Recombinant Campylobacter hominis ATP synthase subunit c (atpE)

What is the role of ATP synthase subunit c in Campylobacter hominis?

ATP synthase subunit c forms the c-ring in the F₀ domain of ATP synthase, which functions as a proton channel across the bacterial membrane. This subunit is crucial for energy conversion, as it utilizes the proton motive force to drive the rotational catalysis that generates ATP. In Campylobacter species, ATP synthesis is particularly important for survival under the microaerobic conditions they typically inhabit.

Studies of filamentous Campylobacter cells have demonstrated greater intracellular ATP content (2.66 to 17.4 fg) compared to spiral forms (0.99 to 1.7 fg), suggesting that ATP production capacity may be linked to stress adaptation and survival mechanisms in Campylobacter . The ATP synthase complex plays a critical role in maintaining this energy balance, particularly when the organism faces environmental stress.

How does the atpE gene conservation compare across Campylobacter species?

While the atpE gene sequence shows substantial conservation across Campylobacter species, significant variations exist that impact protein function and potentially antibiotic susceptibility. Similar to observations in the cmeRABC operon, where certain alleles associated with antimicrobial resistance have been found to cross species boundaries through recombination events, atpE gene sharing may occur between C. jejuni and C. coli .

This conservation pattern differs from other genes like cmeB, where most alleles segregate by species but those conferring high resistance to antibiotics may cross species boundaries. Researchers should consider these phylogenetic relationships when developing recombinant expression systems or designing experiments to study ATP synthase function.

What are the challenges in expressing recombinant Campylobacter hominis ATP synthase subunit c?

The expression of recombinant Campylobacter hominis ATP synthase subunit c presents several technical challenges that require careful methodological consideration:

• Hydrophobicity: As a membrane protein component, subunit c is highly hydrophobic, often leading to aggregation and inclusion body formation during expression.

• Toxicity: Overexpression may disrupt host cell membrane potential, causing toxicity to the expression host.

• Microaerobic requirements: Campylobacter-derived proteins may fold differently under standard aerobic expression conditions used in E. coli systems.

• Codon usage: Significant differences in codon preference between Campylobacter and common expression hosts can reduce expression efficiency.

To overcome these challenges, researchers should consider using specialized expression systems with inducible promoters, fusion tags to enhance solubility, and controlled growth conditions that mimic Campylobacter's preferred microaerobic environment (approximately 7% O₂, v/v) as described in protocols for Campylobacter growth .

How can site-directed mutagenesis of recombinant Campylobacter ATP synthase subunit c inform antibiotic resistance mechanisms?

Site-directed mutagenesis of the atpE gene provides critical insights into both ATP synthase function and potential antibiotic resistance mechanisms. By systematically modifying key residues in the c-subunit, researchers can:

• Identify critical amino acids involved in proton translocation

• Map the binding sites for antibiotics that target ATP synthase

• Characterize resistance mutations that emerge under selective pressure

The methodological approach should involve:

• PCR amplification of the atpE gene using high-fidelity polymerase

• Introduction of specific mutations using overlap extension PCR techniques

• Confirmation of mutations by sequencing

• Expression of wild-type and mutant proteins under identical conditions

• Functional characterization through ATP synthesis assays

This approach parallels methods used to study other Campylobacter virulence factors, such as the generation of cdtC knockout mutants, where a chloramphenicol resistance cassette was inserted into the target gene following PCR amplification and cloning into a suitable vector .

How does ATP synthase subunit c contribute to Campylobacter survival under stress conditions?

ATP synthase functionality directly impacts Campylobacter's ability to survive environmental stresses. Research methodologies to investigate this relationship should include:

• Generating atpE knockdown or knockout strains using techniques similar to those used for cdtC mutation

• Comparing ATP levels in wild-type and mutant strains under various stress conditions

• Assessing morphological changes (such as filamentation) in response to energy limitation

• Measuring survival rates in water at different temperatures (4°C and 37°C)

Filamentation in Campylobacter has been linked to enhanced survival in water at both 4°C and 37°C compared to spiral cells . Researchers should examine whether ATP synthase activity correlates with this morphological adaptation by monitoring intracellular ATP content in different morphological forms and under various stress conditions.

What structural and functional differences exist between ATP synthase subunit c from C. hominis compared to other Campylobacter species?

A comprehensive structural and functional analysis requires:

• Sequence alignment of atpE genes from multiple Campylobacter species

• Homology modeling based on known ATP synthase structures

• Recombinant expression of c-subunits from different species

• Comparative biochemical analysis of purified proteins

Key differences may exist in:

• The number of essential ion-binding sites

• Proton affinity and translocation efficiency

• Interaction interfaces with other ATP synthase subunits

• Susceptibility to inhibitors and antibiotics

Similar comparative approaches have revealed important differences in virulence factors between Campylobacter species, such as the species-specific distribution of certain genes in the cmeRABC operon that contributes to antimicrobial resistance .

