Recombinant Campylobacter jejuni subsp. jejuni serotype O:6 ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase in Campylobacter jejuni?

ATP synthase in Campylobacter jejuni belongs to the F-type ATP synthase family, which consists of two main sectors: F1 (containing the catalytic sites) and Fo (embedded in the membrane). The ATP synthase functions as a rotary molecular motor that uses the energy stored in a transmembrane ion gradient to synthesize ATP from ADP and inorganic phosphate .

The F1 sector contains three catalytic sites primarily located in the β-subunits, with α-subunits contributing essential arginine residues. These sites cycle between empty (βE), occupied by Mg-ADP and phosphate (βDP), and containing Mg-ATP (βTP) states. The Fo sector contains the membrane-embedded c-ring that facilitates ion translocation across the membrane .

When functioning as an ATP synthase, ion flow through the Fo sector drives rotation of the c-ring and central stalk, which induces conformational changes in the F1 sector catalytic sites, facilitating ATP synthesis. Conversely, when operating in ion pump mode, ATP hydrolysis drives rotation and ion transport .

How does the proton motive force relate to ATP synthase function in C. jejuni?

The proton motive force (pmf) is crucial for ATP synthesis in C. jejuni. Research demonstrates that the membrane potential (Δψ) constitutes the major component of the pmf and is essential for both ATP synthesis and bacterial motility .

For C. jejuni, a relatively high membrane potential is generated only under specific conditions: either when formate serves as an electron donor or when oxygen functions as an electron acceptor, each in combination with an appropriate acceptor or donor respectively. This membrane potential drives proton translocation through the Fo sector of ATP synthase, enabling ATP synthesis .

It's important to note that C. jejuni can generate ATP through both oxidative phosphorylation (using the pmf) and substrate-level phosphorylation via the enzyme AckA. This metabolic flexibility allows adaptation to varying environmental conditions, particularly important given the microaerophilic nature of this pathogen .

What experimental approaches are used to express and purify recombinant ATP synthase subunit c from C. jejuni?

For recombinant expression of C. jejuni ATP synthase subunit c (atpE), researchers typically employ heterologous expression systems such as E. coli. The methodological approach involves:

• Gene cloning: The atpE gene is amplified from C. jejuni genomic DNA using PCR with specific primers that incorporate appropriate restriction sites.

• Vector construction: The amplified gene is cloned into an expression vector containing an inducible promoter (commonly T7) and affinity tags (such as His-tag) to facilitate purification.

• Transformation and expression: The construct is transformed into an E. coli expression strain (commonly BL21(DE3) or derivatives), grown to appropriate density, and protein expression is induced.

• Membrane protein solubilization: As subunit c is a hydrophobic membrane protein, detergent solubilization using agents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) is necessary.

• Affinity purification: The tagged protein is purified using affinity chromatography (often Ni-NTA for His-tagged proteins), followed by size exclusion chromatography to obtain highly pure protein.

This experimental approach must be carefully optimized considering the hydrophobic nature of subunit c and potential toxicity when overexpressed .

How do researchers determine the rotational states and catalytic cycle of C. jejuni ATP synthase?

Investigating the rotational states and catalytic cycle of C. jejuni ATP synthase requires sophisticated biophysical techniques. While specific data for C. jejuni is limited in the provided search results, researchers typically employ the following approaches based on studies of other bacterial ATP synthases:

• Cryo-electron microscopy (cryo-EM): High-resolution cryo-EM has been instrumental in resolving rotational states in bacterial F-ATPases. For instance, in bacterial ATP synthase from thermophilic Bacillus PS3, researchers identified discrete rotational states that represent different steps in the catalytic cycle .

• Single-molecule fluorescence spectroscopy: By labeling subunits (particularly the γ-subunit) with fluorescent probes, researchers can directly observe rotation. In bacterial systems, the γ-subunit has been shown to rotate at >130 Hz, indicating synthesis or hydrolysis of approximately 400 ATP molecules per second .

• Biochemical assays: Researchers monitor ATP synthesis and hydrolysis rates under various conditions (pH, ion gradients, inhibitors) to correlate structural states with enzymatic activity.

• Site-directed mutagenesis: Strategic mutations in key residues help identify their roles in the catalytic mechanism and conformational changes during rotation.

For C. jejuni specifically, these approaches would need to account for its unique adaptations to microaerophilic environments and its particular energetic requirements .

What is known about the ion specificity of the C. jejuni ATP synthase c-ring?

