Recombinant Wolbachia sp. subsp. Brugia malayi ATP synthase subunit c (atpE)

What is the basic structure of Wolbachia sp. subsp. Brugia malayi ATP synthase subunit c (atpE)?

Wolbachia sp. subsp. Brugia malayi ATP synthase subunit c (atpE) is a 75-amino acid protein with a highly hydrophobic composition, consistent with its role as a membrane component of the F0 sector of ATP synthase. The complete amino acid sequence is MDLVALKFIAIGLSVLGILGAGLGVANIFSTMLSGLARNPESEGKMKIYVYVGAGMVEFT GLLAFVLAMLLMFVA . This protein contains transmembrane domains that are critical for forming the c-ring structure within the ATP synthase complex. The protein's UniProt ID is Q5GSH7, and it has several synonyms including Wbm0459, F-type ATPase subunit c, and Lipid-binding protein .

How is the activity of ATP synthase subunit c (atpE) linked to the metabolic relationship between Wolbachia and Brugia malayi?

ATP synthase subunit c forms part of the F0F1-ATP synthase, a critical enzyme complex in energy production. Research demonstrates that Wolbachia may function as a metabolic symbiont by supplementing mitochondrial energy production in filarial nematodes like Brugia malayi . The F0 component containing atpE creates a proton gradient that drives ATP synthesis. In low oxygen and low glucose (LOLG) conditions, metabolic modeling shows that Wolbachia exports the maximum amount of ATP possible (100 units) into the B. malayi cytosol . This metabolic interplay is essential to the worm's survival, as Wolbachia provides ATP to the host, particularly under nutrient-limited conditions, highlighting why the atpE protein is of significant research interest.

What is the evolutionary significance of ATP synthase subunit c in Wolbachia compared to other bacterial species?

ATP synthase subunit c is highly conserved across bacterial species, reflecting its fundamental role in energy metabolism. In Wolbachia, this protein demonstrates adaptations specific to its endosymbiotic lifestyle. While this exact evolutionary divergence isn't explicitly detailed in the provided references, research on metabolic interactions suggests that Wolbachia's ATP synthase components have evolved to optimize energy sharing with its host. The bacterium has retained this essential metabolic machinery despite genome reduction common in obligate endosymbionts, indicating its critical importance. Comparative studies of atpE sequences across Wolbachia strains found in different host organisms would provide valuable insights into selective pressures and co-evolutionary patterns.

What expression systems are most effective for producing recombinant Wolbachia atpE protein?

E. coli has been demonstrated as an effective expression system for producing recombinant Wolbachia sp. subsp. Brugia malayi ATP synthase subunit c (atpE) . The recombinant protein is typically expressed with an N-terminal His-tag to facilitate purification. While E. coli is the predominant system described in the literature, researchers should consider the hydrophobic nature of this membrane protein when optimizing expression conditions, including temperature, induction parameters, and strain selection. Alternative expression systems such as yeast might be considered, particularly given that Wolbachia can grow in Saccharomyces cerevisiae under specific conditions , though this would require optimization for membrane protein expression.

What purification strategies yield the highest purity and functional activity of recombinant atpE protein?

Purification of His-tagged atpE protein typically employs immobilized metal affinity chromatography (IMAC). Based on available information, the recombinant protein can be purified to greater than 90% purity as determined by SDS-PAGE . For membrane proteins like atpE, consider these methodological approaches:

• Solubilization using appropriate detergents (e.g., n-dodecyl β-D-maltoside or CHAPS)

• Buffer optimization to maintain protein stability

• Sequential chromatography steps (IMAC followed by size exclusion or ion exchange)

• Storage in stabilizing buffers containing glycerol

The final product is typically provided as a lyophilized powder that can be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol for long-term storage at -20°C/-80°C .

What are the critical factors for maintaining stability of purified recombinant atpE protein?

Several factors are critical for maintaining stability of purified recombinant atpE protein:

• Storage conditions: Store at -20°C/-80°C upon receipt; aliquoting is necessary for multiple uses to avoid repeated freeze-thaw cycles .

• Buffer composition: The protein is typically stored in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 . The trehalose acts as a stabilizing agent for the lyophilized protein.

• Reconstitution protocol: Centrifuge the vial briefly before opening to bring contents to the bottom. Reconstitute in deionized sterile water to 0.1-1.0 mg/mL .

• Glycerol addition: Addition of 5-50% glycerol (final concentration) is recommended for long-term storage, with 50% being the default concentration .