What expression systems are most effective for producing functional recombinant Campylobacter hominis ATP synthase subunit c?

The choice of expression system significantly impacts the yield and functionality of recombinant ATP synthase subunit c. Based on research with other Campylobacter proteins, the following approaches are recommended:

Expression Host Options:

• E. coli C41(DE3) or C43(DE3) strains - Specifically engineered for membrane protein expression

• Cell-free expression systems - Bypass toxicity issues associated with membrane protein overexpression

• Homologous expression in Campylobacter - More complex but may provide proper folding environment

Vector Considerations:

• Use vectors with tightly controlled inducible promoters (T7, araBAD, or tac)

• Include fusion partners that enhance solubility (MBP, SUMO, or Thioredoxin)

• Incorporate a cleavable purification tag (His6, Strep-tag II)

Growth Conditions:

• Induction at lower temperatures (16-20°C) to slow expression and improve folding

• Microaerobic conditions when possible (approximately 7% O₂, v/v) created using evacuation/replacement techniques

• Supplementation with membrane-stabilizing additives

The methodology should be tailored to experimental goals, with E. coli systems preferred for structural studies requiring high yield, while homologous expression may be more appropriate for functional studies.

What purification strategies maximize recovery of correctly folded recombinant ATP synthase subunit c?

The purification of membrane proteins like ATP synthase subunit c requires specialized techniques to maintain native structure and function:

Solubilization Protocol:

• Test multiple detergents (DDM, LDAO, or Fos-choline) at various concentrations

• Optimize solubilization temperature and time (typically 4°C for 1-2 hours)

• Include stabilizing agents (glycerol, specific lipids) in buffers

Purification Steps:

• Initial capture using affinity chromatography based on fusion tag

• Size exclusion chromatography to remove aggregates and detergent micelles

• Optional ion exchange step for increased purity

Quality Control Assessments:

• SDS-PAGE and Western blot analysis to confirm purity

• Circular dichroism spectroscopy to verify secondary structure

• Functional reconstitution assays to confirm activity

When optimizing these protocols, researchers should consider the stability of Campylobacter proteins under different conditions, as studies have shown that morphological changes in Campylobacter are influenced by environmental factors like medium composition and oxygen levels .

How can researchers validate the proper assembly of ATP synthase subunit c into functional complexes?

Validating proper assembly of ATP synthase subunit c into functional complexes requires multiple complementary approaches:

Biochemical Validation:

• Blue native PAGE to visualize intact complexes

• Crosslinking studies to capture subunit interactions

• Analytical ultracentrifugation to determine complex stoichiometry

Functional Validation:

• ATP synthesis assays using reconstituted proteoliposomes

• Proton translocation measurements with pH-sensitive fluorescent dyes

• ATP hydrolysis assays (reverse reaction) as a proxy for complex assembly

Structural Validation:

• Negative-stain electron microscopy to visualize c-ring formation

• Mass spectrometry to confirm subunit composition

• Cryo-electron microscopy for high-resolution structural analysis

These validation methods should be performed under conditions that mimic the microaerobic environment where Campylobacter naturally functions , as oxygen levels can affect protein folding and complex assembly.

How can researchers differentiate between effects due to mutations in ATP synthase subunit c versus other components?

Distinguishing the specific contributions of ATP synthase subunit c mutations from effects caused by other components requires a systematic experimental design and careful data analysis:

Experimental Approach:

• Generate isogenic strains with single mutations in atpE

• Create control strains with mutations in other ATP synthase subunits

• Perform complementation studies with wild-type atpE

• Conduct in vitro reconstitution with purified components

Analysis Methods:

• Comparative phenotyping under various growth conditions

• Measurement of ATP synthesis rates normalized to enzyme concentration

• Determination of proton translocation efficiency

• Structural analysis of isolated c-rings

This differentiation is particularly important as ATP synthase function may influence virulence factor expression through energy availability, similar to how ribosome methylation can modulate the expression of multiple virulence factors in C. jejuni .

What statistical approaches are recommended for comparative studies of wild-type versus mutant ATP synthase variants?

