Recombinant Haemophilus ducreyi ATP synthase subunit c (atpE)

What is the structural organization of Haemophilus ducreyi ATP synthase subunit c (atpE)?

Haemophilus ducreyi ATP synthase subunit c is a small, hydrophobic membrane protein component of the F0 portion of ATP synthase. It forms an oligomeric ring structure embedded in the bacterial membrane that serves as the proton channel for the ATP synthase complex. The protein typically contains two transmembrane α-helices connected by a polar loop, with a conserved carboxylic acid residue (usually aspartic or glutamic acid) that is essential for proton translocation. While specific structural data for H. ducreyi atpE is limited, it likely shares structural homology with other bacterial c-subunits, containing approximately 70-80 amino acids organized into a hairpin-like structure that assembles into a ring of 10-15 subunits .

How is the atpE gene organized in the Haemophilus ducreyi genome?

The atpE gene in H. ducreyi is part of the atp operon, which encodes the various subunits of the ATP synthase complex. This operon organization is consistent with other bacteria where ATP synthase genes are typically arranged in a single operon. The H. ducreyi genome (GenBank accession no. AE017143) has been sequenced, allowing for the identification and characterization of the atpE gene . In the context of the complete genome, the atpE gene is located within the energy production and conversion functional category. Given that H. ducreyi is a fastidious organism that can only be grown in media containing glucose, the ATP synthase complex plays a crucial role in its energy metabolism.

What expression systems are most effective for producing recombinant H. ducreyi atpE?

For recombinant expression of H. ducreyi atpE, E. coli-based expression systems are most commonly employed, with several considerations to optimize yield and functionality:

When expressing this hydrophobic membrane protein, it's crucial to optimize detergent conditions for solubilization after expression. For functional studies, reconstitution into liposomes or nanodiscs is often necessary to maintain the native structure and function of the protein. The addition of a histidine tag to the N- or C-terminus facilitates purification but may affect function, requiring verification through complementation studies.

What are the optimal methods for purifying recombinant H. ducreyi atpE?

The purification of recombinant H. ducreyi atpE presents specific challenges due to its hydrophobic nature and membrane localization. The following methodological approach has proven effective:

• Membrane Fraction Isolation:

  • Harvest bacterial cells and lyse using French press or sonication

  • Separate membrane fraction through ultracentrifugation (100,000 × g for 1 hour)

  • Wash membranes to remove peripheral proteins

• Solubilization:

  • Test multiple detergents for optimal solubilization:

    • n-Dodecyl β-D-maltoside (DDM) at 1-2% often provides good results

    • Digitonin (1%) or LMNG (0.5-1%) may preserve native interactions better

  • Incubate at 4°C with gentle rotation for 1-2 hours

• Affinity Chromatography:

  • If His-tagged, use Ni-NTA resin with detergent-containing buffers

  • Include 5-10 mM imidazole in binding buffer to reduce non-specific binding

  • Elute with 250-300 mM imidazole gradient

• Size Exclusion Chromatography:

  • Critical for separating monomeric from oligomeric species

  • Use Superdex 200 or similar matrix with appropriate detergent-containing buffer

• Quality Assessment:

  • SDS-PAGE followed by silver staining (due to poor Coomassie staining of hydrophobic proteins)

  • Western blotting with anti-His antibodies or specific anti-atpE antibodies

  • Mass spectrometry for identity confirmation

This purification process typically yields 0.5-1 mg of purified protein per liter of bacterial culture, with >90% purity as assessed by SDS-PAGE and mass spectrometry.

How can researchers assess the functionality of recombinant H. ducreyi atpE?

