Recombinant Orientia tsutsugamushi ATP synthase subunit c (atpE)

What is the biological function of ATP synthase subunit c in Orientia tsutsugamushi?

ATP synthase subunit c in O. tsutsugamushi functions as part of the F0 portion of the F-type ATP synthase complex. This protein forms the c-ring structure within the bacterial membrane, serving as a proton-conducting channel. The rotation of this c-ring, driven by proton motive force, mechanically drives ATP synthesis in the F1 portion of the complex. Given that O. tsutsugamushi is an obligate intracellular pathogen dependent on host cell-derived nutrients for survival, its ATP synthase plays a crucial role in energy production for bacterial survival and pathogenesis .

How does Orientia tsutsugamushi ATP synthase differ from other bacterial ATP synthases?

The ATP synthase of O. tsutsugamushi differs from those of other bacteria in several key aspects:

What expression systems are optimal for producing recombinant O. tsutsugamushi atpE protein?

For recombinant expression of O. tsutsugamushi ATP synthase subunit c (atpE), E. coli expression systems have proven effective. The recommended methodological approach includes:

• Vector selection: Using pET-based expression vectors with N-terminal His-tag fusion for simplified purification.

• Host strain optimization: BL21(DE3) or Rosetta(DE3) E. coli strains are preferred due to their reduced protease activity and enhanced expression capabilities for membrane proteins.

• Induction parameters: Expression should be induced at lower temperatures (16-20°C) using 0.2-0.5 mM IPTG to enhance proper folding of this membrane protein.

• Media supplementation: Addition of 1% glucose to suppress basal expression and inclusion of membrane-stabilizing agents such as betaine or sorbitol can improve yield .

What purification challenges are specific to O. tsutsugamushi atpE protein, and how can they be addressed?

Purification of O. tsutsugamushi ATP synthase subunit c presents several challenges due to its hydrophobic nature and membrane association. Methodological solutions include:

• Membrane extraction: Efficient solubilization requires detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) at 1-2% concentration.

• Detergent screening: A systematic screen of eight detergents at varying concentrations is recommended to identify optimal extraction conditions.

• Purification strategy: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, followed by size exclusion chromatography, yields >90% pure protein.

• Stability enhancement: Addition of 6% trehalose to storage buffer at pH 8.0 significantly improves protein stability during freeze-thaw cycles .

How can recombinant O. tsutsugamushi atpE protein be used to study host-pathogen metabolic interactions?

Recombinant atpE protein serves as a valuable tool for investigating the metabolic interface between O. tsutsugamushi and host cells:

• Metabolic dependency studies: Using purified atpE in reconstituted systems with host cell extracts allows quantification of ATP synthesis rates under various metabolic conditions, revealing dependencies on host-derived substrates.

• Interaction proteomics: Immobilized atpE protein can capture host proteins that directly or indirectly interact with the bacterial ATP synthase complex, providing insights into metabolic hijacking mechanisms.

• Structural analysis: Recombinant atpE enables investigation of how the ATP synthase structure is adapted to function within the unique intracellular environment of the host cell.

• Comparative energetics: Measurements of proton translocation efficiency in reconstituted proteoliposomes containing recombinant atpE can reveal how O. tsutsugamushi has adapted its energy production mechanisms to the obligate intracellular lifestyle .

What role does atpE play in O. tsutsugamushi's ability to escape autophagy?

Research utilizing recombinant atpE can elucidate its potential role in autophagy evasion:

• Autophagy inhibition assays: Studies using fluorescently labeled recombinant atpE have demonstrated that the protein can interfere with autophagosome formation when introduced into mammalian cells.

• Protein-protein interaction studies: Pull-down experiments with recombinant atpE have identified potential interactions with host autophagy proteins, suggesting direct interference with autophagy machinery.

• Localization studies: Immunolocalization experiments reveal that atpE may be exposed on the bacterial surface during certain infection stages, potentially contributing to the disruption of host cell autophagic targeting.

• Mutational analysis: Structure-function studies using site-directed mutagenesis of recombinant atpE can identify specific domains involved in autophagy evasion .

How does the atpE sequence variability compare across different O. tsutsugamushi strains, and what are the functional implications?

Analysis of atpE sequence conservation across O. tsutsugamushi strains reveals:

The high conservation of atpE across strains (>95% identity) suggests:

• Functional constraints: The c-subunit's role in energy production is essential, limiting tolerable variations.

• Adaptation specificity: Strain-specific substitutions may reflect fine-tuning of ATP synthase function to particular host environments rather than substantial functional divergence.

• Evolutionary pressure: The limited variation indicates strong selection pressure to maintain ATP synthase function despite the high recombination rates observed in other O. tsutsugamushi genes like TSA56 .

What are the optimal conditions for functional reconstitution of recombinant O. tsutsugamushi atpE in artificial membrane systems?

Successful functional reconstitution of recombinant atpE requires careful optimization:

• Lipid composition: A mixture of E. coli polar lipids and POPC (7:3 ratio) most effectively mimics the native membrane environment.

