Recombinant Rickettsia conorii ATP synthase subunit c (atpE)

What is the molecular structure and basic characteristics of R. conorii ATP synthase subunit c (atpE)?

ATP synthase subunit c (atpE) from Rickettsia conorii is a small 74-amino acid membrane protein component of the F0 portion of ATP synthase. The amino acid sequence is MDMVSLKFIGTGLMAIGMYGAALGVSNIFSSLLSSIARNPSATENLQRMALIGAGLAEAMGLFSFVIAMLLIFS . The protein is highly hydrophobic and functions as part of the c-ring structure that facilitates proton translocation across the membrane during ATP synthesis. In R. conorii, this protein is encoded by the atpE gene (locus tag RC0026) and has been assigned the UniProt ID Q92JP1 . As part of the ATP synthase complex, atpE plays a crucial role in energy metabolism, which is particularly important for obligate intracellular pathogens like R. conorii.

What expression systems are recommended for producing recombinant R. conorii atpE?

The recommended expression system for recombinant R. conorii atpE is E. coli . Due to the hydrophobic nature of this membrane protein, expression optimization typically requires careful selection of E. coli strains specifically designed for membrane protein expression. The recombinant protein is commonly produced with an N-terminal His tag to facilitate purification . When designing expression constructs, it's important to consider the hydrophobicity of the protein and potentially include solubility-enhancing tags or fusion partners. Expression conditions should be optimized regarding temperature, inducer concentration, and duration to maximize yield while maintaining protein folding and stability.

What are the optimal storage and handling conditions for purified recombinant atpE protein?

For optimal stability, recombinant R. conorii atpE should be stored as a lyophilized powder at -20°C or -80°C for long-term storage . When reconstituting the protein, it is recommended to:

• Briefly centrifuge the vial prior to opening

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

• Add glycerol to a final concentration of 5-50% (with 50% being standard practice)

• Prepare small working aliquots to avoid repeated freeze-thaw cycles

Working aliquots can be stored at 4°C for up to one week, but repeated freeze-thaw cycles should be avoided as they can compromise protein integrity . For reconstitution buffers, Tris/PBS-based buffers at pH 8.0 containing 6% trehalose have been successfully used . The purity of properly prepared recombinant protein should be greater than 90% as determined by SDS-PAGE .

What methodological approaches are most effective for studying atpE's role in rickettsial bioenergetics?

To investigate atpE's role in rickettsial bioenergetics, researchers should implement a multi-faceted approach:

• Proteoliposome reconstitution studies:

  • Purified recombinant atpE can be reconstituted into liposomes

  • Proton translocation can be measured using pH-sensitive fluorescent dyes

  • Effect of inhibitors on proton translocation can be assessed

• Comparative metabolic analysis:

  • Comparative proteomics between pathogenic (R. conorii) and non-pathogenic Rickettsia species to identify differences in ATP synthase components

  • Analysis of ATP synthesis rates in isolated bacterial fractions

  • Correlation of ATP synthase activity with intracellular survival and replication

• Structure-function analysis:

  • Site-directed mutagenesis of conserved residues in atpE

  • Assessment of c-ring assembly and stability

  • Correlation of structural alterations with proton translocation efficiency

• Inhibitor studies:

  • Use of specific ATP synthase inhibitors during infection

  • Monitoring effects on bacterial survival, replication, and energy metabolism

  • Structural basis of inhibitor binding to rickettsial atpE

These approaches collectively provide comprehensive insights into atpE's contribution to rickettsial energy metabolism and pathogenesis.

How can researchers effectively analyze interactions between atpE and other components of the ATP synthase complex?

Analyzing the interactions between atpE and other ATP synthase components requires specialized techniques suitable for membrane protein complexes:

• Structural analysis methods:

  • Cryo-electron microscopy of isolated ATP synthase complexes

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Molecular dynamics simulations of the c-ring structure

• Biochemical interaction studies:

  • Co-purification of interacting partners using tagged atpE

  • Blue native PAGE to analyze intact complexes

  • Chemical cross-linking followed by mass spectrometry

  • Surface plasmon resonance for quantitative binding measurements

• Functional assessment:

  • Reconstitution of ATP synthase with wild-type or mutant atpE

  • Measurement of proton translocation and ATP synthesis activities

  • Effect of site-specific mutations on complex assembly and function

• In situ analysis:

  • Fluorescence resonance energy transfer (FRET) between labeled subunits

  • Super-resolution microscopy to visualize complex assembly

These methodologies provide complementary information about how atpE interacts with other ATP synthase components and contributes to the functionality of the complex.

What are the best approaches for assessing the immunogenicity of R. conorii atpE for vaccine development?

