Recombinant Coxiella burnetii ATP synthase subunit beta (atpD)

What is the ATP synthase subunit beta (atpD) in Coxiella burnetii and why is it significant for research?

ATP synthase subunit beta (atpD) is a critical component of the F0F1 ATP synthase complex in Coxiella burnetii, the causative agent of Q fever. This protein has a nominal mass of approximately 50490 Da and a calculated pI value of 5.01, making it a mid-sized, slightly acidic protein. Its significance stems from its seroreactivity, as it is recognized by sera from both experimentally infected mice and human Q fever patients, suggesting its potential as a diagnostic marker and vaccine candidate. Specifically, atpD is recognized by mice sera at days 21 and 28 post-infection and by human sera during Q fever infection, indicating its immunogenic properties during the course of infection . The protein's consistent immunoreactivity across different host species highlights its conserved antigenic properties and potential utility in developing universal diagnostic tools for Q fever.

How does atpD compare to other seroreactive proteins identified in Coxiella burnetii?

Among the 20 seroreactive proteins identified in immunoproteomic studies of C. burnetii, atpD (F0F1 ATP synthase subunit beta) demonstrates notable immunological features. When compared with other major seroreactive proteins like GroEL, YbgF, RplL, Mip, OmpH, Com1, and DnaK, atpD shows a distinct seroreactivity profile. Based on data presented in Table 1 of the referenced study, atpD is recognized by mice sera at days 21 and 28 post-infection, similar to several other proteins including GroEL, trigger factor, dihydrolipoyllysine-residue succinyltransferase, fructose-1,6-bisphosphate aldolase, and malate dehydrogenase . Unlike some proteins that show earlier recognition (day 14), such as GroEL, Mip, OmpH, and Com1, atpD appears to elicit antibody responses slightly later in the infection course, suggesting its role in the developing rather than initial immune response. This temporal pattern of seroreactivity provides valuable insights into the immunopathogenesis of Q fever and informs the optimal timing for diagnostic testing targeting this protein.

What are the physical and biochemical properties of recombinant Coxiella burnetii atpD?

Recombinant Coxiella burnetii ATP synthase subunit beta (atpD) exhibits specific physical and biochemical properties that are important for researchers to consider. Based on proteomic analysis, this protein has a nominal mass of 50490 Da and a calculated isoelectric point (pI) value of 5.01, positioning it in the slightly acidic range . The protein was successfully identified through MALDI-TOF mass spectrometry with a high confidence score of 240 (expect value of 2.70E-18), confirming its identity with 26 queries matched and 54% sequence coverage . This robust identification suggests that the recombinant protein maintains structural fidelity to the native form. The atpD protein likely maintains its catalytic role in ATP synthesis in recombinant form, though functional studies specific to the recombinant version are needed to confirm retention of enzymatic activity. For expression systems, E. coli has been successfully employed for recombinant expression of other C. burnetii proteins in the referenced study, suggesting a viable approach for atpD as well.

What are the optimal conditions for expressing recombinant Coxiella burnetii atpD in E. coli?

The expression of recombinant Coxiella burnetii ATP synthase subunit beta (atpD) in Escherichia coli requires careful optimization of several parameters. Based on successful expression of other C. burnetii proteins, researchers should consider using BL21(DE3) or similar E. coli strains designed for high-level protein expression. For the expression vector, pET systems containing T7 promoters have proven effective for controlling expression of bacterial proteins. The referenced study successfully expressed 19 out of 20 identified C. burnetii proteins in E. coli, suggesting atpD is amenable to recombinant expression . Induction conditions typically involve IPTG at concentrations between 0.5-1.0 mM when cultures reach an OD600 of 0.6-0.8, with expression typically conducted at lower temperatures (16-25°C) for 4-16 hours to enhance proper folding. For purification, a hexahistidine tag approach allows for straightforward immobilized metal affinity chromatography (IMAC). Given the slightly acidic nature of atpD (pI 5.01), ion exchange chromatography using positively charged resins at pH values below 5.01 may provide an effective secondary purification step. Protein stability can be enhanced by adding glycerol (10-20%) to storage buffers and maintaining samples at -80°C for long-term storage.

