Recombinant Vibrio cholerae serotype O1 ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Vibrio cholerae serotype O1?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of the F-type ATP synthase in Vibrio cholerae. This small, hydrophobic protein forms the c-ring in the membrane-embedded portion of the ATP synthase complex. The protein consists of 85 amino acids with a molecular weight of approximately 8-9 kDa and functions primarily in proton translocation across the bacterial membrane during ATP synthesis. In Vibrio cholerae serotype O1, atpE contributes to energy production through oxidative phosphorylation, which is essential for bacterial survival and virulence .

How does atpE function within the ATP synthase complex?

The atpE subunit plays a crucial role in the proton-motive force conversion to chemical energy in the form of ATP. The c-ring formed by multiple atpE subunits rotates within the membrane as protons pass through a channel formed between subunit a and the c-ring. Each c-subunit contains a conserved carboxyl group (typically from an aspartic or glutamic acid residue) that can be protonated and deprotonated during rotation. This proton translocation drives the rotation of the central stalk (γ subunit) within the F1 sector, causing conformational changes in the catalytic sites that lead to ATP synthesis.

The functional mechanism involves:

• Proton binding to the c-subunit at the subunit a interface

• Rotation of the c-ring due to proton movement

• Release of the proton on the opposite side of the membrane

• Coupling of the c-ring rotation to the γ subunit rotation

• Catalysis of ATP synthesis in the F1 sector

This process is essential for bacterial energy metabolism, particularly under aerobic conditions where the proton gradient is established by the electron transport chain .

How is recombinant Vibrio cholerae atpE typically produced for research?

Recombinant Vibrio cholerae serotype O1 ATP synthase subunit c (atpE) is typically produced using heterologous expression systems, most commonly in Escherichia coli. The process involves:

• Gene cloning: The atpE gene (85 amino acids) is PCR-amplified from Vibrio cholerae genomic DNA

• Vector construction: The gene is inserted into an expression vector containing an affinity tag (commonly His-tag at the N-terminus)

• Transformation: The recombinant plasmid is transformed into an E. coli expression strain

• Protein expression: Culture conditions are optimized for protein production

• Purification: The protein is isolated using affinity chromatography and additional purification steps

The recombinant protein is typically produced with a His-tag fusion to facilitate purification and is available in lyophilized powder form. After purification, the protein is stabilized with buffer components including Tris/PBS and trehalose (6%) at pH 8.0. For storage, the protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added for long-term storage at -20°C/-80°C .

What is the relationship between atpE and Vibrio cholerae pathogenesis?

While atpE is primarily known for its role in energy metabolism rather than direct virulence, recent research suggests connections between ATP synthase function and bacterial pathogenesis. The relationship includes:

• Energy requirements for virulence: Vibrio cholerae requires efficient energy production during infection to express virulence factors and adapt to the host environment. The temporal expression patterns of virulence genes like tcpA and ctxA during infection are energy-dependent processes that may indirectly rely on ATP synthase function .

• pH adaptation: The ability of V. cholerae to survive the acidic environment of the stomach before reaching the small intestine depends partly on maintaining proton homeostasis, a process in which ATP synthase plays a role.

• Metabolic shifts during infection: During intestinal colonization, V. cholerae undergoes metabolic transitions that alter its energy requirements, potentially affecting ATP synthase expression and activity.

• Response to host environmental cues: ATP synthase gene expression may be regulated in response to host signals, similar to how other V. cholerae genes are induced during infection as demonstrated by recombinase gene fusion studies .

While direct evidence linking atpE specifically to virulence is limited in the provided search results, its fundamental role in bacterial bioenergetics suggests it could be an indirect contributor to pathogenesis or a potential drug target.

How does atpE expression change during different stages of Vibrio cholerae infection?

Although the search results don't provide direct data on atpE expression patterns during infection, we can infer potential expression patterns based on studies of other V. cholerae genes:

Research using recombinase gene fusions has identified numerous V. cholerae genes that are specifically induced during infection in the infant mouse model of cholera. Using methodologies similar to those described for other genes, researchers have found that:

• Temporal expression patterns: Similar to virulence genes like tcpA, which shows biphasic expression in the small intestine, metabolic genes including those in ATP synthesis pathways likely have specific temporal expression patterns during infection .

• Host environmental triggers: The intestinal environment provides specific signals that trigger expression of certain genes. These signals may include changes in pH, oxygen concentration, nutrient availability, and host-derived compounds.

• Regulation networks: Studies have shown that virulence regulators like ToxR, TcpP, and ToxT have different requirements for gene expression during infection versus during in vitro growth. These regulatory networks may similarly affect metabolic genes including atpE .