• Avoidance of freeze-thaw cycles: Repeated freezing and thawing significantly reduces protein integrity and should be avoided .

For working aliquots, storage at 4°C for up to one week is possible, but longer periods require freezing with glycerol protection.

What experimental approaches can be used to study ATP synthase activity in recombinant Wolbachia atpE?

Several experimental approaches can be employed to study ATP synthase activity involving recombinant Wolbachia atpE:

• Reconstitution into liposomes: Purified atpE can be reconstituted into liposomes along with other ATP synthase components to measure proton translocation activity.

• ATP synthesis assays: Using reconstituted proteoliposomes containing the complete ATP synthase complex, ATP synthesis can be measured under different conditions by detecting ATP production via luciferase-based assays.

• Proton gradient measurements: Fluorescent pH-sensitive dyes can be used to monitor proton translocation across membranes containing reconstituted atpE.

• Oxygen consumption analysis: In intact systems, respiration rates can be measured using oxygen electrodes to indirectly assess ATP synthase function.

• Yeast complementation studies: Since Wolbachia can grow in Saccharomyces cerevisiae , developing yeast mutants lacking endogenous ATP synthase components and complementing with Wolbachia atpE could provide functional insights.

These approaches would need to be optimized specifically for the Wolbachia protein, considering its unique properties and the complex metabolic interplay between the bacterium and its host.

How can researchers assess the interaction between atpE and other components of the ATP synthase complex?

Researchers can employ several techniques to assess interactions between atpE and other components of the ATP synthase complex:

• Co-immunoprecipitation: Using antibodies against tagged versions of atpE to pull down interacting proteins, followed by mass spectrometry identification.

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry can identify residues in close proximity between atpE and other subunits.

• Surface plasmon resonance (SPR): This technique can measure binding kinetics between purified atpE and other ATP synthase components.

• Yeast two-hybrid or bacterial two-hybrid assays: Modified versions of these assays can be used to detect membrane protein interactions.

• Native gel electrophoresis: Blue native PAGE can preserve protein complexes and reveal associations between atpE and other components.

• Cryo-electron microscopy: This technique can provide structural information about the entire ATP synthase complex including the c-ring formed by atpE subunits.

When interpreting results, researchers should consider the hydrophobic nature of atpE and optimize detergent conditions to maintain native interactions while preventing non-specific hydrophobic associations.

What methods are available for studying the role of atpE in energy metabolism within the Wolbachia-Brugia malayi symbiotic relationship?

Several advanced methods can be employed to study atpE's role in the Wolbachia-Brugia malayi symbiotic relationship:

Research demonstrates that under low oxygen and low glucose conditions, Wolbachia exports ATP to B. malayi cytosol, highlighting the crucial role of ATP synthase in maintaining this symbiotic relationship .

How does the expression of Wolbachia atpE change under different nutrient conditions, and what are the implications for host-symbiont metabolism?

The expression and activity of Wolbachia ATP synthase components, including atpE, appear to adapt to different nutrient conditions with significant implications for host-symbiont metabolism. Research using metabolic modeling reveals distinct responses under varying oxygen and glucose conditions :

These findings suggest that atpE expression and ATP synthase activity are likely upregulated under nutrient-limited conditions to maximize energy transfer to the host . This metabolic flexibility allows Wolbachia to adapt to changing environments throughout the B. malayi life cycle, potentially explaining why the endosymbiont is essential for worm survival.

What is the relationship between Wolbachia atpE function and the sirtuin-mediated metabolic regulation in infected hosts?

Research has identified a fascinating relationship between Wolbachia and host sirtuin expression, particularly sirt-4, which has implications for ATP synthase function and energy metabolism:

• Wolbachia infection is associated with reduced sirt-4 expression in a strain-specific manner .

• This Wolbachia-induced suppression of sirt-4 appears to alter glutamate dehydrogenase (GDH) expression and ATP levels . In wildtype Wolbachia-infected flies, there was a 26% increase in gdh expression compared to uninfected controls .

• GDH is a direct target of SIRT-4 and is involved in glutamine metabolism and ATP homeostasis .

• The sirtuin x Wolbachia interaction impacts host glucose metabolism, with high glucose levels detected in Wolbachia-infected flies .