What purification strategies yield the highest purity and activity for recombinant atpD?

Achieving high purity and activity for recombinant Coxiella burnetii ATP synthase subunit beta (atpD) requires a multi-step purification strategy. Initially, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-based resins provides an effective first step when working with His-tagged recombinant atpD. For optimal binding, researchers should maintain slightly alkaline conditions (pH 7.5-8.0) with 20-50 mM imidazole in the binding buffer to reduce non-specific interactions. Elution is typically performed with a step or linear gradient of imidazole (100-500 mM). Given atpD's pI value of 5.01 , anion exchange chromatography at pH 7.0-8.0 serves as an excellent secondary purification step, as the protein will carry a negative charge in this pH range. Size exclusion chromatography as a final polishing step helps separate monomeric atpD from aggregates and improves homogeneity. Throughout purification, researchers should maintain reducing conditions (1-5 mM DTT or 0.5-2 mM β-mercaptoethanol) to prevent disulfide-bond formation and add glycerol (5-10%) to enhance stability. For activity preservation, inclusion of cofactors such as Mg²⁺ (1-5 mM) may be beneficial as these are important for ATP synthase function. Purification success can be monitored through SDS-PAGE, Western blotting, and enzyme activity assays.

How can researchers develop reliable serological assays using recombinant atpD for Q fever diagnosis?

Developing reliable serological assays using recombinant Coxiella burnetii ATP synthase subunit beta (atpD) for Q fever diagnosis requires a systematic approach. First, researchers should express and purify recombinant atpD with high purity (>95%) as assessed by SDS-PAGE and mass spectrometry. The immunoproteomic study referenced showed that atpD is recognized by sera from both experimentally infected mice (at days 21 and 28 post-infection) and human Q fever patients, confirming its potential as a diagnostic antigen . For ELISA development, coat microplates with optimized concentrations of recombinant atpD (typically 1-5 μg/ml) in carbonate buffer (pH 9.6) overnight at 4°C. Block with 1-5% BSA or milk proteins to prevent non-specific binding. Patient sera should be tested at multiple dilutions (typically starting at 1:100) to establish optimal working dilutions that maximize signal-to-noise ratio. Incorporate appropriate controls including sera from confirmed Q fever cases, healthy individuals, and patients with other febrile illnesses to assess cross-reactivity. When developing microarray-based assays similar to those referenced in the study, protein concentrations and spotting buffers should be optimized to ensure uniform protein deposition and accessibility of epitopes . Validation should involve testing against a panel of well-characterized sera with known clinical outcomes, calculating sensitivity, specificity, and predictive values. Comparison with existing commercial assays and correlation with other diagnostic methods (PCR, culture) will strengthen the assay's validity.

How does atpD contribute to Coxiella burnetii pathogenesis and host immune response?

The role of ATP synthase subunit beta (atpD) in Coxiella burnetii pathogenesis and host immune response involves complex interactions within the infection microenvironment. As a component of ATP synthase, atpD primarily functions in energy metabolism, generating ATP through oxidative phosphorylation, which is crucial for bacterial survival and replication within the acidic parasitophorous vacuole of host cells. Immunoproteomic evidence demonstrates that atpD is recognized by sera from both experimentally infected mice (days 21 and 28 post-infection) and human Q fever patients, indicating its accessibility to the host immune system during infection . This seroreactivity suggests that atpD might be exposed on the bacterial surface or released during infection, potentially through bacterial lysis or secretion mechanisms. The temporal pattern of antibody recognition—appearing later (21-28 days) rather than earlier in infection—indicates that atpD may contribute to the developing rather than initial immune response, possibly playing a role in chronic infection establishment. Unlike proteins such as OmpH, Com1, and Mip that show earlier recognition (day 14 post-infection) , atpD's later recognition pattern may reflect differences in protein abundance, accessibility, or immunogenicity during the infection course. Understanding these dynamics is crucial for elucidating the complete picture of C. burnetii pathogenesis and host-pathogen interactions.

What is the potential of recombinant atpD as a component in subunit vaccines against Q fever?