A methodological approach to study atpE expression during infection would involve:

What potential applications exist for targeting atpE in antimicrobial development?

ATP synthase represents an attractive target for antimicrobial development due to its essential role in bacterial metabolism. Specific strategies for targeting Vibrio cholerae atpE include:

• Small molecule inhibitors: Developing compounds that specifically bind to the c-subunit of ATP synthase could disrupt proton translocation and energy production. The unique structure of bacterial ATP synthase c-subunits compared to their eukaryotic counterparts offers potential for selective targeting.

• Peptide-based inhibitors: Based on the understanding of the protein-protein interactions within the ATP synthase complex, peptides could be designed to disrupt the assembly or function of the c-ring.

• Epitope-targeted antibodies: Similar to approaches used with cholera toxin, where modified toxins have been created for vaccine development , engineered atpE proteins could potentially serve as antigens for immunological targeting.

• Combination approaches: ATP synthase inhibitors could potentially enhance the efficacy of existing antibiotics by compromising bacterial energy production, similar to how TolC protein has been identified as important for phage binding and infection .

Research challenges in this area include:

What are the optimal conditions for expressing and purifying recombinant Vibrio cholerae atpE?

Based on established protocols for hydrophobic membrane proteins and the specific information provided about recombinant Vibrio cholerae atpE, the following optimized expression and purification conditions are recommended:

Expression System Optimization:

Purification Protocol:

• Cell lysis using French press or sonication in buffer containing:

  • 50 mM Tris-HCl, pH 8.0

  • 300 mM NaCl

  • 5% glycerol

  • 1 mM PMSF

  • Appropriate detergent (e.g., 1% DDM or LDAO)

• Membrane fraction isolation by ultracentrifugation (100,000 × g, 1 hour)

• Solubilization of membrane proteins in:

  • 50 mM Tris-HCl, pH 8.0

  • 300 mM NaCl

  • 1% DDM or 2% LDAO

  • 5% glycerol

• Affinity purification using Ni-NTA resin with:

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.1% detergent, 20 mM imidazole

  • Wash buffer: Same with 40 mM imidazole

  • Elution buffer: Same with 250-300 mM imidazole

• Size exclusion chromatography in buffer containing:

  • 20 mM Tris-HCl, pH 8.0

  • 150 mM NaCl

  • 0.05% detergent

  • 5% glycerol

• Stabilization with 6% trehalose before lyophilization

The purified protein should achieve >90% purity as determined by SDS-PAGE and can be stored as recommended in the product specifications .

How can recombinant atpE be used in structural studies?

Recombinant Vibrio cholerae atpE can be employed in various structural biology techniques to elucidate its three-dimensional structure and functional mechanisms:

X-ray Crystallography:

• Protein preparation: The purified atpE should be concentrated to 5-15 mg/mL in a detergent-stabilized form.

• Crystallization screening: Use membrane protein-specific sparse matrix screens with different detergents and lipids as additives.

• Crystal optimization: Refine conditions based on initial hits, focusing on detergent type, pH, salt concentration, and precipitants.

• Data collection: Collect diffraction data at synchrotron radiation sources.

• Structure determination: Phase determination may require heavy atom derivatives or molecular replacement using homologous structures.

Cryo-Electron Microscopy:

• Sample preparation: Reconstitute atpE into nanodiscs or liposomes to mimic the native membrane environment.

• Grid preparation: Apply sample to glow-discharged grids and vitrify by rapid freezing.

• Data collection: Collect images on a high-resolution cryo-EM instrument.

• Image processing: Perform particle picking, 2D classification, 3D reconstruction, and refinement.

NMR Spectroscopy:
For this small membrane protein (85 amino acids), solution NMR can be particularly valuable:

• Isotopic labeling: Express atpE in minimal media with 15N and 13C sources.

• Sample preparation: Solubilize in detergent micelles or bicelles.

• Spectral acquisition: Collect 2D and 3D spectra for backbone and side-chain assignments.

• Structure calculation: Generate structural models based on distance constraints.

Methodological Considerations:

What experimental approaches can be used to study atpE function in Vibrio cholerae pathogenesis?

To investigate the potential role of atpE in Vibrio cholerae pathogenesis, researchers can employ several complementary approaches:

Genetic Manipulation Strategies:

• Conditional knockdown systems:
  Since complete deletion of atpE would likely be lethal, inducible antisense RNA or CRISPR interference (CRISPRi) approaches can be used to reduce expression at specific stages of infection.