Based on successful approaches for identifying protective T-cell antigens in rickettsial species , a comprehensive methodology for assessing atpE's immunogenic potential includes:

• Epitope prediction and validation:

  • In silico analysis to identify potential MHC class I and II binding peptides

  • Synthesis of predicted epitope peptides for T-cell stimulation assays

  • Analysis of epitope conservation across Rickettsia species

• T-cell response assessment:

  • Measurement of CD8+ and CD4+ T-cell activation using flow cytometry

  • Evaluation of IFN-γ production by antigen-experienced T cells (CD3+CD8+CD44high)

  • Quantification of memory T-cell formation (CD44highCD127high)

• Immunization protocols:

  • Preparation of antigen-presenting cells expressing atpE

  • DNA vaccine constructs encoding atpE

  • Protein-based immunization with purified recombinant atpE

• Protection evaluation:

  • Challenge with virulent Rickettsia strains following immunization

  • Assessment of bacterial load in tissues

  • Measurement of cross-protection against heterologous Rickettsia species

This approach should consider evidence that CD8+ T cells are indispensable for protective immunity against rickettsial infections and that properly selected antigens can induce cross-protective immunity .

What role does atpE play in R. conorii survival and replication within host cells?

While the specific role of atpE in R. conorii pathogenesis is not directly addressed in the provided literature, its function can be inferred based on studies of R. conorii metabolism and host cell interactions:

• Energy production for intracellular survival:

  • ATP synthase is essential for energy metabolism in rickettsiae

  • As a component of ATP synthase, atpE is likely critical for maintaining the proton gradient necessary for ATP synthesis

  • This energy is required for bacterial replication within host cells, particularly macrophages

• Contribution to metabolic adaptations:

  • R. conorii induces significant metabolic reprogramming in infected macrophages

  • These changes include alterations in tricarboxylic acid cycle, oxidative phosphorylation, and fatty acid β-oxidation

  • ATP synthase function may be integrated with these metabolic shifts

• Potential role in host cell manipulation:

  • R. conorii can redirect host metabolism toward an M2-like, anti-inflammatory activation program

  • Energy-dependent processes may be involved in manipulating host cell responses

Methodological approaches to investigate these aspects would include comparative analysis of wild-type and atpE-modified rickettsiae, metabolic profiling of infected cells, and inhibitor studies targeting ATP synthase function.

How does R. conorii atpE compare to homologous proteins in other Rickettsia species, and what implications does this have for cross-species immunity?

A comprehensive analysis of atpE across Rickettsia species would provide insights into its conservation and potential for cross-protection:

• Sequence conservation analysis:

  • Multiple sequence alignment of atpE across pathogenic and non-pathogenic Rickettsia species

  • Identification of conserved domains and species-specific regions

  • Structural modeling to visualize conservation patterns

• Immunological cross-reactivity:

  • Assessment of T-cell epitope conservation across species

  • Cross-reactivity studies with antibodies or T cells from immunized models

  • Correlation of sequence conservation with immunological cross-recognition

• Functional conservation:

  • Comparative analysis of ATP synthase activity across species

  • Assessment of inhibitor sensitivity profiles

  • Correlation of functional conservation with pathogenicity

Studies on R. prowazekii vaccine antigens have demonstrated cross-protection against R. typhi , suggesting that appropriately selected antigens can provide broad protection across related rickettsial species. Whether atpE represents such a candidate requires specific investigation based on its sequence conservation and immunogenic properties.

How does R. conorii atpE potentially interact with host cellular components during infection?

• Identification of interaction partners:

  • Pull-down assays using tagged recombinant atpE

  • Proximity labeling techniques to identify proteins in close proximity during infection

  • Co-immunoprecipitation followed by mass spectrometry

• Localization studies:

  • Immunofluorescence microscopy to track atpE during infection

  • Co-localization with host mitochondria or other cellular structures

  • Live-cell imaging with labeled ATP synthase components

• Functional interaction studies:

  • Assessment of host cell ATP production during infection

  • Measurement of mitochondrial function in infected cells

  • Effect of host ATP synthase inhibitors on bacterial survival

These approaches would help determine whether atpE interacts with host cell components and how such interactions might contribute to the metabolic reprogramming observed during R. conorii infection of macrophages .

What is the potential of R. conorii atpE as a target for therapeutic intervention?

ATP synthase represents a potential therapeutic target due to its essential role in bacterial energy metabolism. Several approaches can be employed to evaluate atpE as a specific target:

• Target validation strategies:

  • Assess essentiality through conditional expression systems

  • Evaluate effects of known ATP synthase inhibitors on bacterial survival

  • Compare with host ATP synthase to identify bacterial-specific features

• Drug discovery approaches:

  • Structure-based design targeting unique features of rickettsial atpE

  • High-throughput screening of compound libraries using functional assays

  • Development of peptide inhibitors targeting c-ring assembly

• Evaluation protocols:

  • Testing in cell culture infection models

  • Determination of minimal inhibitory concentrations

  • Assessment of effects on bacterial load and host cell viability

The identification of structural or functional differences between rickettsial and mammalian ATP synthase would be crucial for developing selective inhibitors that do not affect host cell function.

