Recombinant Wolbachia sp. subsp. Drosophila simulans ATP synthase subunit c (atpE)

What is Wolbachia and why is it significant in Drosophila research?

Wolbachia are α-proteobacteria that are among the most abundant intracellular bacteria on earth. These endosymbionts infect approximately 65% of insect species worldwide, including Drosophila simulans, where they manipulate host reproduction to favor their own survival . Wolbachia are particularly significant in research because they can influence host fitness, induce cytoplasmic incompatibility, and cause male lethality, male-to-female transformation, and parthenogenesis . The study of Wolbachia proteins like ATP synthase subunit c (atpE) provides valuable insights into host-symbiont interactions and bacterial energy metabolism.

What is the ATP synthase subunit c (atpE) in Wolbachia?

ATP synthase subunit c (atpE) is a critical component of the F0F1-ATP synthase complex in Wolbachia sp. subsp. Drosophila simulans. This protein consists of 75 amino acids with the sequence: MDLVALKFIAIGLAVFGMLGAGLGIANIFSAMLNGIARNPESEGKMKSYVYIGAAMVEIMGLLAFVLAMLLIFAA . As part of the F0 domain, it forms the membrane-embedded proton channel that drives ATP synthesis, making it essential for bacterial energy production and survival within host cells.

How does recombinant technology help in studying Wolbachia proteins?

Recombinant technology allows researchers to isolate and study specific Wolbachia proteins outside their natural bacterial environment. For highly intracellular bacteria like Wolbachia that are difficult to culture independently, recombinant expression provides a means to produce sufficient quantities of specific proteins like atpE for structural, functional, and immunological studies. The commonly used approach involves expressing the target protein in E. coli with an affinity tag (such as His-tag) for simplified purification . This methodology enables detailed biochemical characterization that would be challenging when working with native Wolbachia proteins.

What expression systems are recommended for producing recombinant Wolbachia atpE protein?

The most effective expression system for recombinant Wolbachia atpE production is E. coli, as evidenced by successful expression of the full-length protein (amino acids 1-75) with an N-terminal His-tag . When designing an expression strategy, researchers should consider:

For most basic research applications, the E. coli system is sufficient and has been demonstrated to produce atpE protein with greater than 90% purity as determined by SDS-PAGE .

What purification methods yield highest purity for recombinant Wolbachia atpE?

For His-tagged recombinant atpE, a multi-step purification protocol is recommended:

• Initial capture using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin

• Followed by size exclusion chromatography (SEC) to remove aggregates and non-specifically bound proteins

• Optional ion exchange chromatography as a polishing step if higher purity is required

This methodology consistently yields atpE protein with >90% purity . For membrane proteins like atpE, inclusion of mild detergents (0.1% DDM or 0.05% LMNG) in all buffers is crucial to maintain protein solubility. Final purity should be assessed by SDS-PAGE, and identity confirmed by Western blot using anti-His antibodies or mass spectrometry.

How should researchers optimize storage conditions for recombinant Wolbachia atpE?

Optimal storage of recombinant Wolbachia atpE requires specific conditions to maintain stability and activity:

• Store lyophilized powder at -20°C/-80°C upon receipt

• After reconstitution, prepare working aliquots to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• For reconstitution, use Tris/PBS-based buffer with 6% Trehalose, pH 8.0

The addition of trehalose is particularly important as it acts as a cryoprotectant that helps maintain protein stability during freeze-thaw cycles. Long-term storage should be at -80°C in small aliquots to minimize degradation from multiple freeze-thaw events.

How does Wolbachia atpE contribute to bacterial survival within host cells?

ATP synthase subunit c (atpE) plays a critical role in Wolbachia's energy metabolism within host cells. As an obligate intracellular bacterium, Wolbachia relies heavily on energy production to maintain essential functions. Research suggests that atpE, as part of the ATP synthase complex, has adapted to function optimally within the unique intracellular environment of the host.

Several studies on related intracellular bacteria have shown that ATP synthase components can be regulated in response to nutrient availability from the host. For Wolbachia, which lacks essential amino acid biosynthetic pathways, the ATP synthase function may be particularly crucial for generating energy needed to acquire nutrients from host cells through specialized transport mechanisms . The ATP generated may power protein translocation systems that facilitate amino acid acquisition from host proteolytic pathways .

What structural features distinguish Wolbachia atpE from other bacterial ATP synthase c subunits?

Comparative structural analysis of the Wolbachia atpE protein reveals several distinctive features:

The sequence "IAIGLAVFGMLGAGLGIAN" contains the characteristic hydrophobic motif found in ATP synthase c subunits, but with specific amino acid substitutions that may reflect adaptation to the Wolbachia-Drosophila intracellular environment .

How does recombinant atpE protein interact with host immune responses in experimental settings?

While the search results don't directly address atpE's role in host immune responses, insights can be drawn from related Wolbachia surface protein studies. The Wolbachia surface protein (Wsp) has been demonstrated to play an antigenic role in stimulating immune responses in vertebrate animals infected by filarial worms carrying Wolbachia .

By analogy, researchers investigating atpE should consider:

• Although primarily a membrane-embedded protein, some portions of atpE may be exposed to the host cell cytoplasm

• These exposed epitopes could potentially be recognized by host pattern recognition receptors

• Experimental designs should include immunoprecipitation studies to identify potential host proteins that interact with atpE

• Cell-based assays using recombinant atpE to measure NF-κB activation or cytokine production could reveal immunomodulatory properties

The interaction between Wolbachia proteins and host immunity remains an important research frontier, particularly given Wolbachia's potential applications in controlling vector-borne diseases.

What controls should be included when studying recombinant Wolbachia atpE function?

When designing experiments to study recombinant Wolbachia atpE function, several controls are essential:

• Negative controls:

  • Empty vector-transformed E. coli lysates processed identically to atpE-expressing samples

  • Purified irrelevant protein of similar size and with same tag as atpE

  • Heat-denatured atpE protein to confirm activity-dependent effects

• Positive controls:

  • Well-characterized ATP synthase c subunit from model organisms (E. coli, B. subtilis)

  • Known inhibitors of ATP synthase (oligomycin, DCCD) should block activity

• Validation controls:

  • Site-directed mutants affecting key functional residues

  • Complementation assays in ATP synthase-deficient bacterial strains

These controls help distinguish protein-specific effects from artifacts and provide benchmarks for functional activity. For membrane proteins like atpE, detergent effects must be carefully controlled by including detergent-only conditions in all experiments.

How can researchers verify the correct folding and function of recombinant Wolbachia atpE?

Verifying correct folding and function of recombinant atpE requires multiple complementary approaches:

• Structural assessment:

  • Circular dichroism (CD) spectroscopy to confirm predicted secondary structure (predominantly α-helical)

  • Size exclusion chromatography to verify monodispersity

  • Limited proteolysis to test for compact, folded structure

• Functional assays:

  • Reconstitution into liposomes and measurement of proton translocation

  • Assembly with other ATP synthase components to form functional F0 complex

  • Patch clamp studies to verify ion channel activity in artificial membranes

• Interaction studies:

  • Binding assays with known ATP synthase partners (subunits a and b)

  • Cross-linking experiments to capture native-like oligomeric states

Proper folding is particularly critical for membrane proteins like atpE, as misfolding can lead to aggregation and loss of functionality. The highest standard of validation would be complementation of an atpE-deficient bacterial strain, demonstrating that the recombinant protein can functionally replace the native protein.

What are the major technical challenges when working with recombinant Wolbachia membrane proteins?

Working with recombinant Wolbachia membrane proteins like atpE presents several technical challenges:

• Expression issues:

  • Toxicity to expression host due to membrane integration

  • Inclusion body formation requiring refolding protocols

  • Low yield compared to soluble proteins

• Purification difficulties:

  • Selecting appropriate detergents that maintain native structure

  • Detergent micelles can interfere with biophysical characterization

  • Protein aggregation during concentration steps

• Functional reconstitution:

  • Transfer from detergent to lipid environment

  • Achieving correct orientation in liposomes

  • Verifying native-like behavior in artificial systems

Research in related bacterial systems suggests that using specialized E. coli strains (like C41/C43) designed for membrane protein expression and careful optimization of induction conditions (lower temperature, reduced IPTG concentration) can improve yields of functional atpE . Additionally, fusion partners like MBP can enhance solubility, though they must be removed for certain functional studies.

How does Wolbachia atpE activity relate to mitochondrial function in host cells?

ATP synthase in Wolbachia and mitochondria share evolutionary origins, both deriving from bacterial ancestors. The relationship between Wolbachia atpE and host mitochondrial function reveals interesting parallels:

• Both systems use proton gradients across membranes to synthesize ATP

• The proteins have similar structures but distinct sequences, reflecting their divergent evolution

• They may compete for resources within the host cell, particularly in energy-limited environments

Research has shown that Wolbachia infection influences host mitochondrial function. For example, genome-wide RNAi screening identified that knockdown of host genes influencing mitochondrial function dramatically affected Wolbachia titer . This suggests a metabolic interplay between bacterial and host energy production systems. Researchers studying atpE should consider designing experiments that examine coordination or competition between these parallel ATP-generating systems.

What experimental approaches can elucidate the role of atpE in Wolbachia's reliance on host proteolysis?

Research has revealed that Wolbachia depends heavily on host proteolysis via ubiquitination and ERAD pathways, potentially as a mechanism for provisioning amino acids due to Wolbachia's lack of essential amino acid biosynthetic pathways . To investigate atpE's role in this process:

• Inhibitor studies:

  • Test whether proteolysis inhibitors affect ATP synthase activity in Wolbachia-infected cells

  • Measure ATP levels in infected cells with and without proteolysis inhibitors

• Protein interaction studies:

  • Use co-immunoprecipitation with tagged atpE to identify interactions with host proteolysis machinery

  • Perform proximity labeling (BioID or APEX) with atpE to map its proximity to host proteins

• Metabolic labeling:

  • Use isotope-labeled amino acids to track incorporation into newly synthesized atpE

  • Compare amino acid incorporation rates under conditions of normal and inhibited host proteolysis

• Cellular localization:

  • Perform immunofluorescence microscopy to determine if atpE-containing ATP synthase complexes localize near host protein degradation centers

  • Use electron microscopy to examine the spatial relationship between Wolbachia membranes containing atpE and host ER/proteasomes

These approaches can help determine whether ATP generation via atpE is a critical link in Wolbachia's exploitation of host proteolysis pathways.

How might genetic variation in Wolbachia strains affect atpE structure and function?

Genetic variation in Wolbachia has significant implications for atpE structure and function, particularly given Wolbachia's extensively recombinogenic genome . Evidence from Wolbachia surface protein (wsp) studies reveals a complex pattern of recombination both within and between supergroups , suggesting similar processes may affect atpE.

The impact of genetic variation on atpE can be studied through:

• Comparative sequence analysis:

  • Alignment of atpE sequences from different Wolbachia strains (wMel, wRi, wHa, etc.)

  • Identification of conserved regions likely essential for function versus variable regions

• Strain-specific functional differences:

  • ATP synthesis rates in different Wolbachia strains

  • Differential sensitivity to inhibitors across strains

• Host-specific adaptations:

  • Comparison of atpE from Wolbachia strains in different host species

  • Correlation of sequence variations with host metabolic characteristics

Research on Wolbachia-associated fitness benefits in Drosophila simulans has demonstrated that the same Wolbachia strain (wHa) can have dramatically different effects depending on host genetic background . This host-by-symbiont interaction suggests that proteins like atpE may have co-evolved with host cellular machinery, potentially resulting in strain-specific functional adaptations.

What role might atpE play in Wolbachia-mediated virus resistance in hosts?

Wolbachia infection in insects confers resistance to RNA viruses, though the mechanism remains incompletely understood. Recent research suggests that Wolbachia's reliance on the ERAD pathway and disruption of ER morphology may contribute to this antiviral effect . As a key component of Wolbachia's energy generation system, atpE may influence this process through:

• Energy provision for Wolbachia-mediated modification of cellular environments

• Direct or indirect interactions with host ER membranes

• Contribution to metabolic competition that may disadvantage viral replication

Future experimental approaches to investigate this connection could include:

• RNAi knockdown of atpE in Wolbachia followed by viral challenge of host cells

• Structural studies of atpE-containing membrane complexes in proximity to ER membranes

• Metabolic profiling of Wolbachia-infected cells with modified atpE expression during viral infection

These studies would help determine whether atpE function is directly linked to Wolbachia's antiviral effects or represents an independent aspect of Wolbachia biology.

How can CRISPR-Cas9 technology be adapted to study atpE function in Wolbachia?

While genetic manipulation of obligate intracellular bacteria like Wolbachia presents significant challenges, CRISPR-Cas9 technology offers promising approaches:

• Delivery systems for intracellular bacteria:

  • Packaging Cas9 and guide RNAs in cell-penetrating peptides

  • Using bacteriophage-based delivery systems adapted for Wolbachia

  • Developing specialized transfection protocols for infected insect cells

• Experimental design:

  • Creating knockdown rather than knockout strains due to atpE's likely essential nature

  • Using CRISPRi (CRISPR interference) to reduce expression without complete elimination

  • Employing inducible systems to control timing of atpE disruption

• Validation approaches:

  • qPCR to confirm reduced transcript levels

  • Western blotting to verify protein reduction

  • ATP production assays to measure functional consequences

• Control considerations:

  • Off-target effects must be carefully assessed

  • Parallel targeting of non-essential genes as technical controls

  • Complementation with recombinant atpE to confirm specificity

The recent success in creating genetic tools for related intracellular bacteria suggests this approach is becoming increasingly feasible for Wolbachia research.

What high-throughput screening approaches could identify inhibitors of Wolbachia atpE?

Developing specific inhibitors of Wolbachia atpE could advance both basic research and potential applications in controlling Wolbachia-dependent parasites. Recommended high-throughput screening approaches include:

The most promising candidates would show:

• Specific binding to Wolbachia atpE over host ATP synthase components

• Activity in cellular infection models

• Minimal toxicity to host cells

• Efficacy across multiple Wolbachia strains

This approach could yield valuable research tools for dissecting atpE function and potentially lead to therapeutics for filarial diseases where Wolbachia endosymbionts are essential for parasite survival.