While the direct interaction between sirt-4 and atpE hasn't been explicitly characterized, this evidence suggests a potential metabolic feedback loop: Wolbachia ATP synthase (containing atpE) produces ATP that influences host metabolism, while host sirtuin levels may in turn regulate Wolbachia metabolism and replication. This relationship represents an important area for further investigation to understand how ATP synthase components contribute to the complex metabolic crosstalk between host and endosymbiont.

How does the function of Wolbachia atpE in Brugia malayi compare to its role in other host systems?

ATP synthase subunit c (atpE) serves a fundamental role in energy production across different host-Wolbachia systems, but studies suggest host-specific adaptations:

• In Brugia malayi: Metabolic modeling indicates that Wolbachia ATP synthase contributes significant ATP to the host, especially under nutrient-limited conditions . The endosymbiont appears to supplement mitochondrial energy production, which may explain why Wolbachia is essential for this filarial worm's survival.

• In Drosophila: Research shows that Wolbachia influences host metabolism through interaction with sirtuins, affecting glucose homeostasis and ATP levels . The fact that sirt-4 over-expression reduces Wolbachia ovarian titer suggests a regulatory feedback mechanism involving energy metabolism.

• In Saccharomyces cerevisiae: Experimental evidence shows that Wolbachia can establish infection in yeast, but eventually leads to host cell death during the stationary phase . This suggests that the energy dynamics and ATP synthase function may differ in non-natural hosts.

What is the potential of Wolbachia atpE as a drug target for treating lymphatic filariasis?

Wolbachia atpE represents a promising drug target for treating lymphatic filariasis for several compelling reasons:

• Essentiality: As a component of ATP synthase, atpE is likely essential for Wolbachia survival. Metabolic modeling identified ATP synthase reactions among the 102 reactions essential to B. malayi survival .

• Host dependence: B. malayi relies on Wolbachia for energy supplementation, particularly under nutrient-limited conditions . Disrupting this relationship by targeting atpE could effectively kill the worm.

• Selectivity: Differences between bacterial F-type ATP synthases and human mitochondrial ATP synthases could provide selectivity for targeting Wolbachia while minimizing host toxicity.

• Precedent: Other ATP synthase inhibitors have proven effective as antimicrobials, including bedaquiline for tuberculosis treatment, suggesting this approach is pharmacologically viable.

• Critical metabolic position: ATP synthase represents a bottleneck in energy production, and its inhibition would have cascading effects on multiple essential processes in both Wolbachia and its dependent host.

A drug development strategy could involve high-throughput screening of compound libraries against recombinant atpE or reconstituted ATP synthase complexes, followed by validation in Wolbachia-infected cell cultures and eventually animal models of filariasis.

How can heterologous expression systems be optimized for structural and functional studies of Wolbachia atpE?

Optimizing heterologous expression systems for Wolbachia atpE requires addressing several challenges associated with membrane protein expression:

• E. coli expression systems:

  • Use specialized strains (C41/C43, Lemo21) designed for membrane protein expression

  • Optimize induction conditions (lower temperatures, reduced IPTG concentrations)

  • Consider fusion partners (MBP, SUMO) to enhance solubility

  • Test different detergents for efficient extraction (DDM, LDAO, CHAPS)

• Yeast expression systems:

  • S. cerevisiae or P. pastoris can provide a eukaryotic environment

  • Particularly relevant given Wolbachia's ability to grow in S. cerevisiae

  • Requires special media supplementation (ammonium ferric citrate, bovine fetal serum)

  • Consider inducible promoters (GAL1, AOX1) for controlled expression

• Cell-free expression systems:

  • Avoid cellular toxicity issues

  • Allow direct incorporation into nanodiscs or liposomes

  • Can be supplemented with detergents or lipids during translation

• Purification considerations:

  • Gentle solubilization to maintain native structure

  • Stability screening with differential scanning fluorimetry

  • Use of lipid nanodiscs to maintain native-like environment

For structural studies, expression constructs should be designed with crystallography or cryo-EM in mind, potentially including thermostabilizing mutations or removable fusion proteins to facilitate crystal contacts while maintaining function.

What are the latest approaches for studying the protein-protein interactions between atpE and other ATP synthase components in the context of the Wolbachia-Brugia malayi symbiosis?