To assess the functionality of recombinant H. ducreyi atpE, researchers should employ multiple complementary approaches:

• Proton Translocation Assays:

  • Reconstitute purified atpE into liposomes containing pH-sensitive fluorescent dyes (e.g., ACMA or pyranine)

  • Monitor fluorescence changes upon addition of valinomycin and K+ to generate a membrane potential

  • Compare proton translocation rates with positive controls

• ATP Synthesis Reconstitution:

  • Co-reconstitute atpE with other purified ATP synthase subunits

  • Measure ATP synthesis rates using luciferase-based assays

  • Assess the effect of proton gradient uncouplers to confirm ATP synthesis is coupled to proton movement

• Complementation Studies:

  • Express H. ducreyi atpE in E. coli atpE deletion strains

  • Assess restoration of growth on non-fermentable carbon sources

  • Compare growth rates and ATP production with wild-type strains

• Binding Studies with ATP Synthase Inhibitors:

  • Use isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR)

  • Measure binding affinities with known ATP synthase inhibitors (e.g., oligomycin, DCCD)

  • Compare binding parameters with characterized ATP synthase c-subunits

These methodological approaches provide comprehensive functional assessment and should be conducted at different pH values (pH 5.5-8.0) and temperature ranges relevant to H. ducreyi's environmental conditions during infection (33-37°C).

How does H. ducreyi atpE contribute to bacterial survival during infection?

H. ducreyi faces significant challenges during human infection, including nutrient limitation, pH changes, and host immune responses. The ATP synthase c-subunit plays a crucial role in energy generation and adaptation:

• Adaptation to Nutrient Stress:
  During human infection, H. ducreyi exhibits a transcriptional profile distinct from in vitro growth conditions, with upregulation of alternative carbon utilization pathways . While specific atpE expression data during infection is limited, ATP synthase likely plays a critical role in energy conservation when preferred carbon sources are depleted. H. ducreyi upregulates pathways for L-ascorbate utilization and metabolism during infection , requiring efficient ATP synthesis to support these alternative pathways.

• Anaerobic Adaptation:
  H. ducreyi upregulates genes associated with anaerobiosis during human infection . ATP synthase functionality must adapt to these low-oxygen conditions, potentially involving modifications in c-subunit structure or interactions to maintain proton gradient utilization under anaerobic conditions.

• pH Homeostasis:
  In the human abscess environment, H. ducreyi encounters pH fluctuations. The c-subunit's proton translocation function is directly implicated in maintaining internal pH homeostasis. The conserved carboxylic acid residue in atpE undergoes protonation/deprotonation cycles that are essential for proton transport and may be critical for survival in the varying pH of human lesions.

• Connection to Virulence Regulation:
  H. ducreyi's CpxRA two-component system regulates multiple virulence determinants . While direct regulation of atpE by CpxRA has not been established, the energy production by ATP synthase may indirectly impact virulence factor expression through available ATP pools. The CpxRA system primarily functions as a phosphatase in vivo to maintain virulence determinant expression , a process requiring energy from ATP synthase.

What structural features distinguish H. ducreyi atpE from other bacterial ATP synthase c-subunits?

While specific structural data for H. ducreyi atpE remains limited, comparative analysis with related bacterial species reveals several distinctive features:

The distinctive structural features of H. ducreyi atpE may contribute to its adaptation to the human host environment. The protein likely maintains the core functional elements necessary for proton translocation while having evolved specific adaptations for H. ducreyi's unique metabolic requirements. These adaptations may be particularly important given the bacterium's need to survive in the hostile environment of human lesions, where it must adapt to nutrient stress and anaerobiosis .

How does the expression of atpE change under different environmental conditions faced by H. ducreyi?

H. ducreyi encounters various environmental stresses during infection, and its ATP synthase subunit c expression likely responds to these challenges:

• Nutrient Availability Effects:
  H. ducreyi is a fastidious organism that can only be grown in media containing glucose . RNA-Seq analysis shows that during human infection, H. ducreyi upregulates genes involved in alternative carbon utilization pathways . Though not specifically identified in the available studies, atpE expression would logically be coordinated with these metabolic shifts to maintain energy production under nutrient limitation.

• Growth Phase-Dependent Regulation:
  RNA-Seq comparison of mid-log, transition, and stationary phases shows distinct transcriptional profiles . ATP synthase genes often show growth phase-dependent regulation in bacteria, with expression typically decreasing in stationary phase to conserve resources. The in vivo transcriptome of H. ducreyi is distinct from all in vitro growth phases , suggesting unique regulatory patterns in the host environment.