Experimental Design Considerations:

• Minimum of 3-5 biological replicates per condition

• Technical triplicates within each biological replicate

• Appropriate controls for each experimental variable

• Randomization and blinding where applicable

Statistical Methods:

• Normality testing (Shapiro-Wilk) to determine appropriate tests

• ANOVA with post-hoc tests for multi-group comparisons

• Student's t-test or Mann-Whitney U for two-group comparisons

• Linear mixed effects models for complex experimental designs with multiple variables

Data Visualization:

• Box plots showing distribution of values

• Scatter plots with error bars showing individual data points

• Heat maps for multi-parameter analyses

When analyzing ATP synthase functionality, researchers should account for variables such as growth phase and cellular morphology, as studies have shown that filamentous Campylobacter cells exhibit different physiological properties compared to spiral forms .

How should researchers address discrepancies between in vitro and in vivo ATP synthase activity measurements?

Resolving discrepancies between in vitro and in vivo measurements requires systematic investigation of potential confounding factors:

Sources of Discrepancy:

• Different ionic conditions between buffer systems and cellular environment

• Lipid composition effects on membrane protein function

• Interaction with cellular components absent in purified systems

• Post-translational modifications present only in vivo

Resolution Approach:

• Stepwise complexity addition to in vitro systems (pure proteins → proteoliposomes → membrane vesicles)

• Comparative analysis across multiple measurement techniques

• Assessment under various environmental conditions (pH, ion concentration, temperature)

• Mathematical modeling to account for system differences

This approach parallels methods used to understand complex phenotypes in Campylobacter, such as the investigation of antibiotic resistance mechanisms, which require both phenotypic and genotypic characterization .

How can recombinant ATP synthase subunit c be used to study antibiotic resistance mechanisms in Campylobacter?

ATP synthase represents a potential antibiotic target and may be involved in resistance mechanisms through several pathways:

Research Methodologies:

• Screening for mutations in atpE in antibiotic-resistant clinical isolates

• Introducing identified mutations into laboratory strains via site-directed mutagenesis

• Assessing cross-resistance between ATP synthase inhibitors and other antibiotic classes

• Measuring ATP synthase activity in resistant versus susceptible strains

Relevant Applications:

• Identifying novel resistance mechanisms to complement known pathways involving fluoroquinolone resistance (gyrA mutations) and β-lactam resistance (β-lactamase production)

• Investigating energy-dependent efflux systems that require ATP, such as the cmeRABC-encoded efflux pump that confers resistance to fluoroquinolones and macrolides

• Exploring potential synergistic drug combinations targeting both ATP synthesis and other cellular processes

This research direction is particularly valuable given the rising concerns about antibiotic resistance in Campylobacter, with studies in South America showing widespread resistance to ciprofloxacin and other antimicrobials .

What is the potential of ATP synthase subunit c as a target for new antimicrobial agents against Campylobacter infections?

ATP synthase subunit c represents a promising antimicrobial target due to its essential role in energy metabolism:

Target Validation Approach:

• Demonstrate essentiality through conditional knockdown studies

• Confirm druggability through structure-based analysis

• Assess conservation across resistant clinical isolates

• Evaluate potential for resistance development

Drug Discovery Pipeline:

• High-throughput screening using recombinant ATP synthase activity assays

• Structure-based virtual screening for c-subunit binding compounds

• Medicinal chemistry optimization of lead compounds

• In vitro and in vivo efficacy testing against Campylobacter

Advantage Assessment:

• Comparison with current first-line treatments (fluoroquinolones and macrolides)

• Evaluation against resistant strains, including those with high-level resistance to ciprofloxacin and erythromycin

• Determination of specificity compared to human ATP synthase

This approach could address the critical need for new antibiotics effective against Campylobacter strains with resistance to current treatments, which are increasingly prevalent in both human and animal isolates .

How does ATP synthase activity contribute to Campylobacter adaptation to different environmental conditions?

ATP synthase function is central to Campylobacter's ability to adapt to various environmental stresses:

Research Directions:

• Measure ATP synthase expression and activity during exposure to:

  • Oxidative stress conditions

  • Nutrient limitation

  • Temperature shifts

  • pH changes

  • Antibiotic challenge

• Correlate ATP synthase activity with:

  • Morphological changes (filamentation)

  • Biofilm formation capacity

  • Survival in water or food matrices

  • Virulence factor expression

Studies have shown that filamentous Campylobacter cells contain significantly higher intracellular ATP (2.66 to 17.4 fg) compared to spiral forms (0.99 to 1.7 fg) and demonstrate enhanced survival in water at both 4°C and 37°C . This suggests ATP synthase activity may be critical for adaptation to extra-intestinal environments.

Furthermore, energy availability likely influences other adaptation mechanisms, including the expression of virulence factors and the formation of biofilms, similar to how ribosome methylation affects multiple aspects of C. jejuni pathogenesis .