Recombinant Coxiella burnetii ATP synthase subunit beta (atpD) shows promising characteristics as a potential component in subunit vaccines against Q fever. Its consistent recognition by both murine and human immune systems, as demonstrated in the immunoproteomic study, suggests broad immunogenicity across species . The protein elicits antibody responses at days 21 and 28 post-infection in mice, indicating it triggers a sustained immune response rather than just an early, transient reaction . This temporal pattern aligns well with the development of protective immunity. For vaccine development, atpD could be combined with other identified immunodominant antigens such as GroEL, YbgF, RplL, Mip, OmpH, Com1, and DnaK to create a multi-epitope vaccine that targets multiple aspects of the pathogen's biology. Such an approach might provide more comprehensive protection than single-antigen formulations. When designing delivery systems, researchers should consider adjuvant selection carefully—alum-based adjuvants might be suitable for antibody responses, while TLR agonists could enhance cell-mediated immunity, which is crucial for intracellular pathogens like C. burnetii. In animal models, vaccine efficacy should be evaluated through challenge studies measuring bacterial burden reduction in target organs (particularly spleen, as C. burnetii shows high loads in splenic tissue) . Immunological correlates of protection should be assessed through antibody titers, neutralization assays, and T cell response profiling, with particular attention to Th1-biased responses which are generally protective against intracellular bacteria.

How can structural analysis of atpD contribute to understanding ATP synthase function in Coxiella burnetii?

Structural analysis of ATP synthase subunit beta (atpD) from Coxiella burnetii provides critical insights into the functional mechanisms of this essential enzyme complex within the context of this obligate intracellular pathogen's unique lifestyle. Using X-ray crystallography or cryo-electron microscopy, researchers can determine the three-dimensional structure of atpD at atomic resolution, revealing nucleotide-binding domains and catalytic sites that are essential for ATP synthesis. Comparative structural analysis between C. burnetii atpD and homologs from other organisms could identify unique structural features that may contribute to the pathogen's adaptation to the acidic parasitophorous vacuole environment. The calculated pI value of 5.01 for C. burnetii atpD suggests potential adaptations for functioning in acidic conditions compared to homologs from neutralophilic bacteria. Structural analysis should focus on identifying potential conformational changes during the catalytic cycle, particularly how proton translocation couples with ATP synthesis under the unique physiological conditions of C. burnetii's intracellular niche. Molecular dynamics simulations can complement experimental structural data by modeling protein dynamics under various pH conditions and in the presence of ligands or inhibitors. Structure-guided mutagenesis experiments targeting key residues identified through structural analysis can provide functional validation by assessing how specific mutations affect ATP synthesis activity, oligomerization, or stability under stress conditions. These approaches collectively enhance our understanding of how C. burnetii maintains energy homeostasis during infection, potentially revealing novel targets for therapeutic intervention.

What are common challenges in obtaining soluble recombinant atpD and how can they be addressed?

Researchers frequently encounter solubility issues when expressing recombinant Coxiella burnetii ATP synthase subunit beta (atpD), as this energetic machinery protein often forms inclusion bodies in heterologous expression systems. Several strategies can mitigate these challenges. First, modifying expression conditions by lowering temperature (16-20°C), reducing inducer concentration (0.1-0.2 mM IPTG), and using slower growth media can significantly improve solubility by slowing protein synthesis and allowing proper folding. Expression strains engineered to supply rare codons or additional chaperones (such as Arctic Express or Rosetta-gami strains) may enhance proper folding of this C. burnetii protein in E. coli. Fusion tags beyond the standard His-tag, particularly solubility enhancers like SUMO, MBP, or TrxA, can dramatically improve the soluble fraction of recombinant atpD. If inclusion bodies persist despite optimization, controlled solubilization using mild detergents (0.5-1% Triton X-100) or low-concentration denaturants (1-2 M urea) may recover functional protein without complete unfolding. For severe cases requiring full denaturation, a carefully designed refolding strategy is essential—typically involving gradual dialysis with decreasing denaturant concentrations in the presence of ATP, which may serve as a stabilizing ligand during refolding. Co-expression with other ATP synthase subunits, particularly alpha subunits, may improve solubility by allowing natural complex formation that stabilizes the beta subunit. Protein engineers should consider removing flexible or hydrophobic regions identified through bioinformatic analysis if these contribute to aggregation without compromising the core functional domains.

