: By introducing recombinant atpH into host cells or using it to block native protein function, researchers can study how B. henselae modifies host cell energy metabolism. Recent transcriptomic studies comparing intracellular and extracellular B. henselae revealed that oxidative phosphorylation genes (including ATP synthase components) are downregulated in the intracellular environment, suggesting metabolic adaptation during infection .

What protein-protein interactions involve ATP synthase subunit delta during B. henselae infection?

During B. henselae infection, ATP synthase subunit delta participates in several critical protein-protein interactions:

• Intra-complex interactions: atpH interacts with other ATP synthase subunits to maintain structural integrity of the F1F0 complex. RNAi silencing of F1 α subunit or novel associated proteins has been shown to be essential for the viability of cells and important for the structural integrity of the F0F1-ATP synthase complex in related systems .

• Interaction with host mitochondrial proteins: Research suggests that bacterial ATP synthase components may interact with host cell mitochondrial proteins, potentially affecting energy production. Studies have shown that under conditions of oxidative stress, F-ATP synthases can be converted into Ca²⁺-dependent channels with properties matching those of the mitochondrial permeability transition pore (PTP) .

• Potential interactions with immunoregulatory proteins: B. henselae VirB/VirD4 Type IV Secretion System (T4SS) has been shown to modulate host immune responses through the action of Bartonella effector proteins (Beps) . While direct interaction with ATP synthase hasn't been established, both systems operate in parallel during infection.

A comprehensive understanding of these interactions requires techniques such as co-immunoprecipitation, proximity labeling (BioID), and cross-linking mass spectrometry to map the dynamic interactome of atpH during different phases of infection.

What experimental conditions are optimal for studying ATP synthase activity in B. henselae?

Optimal experimental conditions for studying B. henselae ATP synthase activity should consider the following parameters:

For accurate activity measurements, researchers should consider:

• Membrane preparation: Isolation of bacterial membranes or reconstitution of purified enzyme in liposomes to maintain the proton gradient necessary for ATP synthesis.

• Activity assays: The most sensitive method is the luciferase-based ATP detection assay for synthesis, or the malachite green assay for ATPase activity (hydrolysis direction).

• Redox state control: As B. henselae ATP synthase activity is sensitive to oxidative stress , experiments should include controls for redox state using reducing agents (DTT, β-mercaptoethanol) or oxidants (H₂O₂) to understand physiological regulation.

Remember that B. henselae cells were found to be sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose, contrary to earlier reports, suggesting that ATP synthase activity is essential even when alternative carbon sources are available .

How does transcriptional regulation of atpH differ between intracellular and extracellular B. henselae?

RNA-seq analysis comparing gene expression of B. henselae within host cells (DH82 cells) to expression by free-living bacteria revealed significant differences in the transcriptional profile of energy metabolism genes :

• Downregulation in intracellular environment: Oxidative phosphorylation genes, including those encoding ATP synthase subunits, were downregulated more than twofold when B. henselae resides intracellularly compared to planktonic bacteria.

• Metabolic shift mechanism: The downregulation appears to be part of a strategy for long-term survival within host cells, resulting in the bacteria entering a dormant or persistent state where energy and resource utilization are minimized.

• Regulatory factors: The stringent response mediator (p)ppGpp, controlled by the bifunctional enzyme SpoT, likely plays a role in this adaptive response. In the intracellular environment, CtrA was upregulated (0.5) while DnaA was downregulated (-1.3), suggesting altered (p)ppGpp levels that subsequently induced downregulation of genes encoding ribosomal proteins .

• Energy conservation: Since ribosomal synthesis is an energy-intensive process, consuming up to 40% of the cell's energy, the downregulation of ribosomal gene expression (including ATP synthase components) in intracellular bacteria represents an energy conservation strategy during persistent infection .

This differential regulation highlights the metabolic adaptability of B. henselae and suggests that atpH expression serves as an indicator of the bacterium's physiological state during infection.

What structural modifications to recombinant B. henselae atpH could enhance its immunogenicity for vaccine development?

Enhancing the immunogenicity of recombinant B. henselae atpH for vaccine development could involve several structural modifications:

• Fusion with adjuvant proteins: Conjugating atpH with immunostimulatory proteins such as flagellin, heat-shock proteins, or cytokines can significantly enhance its ability to activate antigen-presenting cells.