Cutting-edge approaches for studying protein-protein interactions involving Wolbachia atpE include:

• Proximity labeling techniques:

  • BioID or APEX2 fusions to atpE can identify proximal proteins in vivo

  • These methods work in the native cellular environment and can capture transient interactions

  • Particularly valuable for membrane protein complexes like ATP synthase

• Native mass spectrometry:

  • Allows analysis of intact membrane protein complexes

  • Can determine subunit stoichiometry and complex stability

  • Requires careful optimization of detergent or nanodiscs

• Cross-linking mass spectrometry (XL-MS):

  • Uses bifunctional cross-linkers followed by proteomic analysis

  • Provides spatial constraints for protein-protein interfaces

  • Can be performed in situ in intact bacterial cells

• Single-particle cryo-electron microscopy:

  • Enables structural determination of the entire ATP synthase complex

  • Can visualize different conformational states during the catalytic cycle

  • Resolution is now comparable to X-ray crystallography

• Integrative modeling approaches:

  • Combines multiple experimental datasets (crosslinking, EM, evolutionary coupling)

  • Generates comprehensive structural models of protein complexes

  • Particularly useful for dynamic assemblies like ATP synthase

These techniques could be applied to studying atpE interactions in the context of Wolbachia isolated from B. malayi or in the S. cerevisiae model system, which has been shown to support Wolbachia growth . The findings would provide insights into how these interactions contribute to the symbiotic relationship and potentially identify new therapeutic targets.

What are common challenges in working with recombinant Wolbachia atpE and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant Wolbachia atpE:

Successful production of functional atpE requires careful optimization at each step from gene design through expression, purification, and storage. The recommended use of Tris/PBS-based buffer with 6% Trehalose at pH 8.0 provides a starting point that may need further refinement based on specific experimental requirements.

How can researchers validate the structural integrity and functionality of purified recombinant atpE protein?

To validate both structural integrity and functionality of purified recombinant Wolbachia atpE protein, researchers should employ multiple complementary techniques:

Structural Validation:

• SDS-PAGE and Western blotting: Confirms correct molecular weight and immunoreactivity

• Circular dichroism (CD) spectroscopy: Assesses secondary structure content and proper folding

• Thermal shift assays: Evaluates protein stability under different buffer conditions

• Size exclusion chromatography: Determines oligomeric state and homogeneity

• Limited proteolysis: Probes for correctly folded, protease-resistant domains

Functional Validation:

• Reconstitution assays: Incorporation into liposomes or nanodiscs to form functional c-rings

• Proton translocation assays: Using pH-sensitive fluorescent dyes to monitor proton movement

• ATP synthesis measurement: When assembled with other ATP synthase components

• Inhibitor binding studies: Interaction with known ATP synthase inhibitors

• Complementation assays: Ability to rescue ATP synthase-deficient bacterial or yeast strains

Careful validation using multiple approaches ensures that any subsequent experimental findings accurately reflect the native properties of Wolbachia atpE rather than artifacts from misfolded or non-functional protein.

What considerations are important when designing experiments to study the effect of environmental conditions on atpE function?

When designing experiments to study environmental effects on Wolbachia atpE function, researchers should consider:

• Physiologically relevant conditions: Based on metabolic modeling, test conditions that mimic the host environment, including:

  • Varying oxygen levels (high: 580 units; low: 90 units)

  • Different glucose concentrations (high: 250 units; low: 45 units)

  • Presence of fatty acids and amino acids that may affect ATP synthase activity

• pH considerations:

  • The proton gradient is crucial for ATP synthase function

  • Test pH ranges that represent different microenvironments within the host

  • Consider how pH affects protein stability and interaction with other subunits

• Temperature effects:

  • Test temperature ranges encountered during different host life stages

  • Include thermal stability assays to determine protein denaturation temperatures

  • Consider how temperature shifts affect ATP synthesis rates

• Experimental systems:

  • Use reconstituted systems (proteoliposomes) for controlled conditions

  • Consider heterologous systems like S. cerevisiae that support Wolbachia growth

  • Compare results from isolated protein with those from intact bacteria when possible

• Data collection and analysis:

  • Implement factorial experimental designs to assess interaction effects

  • Use response surface methodology to identify optimal conditions

  • Apply appropriate statistical analysis for complex multifactorial experiments

When interpreting results, researchers should consider that Wolbachia demonstrates metabolic flexibility, including the novel glutamate metabolic pathway observed under low oxygen conditions , which may impact ATP synthase activity and regulation.

What are promising research directions for understanding the role of atpE in Wolbachia-host metabolic integration?