• Stress Response Integration:
  H. ducreyi upregulates genes involved in heat shock response and growth arrest during human infection . ATP synthase expression is likely integrated with these stress responses, potentially showing regulatory patterns that balance energy production needs against resource conservation.

• Oxygen Availability Response:
  Human infection induces genes associated with anaerobiosis in H. ducreyi . ATP synthase activity and expression often adapt to oxygen limitation in bacteria, with potential modifications in the c-subunit's structure or function to optimize performance under low-oxygen conditions.

• Regulatory Systems Influence:
  The CpxRA two-component system in H. ducreyi regulates numerous genes, primarily repressing nearly 70% of its targets . While atpE wasn't specifically identified among CpxRA-regulated genes in the available studies, energy production genes are often subject to regulation by stress response systems like CpxRA.

What are the major challenges in expressing and purifying functional recombinant H. ducreyi atpE?

Researchers face several significant challenges when working with recombinant H. ducreyi atpE:

• Membrane Protein Expression Issues:
  As a hydrophobic membrane protein, atpE often causes toxicity to expression hosts and forms inclusion bodies.

  Solution: Use specialized E. coli strains like C41/C43(DE3) designed for membrane protein expression. Optimize expression by testing different induction temperatures (16-30°C), inducer concentrations, and expression durations.

• Oligomeric Assembly Challenges:
  The c-subunit functions as an oligomeric ring, and maintaining proper oligomerization during purification is difficult.

  Solution: Employ mild detergents like digitonin or LMNG that better preserve oligomeric states. Use blue native PAGE to verify the oligomeric state throughout purification.

• Functional Assessment Complexity:
  Verifying the functionality of purified atpE is challenging due to its dependence on other ATP synthase subunits.

  Solution: Develop reconstitution systems with minimal components required for proton translocation, using purified a-subunit and lipid nanodiscs to assess basic functionality.

• Protein-Detergent Complex Stability:
  Maintaining stable protein-detergent complexes during purification and storage is difficult.

  Solution: Screen multiple detergents using thermal stability assays (TSA/nanoDSF) to identify optimal stabilizing conditions. Consider addition of specific lipids (cardiolipin, PE) that may stabilize the native structure.

• Limited Structural Information:
  The lack of specific structural data for H. ducreyi atpE complicates rational experimental design.

  Solution: Use homology modeling based on structurally characterized bacterial c-subunits combined with targeted mutagenesis to identify critical residues and regions.

How can researchers study the interaction between H. ducreyi atpE and other ATP synthase subunits?

To investigate the interactions between H. ducreyi atpE and other ATP synthase subunits, researchers should consider these methodological approaches:

• Co-Immunoprecipitation Assays:

  • Express tagged versions of atpE and other subunits in appropriate expression systems

  • Perform pull-down experiments with anti-tag antibodies

  • Identify interacting partners using Western blotting or mass spectrometry

  • Control experiments should include non-specific binding controls and detergent optimization

• Chemical Cross-Linking Coupled with Mass Spectrometry:

  • Treat purified ATP synthase complex with membrane-permeable cross-linkers

  • Digest cross-linked complexes and analyze by LC-MS/MS

  • Identify cross-linked peptides using specialized software (e.g., pLink, MeroX)

  • This approach provides spatial constraints for interacting residues

• Förster Resonance Energy Transfer (FRET):

  • Generate fusion proteins with appropriate FRET pairs (e.g., CFP/YFP)

  • Measure energy transfer efficiency in reconstituted systems

  • Calculate distances between tagged positions

  • Particularly useful for dynamic interaction studies

• Bacterial Two-Hybrid Systems:

  • Adapt membrane-specific two-hybrid systems (BACTH) for atpE interaction studies

  • Screen for interactions with other ATP synthase subunits

  • Quantify interaction strength through reporter gene expression

  • Validate interactions through mutational analysis

• Cryo-Electron Microscopy:

  • Purify intact ATP synthase complex from H. ducreyi or reconstituted systems

  • Determine structure using single-particle cryo-EM approaches

  • Focus on interactions between c-ring and adjacent subunits

  • Complement with molecular dynamics simulations to understand dynamic aspects

These methodological approaches can be used complementarily to build a comprehensive understanding of how atpE interacts within the ATP synthase complex, providing insights into H. ducreyi-specific adaptations.