How should researchers interpret contradictory serological data when using recombinant atpD in diagnostic assays?

When confronted with contradictory serological data using recombinant Coxiella burnetii ATP synthase subunit beta (atpD) in diagnostic assays, researchers should systematically evaluate several potential factors. First, examine the temporal dynamics of antibody response, as atpD recognition occurs later in infection (days 21-28 post-infection in mice) , which means false negatives might occur in early-stage samples. Cross-reactivity could produce false positives, as ATP synthase is conserved across bacterial species; therefore, proper cutoff values established with diverse control sera (including sera from patients with similar bacterial infections) are essential. Protein quality issues may contribute to inconsistent results—verify recombinant atpD integrity through mass spectrometry, circular dichroism, and activity assays to ensure proper folding and epitope presentation. The referenced study showed that atpD was recognized by both mouse and human sera , suggesting broad antigenicity, but individual variation in immune responses may still occur. Comparison with composite serological panels is advisable—the referenced study identified seven major seroreactive proteins (GroEL, YbgF, RplL, Mip, OmpH, Com1, and DnaK) , suggesting that a multi-antigen approach may provide more reliable diagnosis than atpD alone. Statistical analysis using ROC curves helps establish optimal cutoff values, sensitivity, and specificity for different clinical scenarios. For particularly ambiguous samples, orthogonal testing methods such as PCR, cell culture, or testing against multiple C. burnetii antigens can resolve contradictions. Finally, careful documentation of clinical information including symptom duration, antibiotic treatment history, and exposure risk is essential for proper interpretation of serological data in the context of the known biological behavior of C. burnetii infection.

What strategies can minimize cross-reactivity when using atpD in serological assays for Q fever?

Minimizing cross-reactivity in serological assays using recombinant Coxiella burnetii ATP synthase subunit beta (atpD) requires a multi-faceted approach to enhance specificity. Researchers should first identify and focus on C. burnetii-specific epitopes within the atpD protein through epitope mapping using peptide arrays or phage display techniques, then design constructs that express only these unique regions rather than the whole protein. The referenced study noted that major seroreactive C. burnetii proteins showed "fewer cross-reactions" with sera from patients with rickettsial spotted fever, Legionella pneumonia, or streptococcal pneumonia, suggesting inherent specificity potential . Implementing stringent washing protocols with higher detergent concentrations (0.1-0.5% Tween-20) and salt concentrations (up to 500 mM NaCl) in wash buffers can reduce non-specific binding. Pre-adsorption of test sera with lysates from related bacteria (particularly those with homologous ATP synthase subunits) can remove cross-reactive antibodies before testing with C. burnetii atpD. Statistical approaches including parallel testing of samples against homologous proteins from related species allows mathematical correction for cross-reactivity through differential absorption techniques. The combination of atpD with other C. burnetii-specific antigens in multiplexed formats (such as the microarray approach mentioned in the study ) can provide a more specific signature than single-antigen testing. Finally, confirmatory algorithms incorporating orthogonal testing methods (immunofluorescence, PCR) for samples with borderline results can enhance diagnostic accuracy. These comprehensive approaches help ensure that positive results genuinely reflect C. burnetii exposure rather than cross-reactivity with related bacterial species.

What are promising avenues for structure-function studies of Coxiella burnetii atpD?