Several promising research directions could advance our understanding of atpE's role in Wolbachia-host metabolic integration:

• Comparative metabolomics: Profiling metabolites in Wolbachia-infected versus uninfected hosts under various conditions to identify shifts in energy-related metabolites linked to ATP synthase activity.

• Tissue-specific energy dynamics: Investigating whether atpE expression and ATP production vary across different host tissues, particularly reproductive versus somatic tissues, given Wolbachia's reproductive manipulation phenotypes.

• Life stage-specific regulation: Exploring how atpE expression and ATP synthase activity change throughout host developmental stages, particularly in B. malayi where life stage-specific gene expression data is available .

• Host-symbiont co-evolution: Comparative analysis of atpE sequences and function across Wolbachia strains from different hosts to identify adaptations that optimize energy sharing in specific symbiotic relationships.

• Integration with sirtuin pathways: Further investigation of the Wolbachia-sirtuin interaction , specifically how sirt-4 regulation impacts ATP synthase activity and whether there's direct regulation of atpE expression.

• Mitochondria-Wolbachia interaction: Examining potential synchronization between mitochondrial and Wolbachia ATP production, possibly through shared regulatory mechanisms or metabolite exchange.

These research directions would benefit from integrating computational approaches like metabolic modeling with experimental validation using techniques like isotope labeling to track energy flow between symbiont and host.

How might synthetic biology approaches be applied to manipulate Wolbachia atpE for therapeutic purposes?

Synthetic biology offers innovative approaches to manipulate Wolbachia atpE for therapeutic purposes:

• Engineered atpE variants:

  • Design modified atpE proteins with reduced efficiency to create attenuated Wolbachia strains

  • These could potentially maintain beneficial host effects while reducing pathogenicity

  • Alternatively, super-efficient variants could enhance beneficial metabolic supplementation

• Controllable expression systems:

  • Develop inducible promoters for atpE that respond to external signals

  • Enable dynamic control of Wolbachia energy production in response to host needs

  • Create conditional lethal strains for targeted elimination in specific tissues

• Delivery mechanisms:

  • Engineer outer membrane vesicles containing modified atpE to deliver to existing Wolbachia

  • Develop phage-based systems to introduce engineered atpE genes into Wolbachia

  • Create lipid nanoparticles to deliver modified ATP synthase components

• Drug delivery systems:

  • Use knowledge of atpE structure to design targeted inhibitors

  • Create pro-drugs activated by Wolbachia-specific enzymes near ATP synthase

  • Develop aptamers that specifically bind atpE to deliver therapeutic payloads

• Diagnostic applications:

  • Engineer biosensors using atpE binding domains to detect Wolbachia infection

  • Develop reporter systems linked to ATP synthase activity to monitor treatment efficacy

These approaches could potentially treat not only lymphatic filariasis caused by B. malayi but also other Wolbachia-dependent filarial diseases and potentially modify Wolbachia-insect interactions relevant to vector control.

What interdisciplinary approaches might yield new insights into the structural biology and biochemistry of Wolbachia atpE?

Interdisciplinary approaches that combine multiple techniques and perspectives could dramatically advance our understanding of Wolbachia atpE:

• Integrated structural biology:

  • Combining cryo-EM, X-ray crystallography, and NMR to characterize different states

  • Using molecular dynamics simulations to model proton movement through the c-ring

  • Applying hydrogen-deuterium exchange mass spectrometry to probe dynamics

• Systems biology integration:

  • Connecting atpE function to genome-scale metabolic models like iDC625

  • Using flux balance analysis to predict effects of atpE modifications

  • Integrating transcriptomic and proteomic data across different conditions

• Advanced biophysical approaches:

  • Single-molecule FRET to observe conformational changes during rotation

  • High-speed atomic force microscopy to visualize c-ring rotation in real-time

  • Nanoscale thermophoresis to characterize interactions with inhibitors

• Computational drug design:

  • Virtual screening against atpE structural models

  • Fragment-based drug discovery targeting the c-ring assembly

  • Quantum mechanical simulations of proton transfer mechanisms

• Evolutionary biochemistry:

  • Ancestral sequence reconstruction to trace atpE evolution

  • Deep mutational scanning to map sequence-function relationships

  • Comparative analysis across diverse Wolbachia strains to identify conserved functional elements

By integrating these approaches, researchers could develop a comprehensive understanding of atpE structure, function, and evolution, potentially leading to novel therapeutic strategies for treating Wolbachia-dependent diseases like lymphatic filariasis.