The ion specificity of ATP synthase c-rings varies across bacterial species and represents an important adaptation to different environmental niches. For C. jejuni ATP synthase:

• Ion selectivity: C. jejuni ATP synthase is primarily proton-coupled, using the proton gradient across the membrane to drive ATP synthesis. This is consistent with its role in utilizing the proton motive force generated by the electron transport chain .

• c-ring structure: While the exact stoichiometry of the C. jejuni c-ring has not been specifically detailed in the provided search results, bacterial c-rings typically contain 8-15 c-subunits. Each c-subunit contains an ion-binding site, usually formed by a conserved carboxylate residue (aspartate or glutamate) that participates in proton translocation .

• Energetic implications: The number of c-subunits in the ring determines the ion-to-ATP ratio, directly affecting the bioenergetic efficiency of the enzyme. This ratio represents an evolutionary adaptation to the organism's specific energy requirements and environmental niche .

• Experimental approaches: Determining ion specificity typically involves biochemical assays measuring ATP synthesis/hydrolysis under varying ion gradients, complemented by structural studies using X-ray crystallography or cryo-EM to identify ion-binding sites .

Understanding the ion specificity of C. jejuni ATP synthase is particularly relevant given the pathogen's adaptation to microaerophilic conditions and its requirement for specific electron donors like formate to generate a sufficient membrane potential .

What strategies can be employed to create recombinant strains expressing modified ATP synthase subunit c in C. jejuni?

Creating recombinant C. jejuni strains with modified ATP synthase subunit c requires specific genetic approaches that accommodate this fastidious pathogen:

• Homologous recombination: The most reliable method for chromosomal gene replacement in C. jejuni involves:

  • Creating a construct with the modified atpE gene flanked by homologous regions

  • Including a selectable marker (typically kanamycin or chloramphenicol resistance)

  • Natural transformation of C. jejuni with the linear DNA fragment

  • Selection of recombinants on appropriate media

• Complementation systems: For functional studies, researchers often use:

  • Shuttle vectors capable of replication in both E. coli and C. jejuni

  • Integration vectors targeting neutral loci in the C. jejuni genome

  • Inducible promoter systems to control expression levels

• Challenges and considerations:

  • C. jejuni has relatively low transformation efficiency compared to model organisms

  • The microaerophilic growth requirements complicate standard molecular biology procedures

  • Modifications to essential genes like atpE may affect viability, necessitating conditional expression systems

• Verification methods:

  • PCR and sequencing to confirm correct integration

  • Western blotting to verify protein expression

  • Functional assays to assess ATP synthase activity

These approaches can be used to introduce point mutations, affinity tags, or fluorescent protein fusions to study structure-function relationships in ATP synthase subunit c.

How can researchers assess the impact of atpE mutations on C. jejuni viability and energy metabolism?

Assessing the impact of atpE mutations on C. jejuni viability and energy metabolism requires a multifaceted approach:

• Growth curve analysis:

  • Comparison of growth rates under different conditions (varying oxygen tensions, carbon sources)

  • Assessment of final cell densities and lag phases

  • Determination of minimum inhibitory concentrations for various antibiotics, particularly those targeting energy metabolism

• Membrane potential measurements:

  • Fluorescent probes such as DiSC3(5) or JC-1 to quantify membrane potential

  • Flow cytometry to assess population heterogeneity in membrane potential

  • Correlation of membrane potential with ATP synthase mutations

• ATP quantification:

  • Luciferase-based assays to measure intracellular ATP levels

  • ATP/ADP ratio determination to assess energy charge

  • Comparison between wild-type and mutant strains under different growth conditions

• Respiratory chain analysis:

  • Oxygen consumption measurements using oxygen electrodes

  • Activity assays for key respiratory enzymes (formate dehydrogenase, hydrogenase)

  • Assessment of adaptation to low oxygen tension

• Transcriptomic/proteomic profiling:

  • RNA-seq or microarray analysis to identify compensatory changes in gene expression

  • Proteomic analysis to detect alterations in protein levels, particularly in energy metabolism pathways

  • Targeted RT-qPCR for key genes involved in energy metabolism

• Motility assays:

  • Soft agar motility tests (as motility depends on pmf)

  • Microscopic analysis of swimming behavior

  • Correlation between ATP synthase function and flagellar motility

How does ATP synthase function contribute to C. jejuni pathogenesis and host colonization?

ATP synthase function is critical for C. jejuni pathogenesis through several interconnected mechanisms:

• Energy provision for virulence traits:

  • ATP generation supports the synthesis of virulence factors, including the cytolethal distending toxin (CdtC)

  • Energy-dependent secretion systems require ATP for assembly and operation

  • Bioenergetic support for outer membrane vesicle (OMV) production via the MlaEFD system

• Adaptation to host microenvironments:

  • Flexible energy metabolism enables survival in the oxygen-limited intestinal environment

  • Under low oxygen tension, C. jejuni upregulates alternative electron transport complexes (formate dehydrogenase, hydrogenase) to maintain ATP synthesis

  • This metabolic flexibility allows adaptation to various niches within the host

• Motility and chemotaxis:

  • The proton motive force generated during ATP synthesis drives flagellar rotation

  • Motility is essential for colonization, invasion of epithelial cells, and navigation through intestinal mucus

  • Impaired bioenergetics directly impacts motility and subsequently colonization ability

• Biofilm formation:

  • ATP-dependent processes are critical for biofilm development

  • Disruption of energy metabolism reduces biofilm formation capacity

  • Biofilms contribute to environmental persistence and antibiotic tolerance

• Stress response and survival:

  • Energy-dependent stress response systems protect against host immune defenses

  • ATP-driven efflux pumps contribute to antimicrobial resistance

  • Maintenance of cellular homeostasis under stress conditions requires adequate ATP supply

These interconnected roles make ATP synthase function integral to C. jejuni's ability to establish infection and cause disease .

What is the relationship between ATP synthase function and antibiotic susceptibility in C. jejuni?

The relationship between ATP synthase function and antibiotic susceptibility in C. jejuni is complex and multifaceted:

These interconnections suggest that modulating ATP synthase function could potentially serve as a strategy to enhance antibiotic efficacy against C. jejuni infections .

How can structural differences between human and C. jejuni ATP synthase be exploited for antimicrobial development?

Structural differences between human and C. jejuni ATP synthase offer promising targets for selective antimicrobial development:

• Unique structural features as targets:

  • Similar to mycobacterial ATP synthase, C. jejuni may possess unique regulatory mechanisms including auto-inhibition features not present in human enzymes

  • Structural differences in the c-ring stoichiometry and ion-binding sites could be exploited

  • Species-specific protein-protein interfaces, particularly in the peripheral stalk or OSCP region, may provide selective binding sites

• Experimental approaches for target identification:

  • High-resolution structural analysis using cryo-EM to identify C. jejuni-specific features

  • Comparative structural bioinformatics between human and C. jejuni ATP synthase

  • Molecular dynamics simulations to identify differential binding pockets

  • Targeted mutagenesis to validate the importance of specific structural elements

• Drug discovery strategies:

  • Structure-based virtual screening against identified unique sites

  • Fragment-based approaches targeting C. jejuni-specific interfaces

  • Repurposing screens of approved drugs, particularly those known to interact with other bacterial ATP synthases

  • Phenotypic screening for compounds that specifically inhibit C. jejuni growth under conditions requiring ATP synthase function

• Validation methodologies:

  • Biochemical assays comparing inhibition of purified C. jejuni and human ATP synthase

  • Cell-based assays demonstrating selective toxicity

  • Membrane potential measurements to confirm on-target activity

  • Resistance development studies to validate the target mechanism

This approach is conceptually similar to how bedaquiline selectively targets mycobacterial ATP synthase by exploiting structural differences from the human enzyme, as illustrated in research on Mycobacterium smegmatis ATP synthase .

What methodologies are most effective for analyzing ATP synthase-membrane interactions in C. jejuni?

Analyzing ATP synthase-membrane interactions in C. jejuni requires specialized techniques that address the complexity of membrane protein systems:

• Advanced structural approaches:

  • Cryo-electron microscopy of ATP synthase in nanodiscs or native membrane environments

  • Lipid nanodiscs with defined composition to study specific lipid interactions

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify membrane-interacting regions

  • Cross-linking mass spectrometry to capture dynamic interactions with lipids and other membrane components

• Biophysical techniques:

  • Fluorescence resonance energy transfer (FRET) between labeled ATP synthase and membrane components

  • Solid-state NMR to study protein-lipid interactions in native-like environments

  • Atomic force microscopy to visualize ATP synthase arrangement in membranes

  • Surface plasmon resonance to quantify binding affinities with specific lipids

• Functional assays in reconstituted systems:

  • Proteoliposome reconstitution with varying lipid compositions

  • ATP synthesis/hydrolysis measurements in defined membrane environments

  • Proton pumping assays using pH-sensitive fluorescent probes

  • Patch-clamp electrophysiology to study ion translocation through the Fo complex

• In situ approaches:

  • Super-resolution microscopy to visualize ATP synthase distribution in bacterial membranes

  • Correlative light and electron microscopy (CLEM) to connect function with structure

  • Proximity labeling methods (e.g., APEX2) to identify proteins interacting with ATP synthase in native membranes

  • Lipidomics analysis of membranes associated with purified ATP synthase complexes

An integrative approach combining these methodologies would provide comprehensive insights into how C. jejuni ATP synthase interacts with its membrane environment, particularly important given the unique lipid composition of Campylobacter membranes and the importance of membrane interactions in shaping ATP synthase function and evolution .