Structure-function studies of Coxiella burnetii ATP synthase subunit beta (atpD) represent a fertile ground for uncovering unique adaptations of this obligate intracellular pathogen. Future research should prioritize high-resolution structural determination through X-ray crystallography or cryo-electron microscopy, potentially revealing adaptations that facilitate function in the acidic parasitophorous vacuole where C. burnetii replicates. Computational comparison of atpD's nucleotide-binding domains with homologs from neutralophilic bacteria could identify unique residues that contribute to acid stability or altered catalytic properties. The protein's observed pI value of 5.01 raises interesting questions about its charge distribution and electrostatic interactions under varying pH conditions. Site-directed mutagenesis targeting conserved versus divergent residues, followed by enzymatic activity assays under conditions mimicking the intracellular niche (pH 4.5-5.5), would provide experimental validation of structure-based hypotheses. Protein-protein interaction studies focusing on atpD's association with other ATP synthase subunits could reveal adaptations in complex assembly or stability specific to C. burnetii. Investigation of post-translational modifications through mass spectrometry might uncover regulatory mechanisms unique to this pathogen's energy metabolism. Single-molecule studies using techniques like FRET could provide insights into conformational dynamics during the catalytic cycle. Because atpD shows seroreactivity in both mice and humans , epitope mapping studies correlating structural features with immunogenicity could inform both diagnostic and vaccine development. This multi-disciplinary approach would substantially advance our understanding of how C. burnetii has adapted its central energetic machinery to thrive in its unique intracellular lifestyle.

How can systems biology approaches integrate atpD research into broader understanding of Coxiella burnetii pathogenesis?

Systems biology approaches offer powerful frameworks for integrating ATP synthase subunit beta (atpD) research into a comprehensive understanding of Coxiella burnetii pathogenesis. Researchers should pursue multi-omics integration, combining transcriptomics data tracking atpD expression under various conditions with proteomics quantifying protein levels and post-translational modifications, and metabolomics measuring changes in ATP/ADP ratios and related metabolites. Network analysis positioning atpD within the broader context of C. burnetii's metabolic and virulence networks can reveal unexpected functional connections; for instance, potential relationships between energy metabolism and virulence factor expression. The temporal patterns of atpD recognition by host immune systems (days 21-28 post-infection in mice) should be incorporated into host-pathogen interaction models that track the complete immunological timeline of infection. Computational modeling of C. burnetii's core metabolism under various environmental conditions can predict how atpD function influences bacterial adaptation to the intracellular niche. Comparative systems analyses across C. burnetii strains with varying virulence could identify correlations between atpD sequence variations or expression patterns and pathogenicity. Single-cell approaches examining heterogeneity in atpD expression within C. burnetii populations might reveal subpopulations with distinct metabolic states contributing to persistence. Advanced microscopy techniques like correlative light and electron microscopy (CLEM) can spatiotemporally map atpD localization relative to other cellular components during different infection stages. Finally, perturbation biology using CRISPR interference or small molecule inhibitors targeting atpD can help validate systems-level predictions about this protein's role in pathogenesis. These integrated approaches will position atpD within the complex biological system of C. burnetii infection rather than studying it in isolation.

What potential does atpD have as a target for novel antimicrobial development against Coxiella burnetii?

ATP synthase subunit beta (atpD) presents a compelling target for novel antimicrobial development against Coxiella burnetii due to several advantageous characteristics. As a component of the essential ATP synthase complex, atpD plays a critical role in energy metabolism, making it indispensable for bacterial survival and replication within host cells. The relatively high conservation of atpD's functional domains across bacterial species suggests that effective inhibitors might have broad-spectrum potential while allowing for the design of C. burnetii-specific compounds targeting unique structural features. The immunoproteomic study referenced demonstrated that atpD is recognized by both murine and human immune systems , suggesting it is accessible during infection and potentially druggable. Structure-based drug design approaches should focus on the ATP-binding pocket and catalytic sites, utilizing any structural differences between bacterial and mammalian ATP synthases to enhance selectivity and reduce off-target effects. High-throughput screening campaigns using recombinant atpD in enzymatic assays can identify initial hit compounds, which can then be optimized through medicinal chemistry. Promising compounds should be evaluated in cellular infection models measuring C. burnetii loads in macrophages and other relevant cell types. The temporal pattern of disease in mouse models, where peak bacterial loads occur at day 7 post-infection with subsequent decreases , provides a valuable framework for assessing therapeutic efficacy at different disease stages. Combination therapy approaches pairing atpD inhibitors with current standard-of-care antibiotics (doxycycline, hydroxychloroquine) should be explored for potential synergistic effects, particularly against persistent forms of C. burnetii. The development of such targeted therapeutics could significantly improve treatment options for both acute and chronic Q fever, addressing current challenges in antibiotic efficacy and treatment duration.