• Multimeric presentation: Creating recombinant constructs that present atpH in multimeric forms (dimers, trimers, etc.) can increase avidity for B-cell receptors and enhance antibody responses.

• Epitope enhancement: Computational analysis and directed mutagenesis of predicted B-cell and T-cell epitopes within atpH can create variants with improved binding to MHC molecules or B-cell receptors.

• Glycoengineering: Addition of specific glycosylation patterns to recombinant atpH can enhance uptake by antigen-presenting cells and modulate immune responses.

When evaluating such modifications, researchers should conduct comparative studies examining:

• Antibody titers (quantity and subclass distribution)

• T-cell response profiles (Th1/Th2/Th17 balance)

• Protection in appropriate animal models

• Safety profiles and reactogenicity

Recent research on recombinant B. henselae Pap31 protein demonstrated 72% sensitivity and 61% specificity at a cutoff value of 0.215 for human Bartonelloses , providing a benchmark against which improved atpH constructs could be measured.

How might structural differences in ATP synthase delta subunit contribute to the variable host cell invasion capabilities observed among B. henselae strains?

Different B. henselae strains exhibit variable capabilities in adhering to and invading host cells, with significant differences observed among the Houston-1, JK-40, U-4, and JK-47 strains . While the exact role of ATP synthase delta subunit in this process is not directly established in the available data, several mechanisms can be proposed:

• Energy provisioning for invasion: Structural variations in atpH could affect ATP synthase efficiency, thereby influencing the energy available for invasion processes. The Houston-1 strain showed high invasive capability despite lower adhesion, suggesting efficient energy utilization during invasion .

• Surface exposure possibilities: Although ATP synthase is traditionally considered a cytoplasmic membrane protein, research has demonstrated that F1F0-ATP synthase can translocate to the cell surface in certain conditions . If atpH has variant forms that facilitate such translocation, it could directly participate in host-pathogen interactions.

• Indirect effects via metabolic adaptation: Structural variations in atpH might affect the metabolic flexibility of different strains, influencing their ability to adapt to the intracellular environment. Two-dimensional gel electrophoresis analysis identified ATP synthase subunit alpha as potentially involved in the invasion process .

• Interaction with virulence factors: The ATP synthase complex may interact with known virulence factors such as Bartonella adhesin A (BadA), which shows variation among strains due to frameshift mutations and recombination within repetitive domains . Energy provision by an efficient ATP synthase could support the expression and function of these adhesins.

Comparative structural biology approaches, including protein modeling, molecular dynamics simulations, and experimental structure determination of atpH from different strains, would help elucidate the precise contributions of this subunit to strain-specific invasion capabilities.

What purification strategies yield the highest activity for recombinant B. henselae ATP synthase subunit delta?

Based on reported purification methods for similar ATP synthase subunits, the following optimized protocol would yield high-activity recombinant B. henselae ATP synthase subunit delta:

Step-by-Step Purification Protocol:

• Cell lysis: Use gentle lysis methods (e.g., lysozyme treatment followed by mild sonication) in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT, and protease inhibitors.

• Affinity chromatography: For His-tagged constructs, use Ni-NTA resin with gradient elution (20-250 mM imidazole) to minimize co-purification of contaminants.

• Tag removal: If necessary, cleave affinity tags using specific proteases (TEV or PreScission) followed by reverse affinity chromatography.

• Ion exchange chromatography: Apply sample to anion exchange column (Q-Sepharose) using a 50-500 mM NaCl gradient to separate differentially charged species.

• Size exclusion chromatography: Final polishing step using Superdex 75/200 columns in storage buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 6% trehalose).

• Storage: Aliquot and flash-freeze in liquid nitrogen. Store at -80°C to prevent repeated freeze-thaw cycles, which should be avoided as noted for similar proteins .

Quality Control Metrics:

For reconstitution, we recommend adding the protein to deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol (final concentration) for long-term storage at -20°C/-80°C .

What analytical techniques can differentiate between active and inactive forms of recombinant B. henselae ATP synthase subunits?

Several analytical techniques can effectively differentiate between active and inactive forms of recombinant B. henselae ATP synthase subunits:

Functional Assays:

• ATP synthesis/hydrolysis measurements: Quantify ATP production or hydrolysis rates using luciferase-based assays or phosphate release assays (malachite green).