What controls are essential when studying the effects of atpE inhibition on Wolbachia-host interactions?

When designing experiments to study atpE inhibition effects, researchers should implement these essential controls:

• Target validation controls:

  • Demonstrate specific binding of inhibitors to recombinant atpE

  • Use site-directed mutagenesis to create inhibitor-resistant atpE variants

  • Compare effects of atpE inhibitors with inhibitors of other ATP synthase components

• Host effect controls:

  • Include uninfected host cells/organisms to distinguish direct host effects from Wolbachia-mediated effects

  • Test effects on host ATP synthase to assess target specificity

  • Include Wolbachia-free B. malayi (generated by antibiotic treatment) when available

• Metabolic context controls:

  • Test under different nutrient conditions (HOHG, HOLG, LOHG, LOLG) to account for metabolic flexibility

  • Include metabolite supplementation (e.g., ATP, glutamate) to determine if phenotypes are rescued

  • Monitor multiple metabolic pathways to detect compensatory responses

• Technical controls:

  • Include vehicle controls matching inhibitor solvent

  • Implement concentration gradients to establish dose-response relationships

  • Include time-course sampling to distinguish immediate from adaptive effects

• Genetic controls:

  • When possible, compare inhibitor effects with genetic knockdown/knockout of atpE

  • Test effects in strains with varying Wolbachia densities

  • Consider sirt-4 manipulation given its interaction with Wolbachia metabolism

How can researchers design experiments to differentiate between direct effects of atpE inhibition and secondary metabolic consequences?

Designing experiments to differentiate direct atpE inhibition effects from secondary metabolic consequences requires sophisticated approaches:

• Temporal analysis:

  • Implement high-resolution time-course experiments

  • Direct effects on ATP production should occur rapidly (minutes to hours)

  • Secondary metabolic adaptations typically emerge later (hours to days)

  • Use metabolic flux analysis at different timepoints to track pathway rewiring

• Spatial resolution:

  • Employ subcellular fractionation to isolate Wolbachia from host components

  • Use fluorescent probes to monitor ATP levels and membrane potential in distinct compartments

  • Implement tissue-specific analyses to detect localized versus systemic effects

• Combined inhibitor approaches:

  • Apply atpE inhibitors alongside inhibitors of potential compensatory pathways

  • Test "rescue experiments" with metabolic supplements (ATP, amino acids)

  • Use inhibitor combinations targeting different ATP synthase components

• Multi-omics integration:

  • Combine targeted metabolomics focusing on ATP, ADP, AMP and related energy metabolites

  • Integrate with proteomics to detect compensatory protein expression changes

  • Add transcriptomics to identify regulatory responses

• Isotope labeling strategies:

  • Use 13C-labeled substrates to track metabolic flux changes

  • Implement pulse-chase experiments to distinguish immediate versus delayed effects

  • Analyze isotopologue distributions to identify activated alternative pathways

This experimental framework allows researchers to construct detailed mechanistic models that separate primary effects of atpE inhibition from the cascade of secondary adaptations, providing clearer insights into Wolbachia-host metabolic integration.

What experimental design would best elucidate the impact of environmental stressors on atpE expression and function in the Wolbachia-Brugia malayi system?

A comprehensive experimental design to elucidate environmental stressor impacts on atpE would include:

1. Factorial design with multiple stressors:

• Oxygen levels (hypoxia, normoxia, hyperoxia)

• Nutrient availability (glucose, amino acids, lipids)

• Temperature variations (fever-like, normal, reduced)

• pH changes (acidosis, normal, alkalosis)

• Oxidative stress (H₂O₂, paraquat exposure)

2. Multi-level analysis approach:

3. Time-course sampling:

• Acute response (minutes to hours)

• Adaptive response (hours to days)

• Long-term effects (days to weeks)

4. Systems for study:

• In vitro: Isolated Wolbachia in cell-free systems

• Ex vivo: B. malayi tissues maintained in culture

• In vivo: Animal models of filariasis

• In silico: Update metabolic models like iDC625 with experimental data

5. Validation strategies:

• Genetic: manipulate sirt-4 expression to modulate host-symbiont interactions

• Pharmacological: use specific inhibitors of ATP synthase

• Host comparison: contrast with Wolbachia from other hosts (e.g., insects)

This comprehensive design would generate a systems-level understanding of how environmental stressors affect atpE function, potentially revealing new therapeutic opportunities and insights into the evolutionary adaptations enabling this successful symbiosis.