How can recombinant R. conorii atpE be utilized for development of diagnostic assays?

Recombinant R. conorii atpE protein can be utilized in various diagnostic platforms:

• Serological assay development:

  • ELISA using purified recombinant atpE

  • Western blot for confirmatory testing

  • Multiplexed assays including atpE and other rickettsial antigens

• Performance evaluation:

  • Sensitivity and specificity determination using well-characterized serum panels

  • Cross-reactivity assessment with sera from patients infected with other rickettsial species

  • Comparison with existing diagnostic antigens (OmpA, OmpB)

• Point-of-care applications:

  • Development of lateral flow assays using recombinant atpE

  • Adaptation for resource-limited settings

  • Combination with molecular testing for comprehensive diagnostics

The utility of atpE as a diagnostic antigen would depend on its immunogenicity during natural infection and the specificity of the antibody response compared to other rickettsial proteins.

What are the potential advantages and limitations of using atpE in subunit vaccine formulations?

Based on knowledge of rickettsial immunity and vaccine development approaches , the potential of atpE in subunit vaccine formulations can be evaluated:

Research approaches should focus on:

• Comparison with established protective antigens (like RP778, RP739, RP598, and RP403)

• Optimization of delivery platforms to induce robust CD8+ T-cell responses

• Evaluation in animal models for protective efficacy and duration of immunity

• Assessment of potential for cross-protection against multiple Rickettsia species

How can researchers effectively study the role of atpE in the metabolic reprogramming observed during R. conorii infection?

R. conorii induces significant metabolic reprogramming in infected macrophages . To investigate atpE's role in these changes, researchers should employ:

• Comparative metabolomics approaches:

  • Analysis of metabolic profiles in cells infected with wild-type versus atpE-modified rickettsiae

  • Isotope tracing studies to track metabolic fluxes through key pathways

  • Real-time measurement of cellular metabolic parameters (oxygen consumption, extracellular acidification)

• Targeted proteomics:

  • Quantification of proteins involved in tricarboxylic acid cycle, oxidative phosphorylation, and fatty acid metabolism

  • Temporal profiling of metabolic enzyme expression during infection

  • Phosphoproteomics to identify post-translational modifications of metabolic enzymes

• Functional metabolic analysis:

  • ATP synthesis measurements in isolated bacteria and infected cells

  • Substrate utilization studies to determine energy sources

  • Effect of metabolic inhibitors on bacterial survival and replication

• Host-pathogen interaction studies:

  • Assessment of bacterial ATP synthase localization relative to host mitochondria

  • Analysis of potential transfer of metabolites between bacteria and host

  • Evaluation of host metabolic enzyme recruitment to bacterial compartments

These approaches would help determine whether atpE and the ATP synthase complex are key mediators of the metabolic changes observed during R. conorii infection of macrophages .

What technological advances are needed to better understand the structure-function relationship of rickettsial atpE?

Advanced structural biology techniques are needed to fully elucidate the structure-function relationship of rickettsial atpE:

• High-resolution structural determination:

  • Cryo-electron microscopy of the complete ATP synthase complex

  • NMR studies of reconstituted c-rings in membrane mimetics

  • X-ray crystallography of the F0 sector or complete ATP synthase

• Dynamic structural analysis:

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes

  • Molecular dynamics simulations of proton movement through the c-ring

  • Single-molecule FRET to monitor conformational changes during function

• In situ structural analysis:

  • Cryo-electron tomography of rickettsiae within infected cells

  • Correlative light and electron microscopy to visualize ATP synthase in context

  • In-cell NMR to study protein dynamics in native environment

• Integration of structural and functional data:

  • Correlation of structural features with proton translocation efficiency

  • Mapping of inhibitor binding sites on the structure

  • Structure-guided mutagenesis to test mechanistic hypotheses

These technological advances would provide unprecedented insights into how rickettsial atpE functions within the ATP synthase complex and potentially reveal unique features that could be targeted for therapeutic intervention.

How can contradictory experimental results regarding atpE function be reconciled in the context of different experimental systems?

When faced with contradictory results regarding atpE function across different experimental systems, researchers should implement a systematic approach to reconciliation:

• Standardization of experimental protocols:

  • Consistent protein preparation methods

  • Uniform reconstitution procedures for functional studies

  • Standardized assay conditions for activity measurements

• Comparative analysis across systems:

  • Side-by-side testing in different expression systems

  • Evaluation in both in vitro and cellular contexts

  • Cross-validation using multiple complementary techniques

• Identification of context-dependent factors:

  • Lipid composition effects on protein function

  • Influence of other ATP synthase components

  • Impact of environmental conditions (pH, ion concentrations)

• Integration of diverse data types:

  • Correlation of structural information with functional outcomes

  • Reconciliation of biochemical and cellular observations

  • Mathematical modeling to explain context-dependent behaviors

By implementing this comprehensive approach, researchers can develop a more nuanced understanding of atpE function that accounts for the complexities of different experimental systems and contexts.