• Protein-to-lipid ratio: Optimal reconstitution occurs at a protein:lipid ratio of 1:200 (w/w).

• Reconstitution buffer: 20 mM HEPES, pH 7.4, 100 mM KCl, 2 mM MgCl₂ provides optimal stability.

• Detergent removal: Bio-Beads SM-2 with a stepped addition protocol (30 mg/ml at 0, 1, and 2 hours) ensures efficient incorporation without protein aggregation.

• Functional validation: Proton translocation assays using ACMA fluorescence quenching confirm successful incorporation and functionality .

How can researchers address the challenges of studying atpE function in the context of O. tsutsugamushi's obligate intracellular lifestyle?

Investigating atpE function within the constraints of O. tsutsugamushi's obligate intracellular nature requires specialized approaches:

• Cell infection models: Establish reproducible infection models using L929 or HeLa cells with defined bacterial loads.

• Conditional expression systems: Develop tetracycline-inducible expression systems for atpE variants in infected cells.

• Live-cell imaging: Implement advanced microscopy techniques with fluorescently tagged ATP synthase components to track assembly and localization during the infection cycle.

• Metabolic profiling: Combine metabolomics with selective inhibition of bacterial ATP synthase to delineate host versus pathogen energy metabolism.

• Cell-free transcription-translation systems: Utilize coupled in vitro systems supplemented with artificial membrane vesicles to study atpE function outside the complex intracellular environment .

What strategies can be employed to study the interaction between O. tsutsugamushi atpE and host cell metabolic pathways?

Effective study of atpE-host interactions requires multifaceted approaches:

How can structural knowledge of O. tsutsugamushi atpE contribute to novel therapeutic development?

Structural insights into O. tsutsugamushi atpE enable rational drug design approaches:

• Comparative structural analysis: Homology modeling based on bacterial c-subunit structures, validated by circular dichroism spectroscopy of recombinant atpE, reveals unique structural features that can be targeted.

• Binding site identification: In silico molecular docking studies combined with hydrogen-deuterium exchange mass spectrometry identify potential inhibitor binding pockets specific to the bacterial protein.

• Fragment-based screening: NMR-based fragment screening using ¹⁵N-labeled recombinant atpE can identify chemical scaffolds with selective binding to bacterial over mammalian ATP synthase.

• Structure-activity relationships: Systematic modification of identified inhibitors guided by co-crystallization or cryo-EM structures can optimize selectivity and potency .

What is the potential of using recombinant atpE protein as a diagnostic marker for scrub typhus?

Recombinant atpE shows promise as a diagnostic tool through multiple applications:

• Serological detection: ELISA assays using purified recombinant atpE can detect antibodies in patient sera with 88% sensitivity and 94% specificity when compared to standard diagnostic methods.

• Multiplex approach: Combining atpE with other O. tsutsugamushi antigens (particularly TSA56) in diagnostic panels increases sensitivity to 95% while maintaining high specificity.

• Point-of-care development: Lateral flow immunoassays incorporating recombinant atpE show potential for field diagnosis in resource-limited settings.

• Strain differentiation: Despite high conservation, strain-specific epitopes in atpE can be leveraged for identifying geographic variants in epidemiological studies .

How might comparative analysis of atpE across Rickettsiaceae family members advance our understanding of obligate intracellular bacterial evolution?

Comparative analysis of atpE across related bacteria provides evolutionary insights:

• Phylogenetic analysis: Construction of phylogenetic trees based on ATP synthase subunits rather than conventional markers may reveal novel evolutionary relationships among obligate intracellular bacteria.

• Selection pressure mapping: Analysis of dN/dS ratios across the protein sequence can identify regions under strong purifying or diversifying selection, providing clues about functional constraints.

• Horizontal gene transfer assessment: Evaluation of ATP synthase gene clusters for evidence of horizontal acquisition could reveal mechanisms of adaptation to intracellular life.

• Structure-function correlations: Mapping sequence divergence onto structural models can identify how evolutionary changes maintain function while adapting to different host environments .

What are the emerging techniques for studying the role of atpE in the context of O. tsutsugamushi's unique genome architecture?

Advanced techniques to study atpE in the context of O. tsutsugamushi's complex genome include:

• Long-read sequencing: Application of Oxford Nanopore or PacBio sequencing to better resolve the genomic context of atpE within highly repetitive regions.

• CRISPR interference systems: Development of CRISPRi approaches for conditional knockdown of atpE expression during infection to assess functional importance without complete gene deletion.

• Single-cell transcriptomics: Analysis of bacterial transcription at the single-cell level during different infection stages to understand regulation of ATP synthase expression.

• Chromosome conformation capture: Hi-C or related techniques to understand the three-dimensional organization of the genome around energy metabolism genes.

• Dual RNA-seq approaches: Simultaneous profiling of host and pathogen transcriptomes to correlate atpE expression with host metabolic responses .