What are the most common challenges in purifying functional recombinant ATP synthase subunit c from C. jejuni and how can they be addressed?

Purifying functional recombinant ATP synthase subunit c from C. jejuni presents several challenges due to its hydrophobic nature and small size. Here are the major challenges and approaches to address them:

• Expression challenges:

  • Toxicity to host cells: Use tightly regulated expression systems (e.g., T7-based with glucose repression)

  • Inclusion body formation: Lower induction temperature (16-18°C), reduce inducer concentration

  • Proteolytic degradation: Use protease-deficient host strains, include protease inhibitors

  • Low expression levels: Optimize codon usage for expression host, use strong ribosome binding sites

• Solubilization difficulties:

  • Inefficient extraction: Screen multiple detergents (DDM, LMNG, digitonin) at various concentrations

  • Protein aggregation: Include stabilizing agents (glycerol, specific lipids)

  • Detergent-induced denaturation: Use milder alternatives like styrene maleic acid lipid particles (SMALPs) or nanodiscs

  • Mixed micelle formation: Implement sequential solubilization protocols

• Purification complications:

  • Poor affinity tag accessibility: Position tags at both N- and C-termini to identify optimal configuration

  • Co-purifying contaminants: Include multiple orthogonal purification steps

  • Tag interference with function: Include cleavable tags and verify function post-cleavage

  • Protein precipitation during concentration: Use stabilizing additives, concentrate at low temperatures

• Functional assessment:

  • Reconstitution into liposomes: Optimize lipid composition based on C. jejuni membrane lipids

  • Activity measurement: Develop sensitive assays for proton translocation

  • Structural integrity: Use circular dichroism to confirm secondary structure

  • Oligomerization assessment: Use native PAGE or analytical ultracentrifugation

• Quality control checkpoints:

  • Use mass spectrometry to confirm protein identity and integrity

  • Verify proper folding through limited proteolysis assays

  • Assess homogeneity through size exclusion chromatography

  • Confirm functionality in reconstituted systems

These approaches should be systematically optimized for the specific properties of C. jejuni ATP synthase subunit c, with particular attention to maintaining the native structure and function throughout the purification process .

How can researchers effectively assess the impact of ATP synthase modifications on C. jejuni virulence in laboratory models?

Effectively assessing the impact of ATP synthase modifications on C. jejuni virulence requires carefully designed laboratory models that capture key aspects of pathogenesis:

• In vitro cell culture models:

  • Adhesion and invasion assays using intestinal epithelial cell lines (Caco-2, HT-29)

  • Quantification using differential plating or fluorescent labeling

  • Gentamicin protection assays to distinguish adherent from internalized bacteria

  • Trans-epithelial electrical resistance (TEER) measurements to assess barrier disruption

  • Assessment of host cell cytotoxicity and innate immune responses

• Biofilm formation assessment:

  • Crystal violet staining for quantification

  • Confocal microscopy with fluorescent strains to assess architecture

  • Biofilm formation under various stressors (oxygen limitation, bile salts)

  • Mixed-species biofilms to mimic intestinal environments

• Stress resistance testing:

  • Survival under acid stress (gastric transit simulation)

  • Bile salt resistance (intestinal conditions)

  • Oxidative stress response (host immune defense)

  • Temperature stress (fever response)

• Animal models:

  • Colonization efficiency in chicken models (natural host)

  • Disease progression in specialized mouse models

  • Competitive index assays comparing wild-type and ATP synthase-modified strains

  • In vivo bioluminescence imaging for real-time tracking

• Molecular correlates of virulence:

  • Quantification of cytolethal distending toxin production

  • Assessment of outer membrane vesicle (OMV) production

  • Expression analysis of key virulence genes under host-relevant conditions

  • Proteomics of secreted virulence factors

• Advanced microscopy approaches:

  • Live cell imaging of host-pathogen interactions

  • Super-resolution microscopy to visualize subcellular localization

  • Electron microscopy to assess ultrastructural features

These approaches should be used in combination to provide a comprehensive assessment of how ATP synthase modifications affect the various aspects of C. jejuni virulence, from initial colonization to disease manifestation .