How might structural and functional studies of H. ducreyi atpE contribute to antimicrobial development?

ATP synthase has emerged as a promising target for new antimicrobials, with the c-subunit offering several advantages as a drug target:

• Unique Structural Features:
  The c-subunit of ATP synthase contains essential, highly conserved functional elements while displaying species-specific variations that can be exploited for selective targeting. H. ducreyi atpE likely contains unique structural features adapted to its specialized lifestyle as a human pathogen, potentially providing selective targeting opportunities.

• Precedent for c-Subunit Targeting:
  Several natural compounds, including diarylquinolines (like bedaquiline used against M. tuberculosis) and certain oligomycin derivatives, specifically target the c-subunit of ATP synthase. Structure-function studies of H. ducreyi atpE could identify specific binding sites for similar compounds or novel molecular scaffolds.

• Essential for Survival:
  ATP synthase is generally essential for bacterial survival, particularly under non-fermentative conditions. H. ducreyi has limited metabolic flexibility, growing only in media containing glucose , suggesting strong dependence on efficient energy conservation through ATP synthase.

• Role in Adaptation to Host Environment:
  During human infection, H. ducreyi upregulates genes involved in alternative carbon utilization and adapts to anaerobic conditions . ATP synthase likely plays a crucial role in these adaptations, making it a potential vulnerability when the bacterium is stressed by host conditions.

• Rational Design Opportunities:
  Detailed structural information about H. ducreyi atpE would enable structure-based drug design approaches, including:

  • In silico screening for compounds that bind to critical regions

  • Fragment-based approaches targeting the proton-binding site or subunit interfaces

  • Design of peptidomimetics that disrupt essential protein-protein interactions

These approaches could lead to novel antimicrobials specifically targeting H. ducreyi or potentially broader-spectrum agents against related pathogens.

What research gaps remain in our understanding of H. ducreyi atpE and how might they be addressed?

Despite advances in understanding H. ducreyi biology, significant knowledge gaps remain regarding its ATP synthase c-subunit:

• Structural Characterization:
  The three-dimensional structure of H. ducreyi atpE remains undetermined, limiting our understanding of its specific adaptations.

  Research Direction: Apply cryo-electron microscopy to purified ATP synthase complexes or reconstituted c-rings to determine the structure. Complementary approaches could include NMR studies of specific domains and computational modeling validated by mutagenesis.

• Environmental Regulation:
  How expression and activity of H. ducreyi ATP synthase respond to the changing host environment during infection remains poorly understood.

  Research Direction: Perform transcriptomic and proteomic analyses on H. ducreyi isolated directly from human infection models under well-characterized conditions . Time-course studies would be particularly valuable to understand adaptation dynamics.

• Role in Virulence:
  The connection between energy metabolism through ATP synthase and virulence factor expression in H. ducreyi is incompletely characterized.

  Research Direction: Generate conditional atpE mutants to study the effects of reduced ATP synthase activity on virulence factor expression and function. This could include studies in the human infection model to directly assess the importance of atpE in virulence.

• Post-Translational Modifications:
  Potential post-translational modifications of atpE that might regulate its function in response to stress remain unexplored.

  Research Direction: Apply advanced mass spectrometry techniques to identify modifications on atpE purified from H. ducreyi grown under different stress conditions relevant to human infection.

• Interaction with Host Factors:
  Potential interactions between ATP synthase components and host factors during infection remain unexplored.

  Research Direction: Apply proximity labeling approaches (BioID, APEX) in infection models to identify potential host proteins that interact with bacterial ATP synthase components.

Addressing these knowledge gaps would significantly advance our understanding of H. ducreyi energy metabolism and its role in pathogenesis, potentially revealing new therapeutic approaches for chancroid and related diseases.