• Proton pumping assays: Monitor pH changes in reconstituted proteoliposomes using pH-sensitive fluorescent dyes (ACMA, pyranine).

• Single-molecule rotation assays: For assembled complexes, fluorescence-based rotational assays can detect the mechanical activity of the ATP synthase motor.

Structural Integrity Assessments:

• Limited proteolysis: Active conformations typically show distinct proteolytic patterns compared to inactive forms.

• Thermal shift assays: Differentially scan fluorimetry to assess protein stability, with active forms generally showing higher melting temperatures.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps conformational differences between active and inactive states.

Binding Assays:

• Nucleotide binding studies: Measure affinity for ATP/ADP using isothermal titration calorimetry or fluorescence-based approaches.

• Inhibitor binding: Differential binding of specific inhibitors like oligomycin, DCCD, or azide, which have been shown to inhibit halobacterial ATP synthesis .

Spectroscopic Methods:

• Circular dichroism (CD): Detects changes in secondary structure that may correlate with activity.

• Intrinsic fluorescence: Monitors tertiary structural changes through tryptophan fluorescence.

• FTIR spectroscopy: Can detect subtle conformational differences in protein backbone.

When applying these techniques, researchers should use known activators (Mg²⁺) and inhibitors (Ca²⁺, thiol reagents) of ATP synthase to validate the assays, as these ions have been shown to modulate ATP synthase activity .

How can researchers address the challenge of protein misfolding when expressing recombinant B. henselae ATP synthase subunits?

Researchers can employ several strategies to address protein misfolding challenges when expressing recombinant B. henselae ATP synthase subunits:

Expression Optimization:

• Temperature modulation: Lower induction temperatures (16-20°C) slow protein synthesis, allowing more time for proper folding.

• Inducer concentration adjustment: Reduce IPTG concentration to 0.1-0.3 mM to decrease expression rate.

• Co-expression of chaperones: Include plasmids encoding molecular chaperones (GroEL/ES, DnaK/DnaJ/GrpE) to assist folding.

• Expression strain selection: Use specialized E. coli strains like Origami (enhanced disulfide bond formation) or ArcticExpress (cold-adapted chaperones).

Construct Engineering:

• Domain truncation: Express individual domains if the full-length protein proves recalcitrant.

• Solubility tags: Fusion with MBP, SUMO, or TrxA can significantly enhance solubility.

• Surface entropy reduction: Identify and mutate surface residue clusters to reduce entropy and promote crystallization.

• Codon optimization: Adjust codon usage for the expression host to ensure proper translation kinetics.

Refolding Strategies:

• On-column refolding: Immobilize denatured protein on affinity resin and gradually remove denaturant.

• Pulse refolding: Add denatured protein in pulses to refolding buffer to prevent aggregation.

• Additives: Include arginine, trehalose, or non-detergent sulfobetaines in refolding buffer to suppress aggregation.

Case-Specific Adaptations:
For B. henselae ATP synthase subunits specifically, consider:

• Using a Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which has proven effective for atpF1

• Brief centrifugation prior to opening to bring contents to the bottom

• Including 5-50% glycerol in storage buffers to maintain stability during freeze-thaw cycles

For all optimization efforts, use a multi-parallel approach testing several conditions simultaneously, and implement high-throughput screening methods like differential scanning fluorimetry to rapidly identify stabilizing conditions.

How can recombinant B. henselae ATP synthase subunits be utilized in developing improved diagnostic tests for bartonellosis?

Recombinant B. henselae ATP synthase subunits offer several advantages for developing enhanced diagnostic tests for bartonellosis:

Serological Assay Development:

• ELISA optimization: Recombinant atpH can serve as a highly pure antigen for detecting anti-Bartonella antibodies. This approach could potentially improve upon existing recombinant protein diagnostics, such as the Pap31-based ELISA which showed 72% sensitivity and 61% specificity for human bartonellosis .

• Multi-antigen panels: Combining atpH with other recombinant B. henselae antigens can increase diagnostic sensitivity. For example, including Pap31 fragments (N-terminal, middle, and C-terminal domains) alongside ATP synthase subunits could provide complementary detection capabilities .

• Lateral flow assays: Developing point-of-care tests using recombinant atpH would enable rapid diagnosis in resource-limited settings where bartonellosis may be endemic.

Nucleic Acid-Based Diagnostics:

• PCR target validation: The atpH gene sequence can serve as a specific target for PCR-based diagnosis, potentially offering improved sensitivity over existing targets.

• LAMP assay development: atpH-targeted loop-mediated isothermal amplification could provide sensitive, field-deployable diagnostic capability.

Biomarker Discovery Platform:
Recombinant atpH can be used to identify B. henselae-specific immune signatures by:

• Profiling antibody responses in infected versus non-infected individuals

• Characterizing T-cell epitopes that could serve as diagnostic biomarkers

• Investigating host proteins that interact with bacterial ATP synthase components

When evaluating any new diagnostic approach using recombinant atpH, researchers should conduct comprehensive validation studies comparing results with established methods and calculating sensitivity, specificity, and predictive values across diverse patient populations with suspected bartonellosis.

What biological safety considerations should researchers observe when working with recombinant B. henselae proteins?

When working with recombinant B. henselae proteins, researchers should implement the following biological safety measures:

Risk Assessment and Biosafety Level:

• Recombinant B. henselae proteins should generally be handled at Biosafety Level 2 (BSL-2), as they are derived from a Risk Group 2 pathogen capable of causing human disease.

• While purified recombinant proteins themselves are unlikely to cause infection, the potential for contamination with expression host material requires appropriate precautions.

Laboratory Practices:

• Personal protective equipment: Laboratory coat, gloves, and eye protection should be worn when handling recombinant proteins.

• Engineering controls: Work should be conducted in a certified biosafety cabinet when there is potential for aerosol generation.

• Decontamination: Work surfaces should be decontaminated with appropriate disinfectants (e.g., 70% ethanol or 10% bleach) before and after use.

Special Considerations for B. henselae Proteins:

• Allergenicity potential: Some researchers may develop hypersensitivity to repeatedly handled proteins. Implement procedures to minimize exposure, particularly to aerosolized proteins.

• Immunomodulatory effects: B. henselae proteins may have immunomodulatory properties. For example, BepD of B. henselae has been shown to promote an anti-inflammatory response through STAT3 activation . Handle with appropriate precautions.

• Laboratory-acquired infections: While rare with recombinant proteins alone, laboratory-acquired bartonellosis has been reported in contexts working with live bacteria. Maintain strict separation between recombinant protein work and any work with viable B. henselae.

Documentation and Training:

• Maintain comprehensive standard operating procedures for all work with recombinant B. henselae proteins.

• Ensure all personnel receive specific training on the potential hazards of these materials.

• Document any potential exposures and have procedures in place for post-exposure management.

Remember that product literature for commercially available recombinant B. henselae proteins often includes the warning "Not For Human Consumption!" , reinforcing the need for proper laboratory containment and handling.

How do mutations in ATP synthase genes correlate with B. henselae virulence and clinical presentation?

Strain-Specific Variations:
B. henselae strains show different capabilities in stimulating endothelial cell proliferation and invading host cells, with the Houston-1 strain demonstrating high invasive capability and the JK-40 strain showing strong ability to induce cell proliferation . ATP synthase components, including subunit alpha, have been implicated in the invasion process , suggesting that genetic variations in ATP synthase genes could contribute to these strain-specific differences.

Metabolic Adaptation and Persistence:
Transcriptomic analysis revealed that oxidative phosphorylation genes (including ATP synthase components) are downregulated in intracellular B. henselae compared to extracellular bacteria . Mutations affecting this regulatory response could potentially alter the bacterium's ability to establish persistent infection, thereby influencing clinical presentation.

Energy Production for Virulence Factor Expression:
B. henselae virulence depends on several energy-intensive processes, including:

• Type IV secretion system (T4SS) operation for effector protein translocation

• Expression of adhesins like BadA, which varies among strains due to frameshift mutations

• Induction of angiogenesis and endothelial cell proliferation

Mutations in ATP synthase genes that affect energy production efficiency could indirectly impact these virulence mechanisms, resulting in variable clinical manifestations ranging from mild cat scratch disease to more severe presentations in immunocompromised patients.

Comparative Analysis Framework:
To establish definitive correlations, researchers should conduct comprehensive comparative genomics and molecular epidemiology studies examining:

• ATP synthase gene sequences across B. henselae isolates from different clinical presentations

• Expression levels of ATP synthase components during various stages of infection

• Functional analysis of identified mutations using isogenic mutants