• Site-directed mutagenesis:
  Introducing point mutations that affect proton binding or c-ring stability without completely abolishing function allows for the study of partial loss-of-function phenotypes.

• Recombinase-based in vivo expression analysis:
  Similar to the approach described for other V. cholerae genes , construct an atpE-tnpR fusion to monitor gene expression during infection using a resolvase-based reporter system.

Functional Analysis Methods:

• ATP synthesis assays in membrane vesicles:
  Isolate membrane vesicles from V. cholerae grown under different conditions and measure ATP synthesis rates.

• Proton translocation measurements:
  Use pH-sensitive fluorescent probes to monitor proton movement across membranes in wild-type and atpE-modified strains.

• Metabolic flux analysis:
  Employ isotope-labeled substrates to track changes in metabolic pathways when atpE function is altered.

In vivo Infection Studies:

• Competitive index assays:
  Compare colonization efficiency of wild-type and atpE-modified strains in animal models, similar to approaches used for other V. cholerae genes .

• Temporal gene expression analysis:
  Extract bacteria from different intestinal regions at various time points post-infection to analyze atpE expression patterns, similar to studies of virulence gene expression .

• Host response monitoring:
  Evaluate how alterations in atpE affect host immune responses and disease progression.

How can recombinant atpE be used in vaccine development research?

Recombinant Vibrio cholerae atpE could potentially contribute to vaccine development through several research approaches:

• Antigen identification and characterization:
  Similar to studies with cholera toxin subunits , recombinant atpE can be evaluated for immunogenicity and protective capacity. While ATP synthase components are not traditional vaccine targets due to their conservation and membrane localization, their essential nature makes them interesting candidates for novel approaches.

• Adjuvant development:
  Bacterial ATP synthase components have been explored as potential adjuvants in vaccine formulations due to their ability to interact with host immune components.

• Carrier protein applications:
  Modified atpE could potentially serve as a carrier protein for other Vibrio cholerae antigens, particularly if engineered to expose specific epitopes while maintaining structural integrity.

• Screening platform:
  Immobilized recombinant atpE can be used to screen antibody libraries or evaluate immune responses from infected or vaccinated individuals.

Research methodologies could include:

What experimental systems can be used to study atpE inhibitors for potential therapeutic development?

The development of inhibitors targeting Vibrio cholerae atpE would require multiple experimental systems to progress from initial screening to validation:

In vitro Screening Systems:

• ATP synthesis assays:
  Using inverted membrane vesicles containing ATP synthase complexes to measure ATP production in the presence of potential inhibitors.

• Proton flux measurements:
  Monitoring proton translocation across membranes using pH-sensitive fluorescent dyes to identify compounds that interfere with c-ring function.

• Binding assays:
  Developing radioligand or fluorescence-based binding assays to identify compounds that directly interact with recombinant atpE.

Structural Biology Approaches:

• Co-crystallization:
  Obtaining crystal structures of atpE with bound inhibitors to guide structure-based drug design.

• NMR-based fragment screening:
  Using NMR spectroscopy to identify small molecule fragments that bind to specific sites on atpE.

• In silico docking:
  Computational screening of compound libraries against the atpE structure to identify potential binding molecules.

Cellular and In vivo Validation:

• Bacterial growth inhibition:
  Standard minimum inhibitory concentration (MIC) determination against various V. cholerae strains.

• Cell-based ATP quantification:
  Measuring intracellular ATP levels in bacterial cultures exposed to inhibitors.

• Animal infection models:
  Testing promising compounds in the infant mouse model of cholera infection or other relevant animal models.

How can recombinant atpE be used to study bacterial adaptation to environmental stresses?

ATP synthase function is critical for bacterial adaptation to various environmental stresses, and recombinant atpE can be used to investigate these processes:

• pH adaptation studies:
  Vibrio cholerae must survive transit through the acidic stomach before colonizing the small intestine. The ATP synthase complex plays a role in maintaining pH homeostasis, and recombinant atpE can be used to study structural and functional changes under different pH conditions.

• Oxidative stress response:
  During infection, V. cholerae encounters reactive oxygen species. ATP synthase activity may be modulated during oxidative stress, and recombinant atpE can be used to investigate potential post-translational modifications or structural changes.

• Nutrient limitation adaptation:
  Under nutrient-limited conditions, energy metabolism must be precisely regulated. Recombinant atpE can be used in reconstitution experiments to study how ATP synthase activity adapts to different nutrient environments.

• Temperature response:
  V. cholerae survives in aquatic environments and the human host at different temperatures. Thermal stability studies with recombinant atpE can reveal adaptations to temperature shifts.

Experimental approaches would include: