Recombinant Mycoplasma arthritidis ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) in Mycoplasma arthritidis and what is its significance in research?

ATP synthase subunit c (atpE) is a critical component of the F0F1 ATP synthase complex in M. arthritidis. This protein forms the c-ring structure in the membrane-embedded F0 portion of the ATP synthase and plays an essential role in proton translocation during ATP synthesis.

Unlike many sequenced mycoplasma species that utilize glycolysis, M. arthritidis is a non-glycolytic mycoplasma that relies on arginine catabolism as its major energy source . This fundamental metabolic difference makes its ATP synthase components particularly interesting for research, as they may have adapted to support this alternative energy generation pathway.

The significance of studying recombinant atpE includes:

• Understanding energy metabolism in minimal organisms

• Investigating adaptations of essential cellular machinery to different metabolic strategies

• Exploring potential therapeutic targets, as M. arthritidis causes severe polyarthritis in rats and serves as a model for human rheumatoid arthritis

• Comparing energetic efficiency between glycolytic and non-glycolytic mycoplasmas

How does the genomic context of atpE compare between Mycoplasma arthritidis and other mycoplasma species?

The genome of M. arthritidis is 820,453 bp in size with 635 predicted coding regions . While specific information about the atpE gene organization isn't provided in the available literature, we can make several informed observations:

In most bacteria including mycoplasmas, ATP synthase genes are typically organized in an operon structure. The genomic context of atpE may differ between glycolytic and non-glycolytic mycoplasmas due to their different energy metabolic pathways. For instance, in M. arthritidis, the ATP synthase genes might show coordinated regulation with arginine catabolism genes rather than glycolytic genes.

The complete genome sequence of M. arthritidis revealed two large families of genes that encode phase-variable proteins . While there's no specific indication that atpE belongs to these families, phase variation in components related to energy metabolism could potentially allow adaptation to varying environmental conditions.

A comparative genomic analysis would be valuable to identify specific adaptations in the atpE gene sequence and its regulatory elements that might reflect M. arthritidis' unique energy metabolism requirements.

What is known about the expression of ATP synthase components in Mycoplasma arthritidis compared to other mycoplasmas?

Expression patterns of ATP synthase components likely differ between glycolytic mycoplasmas (like M. pneumoniae) and non-glycolytic mycoplasmas like M. arthritidis that utilize arginine catabolism . These expression differences would reflect their distinct energy generation pathways and requirements.

In M. pneumoniae, the ATP synthase beta subunit (AtpD) has been identified as immunogenic during infection, eliciting antibody responses in infected patients . This raises the possibility that ATP synthase components in M. arthritidis, including atpE, might similarly be expressed during infection and potentially recognized by the host immune system.

The genome sequencing of M. arthritidis revealed the presence of genes required for arginine catabolism , suggesting that the expression of these genes along with ATP synthase components might be coordinated to optimize energy production from this alternative metabolic pathway.

What are the recommended strategies for expressing recombinant Mycoplasma arthritidis atpE in heterologous systems?

Based on successful approaches used for other mycoplasma proteins, the following strategies are recommended for expressing recombinant M. arthritidis atpE:

Expression systems and vectors:

• E. coli BL21(DE3) has been successfully used for expressing recombinant mycoplasma proteins

• Expression vectors providing affinity tags (such as pDEST17 with an N-terminal His-tag) facilitate purification

• For membrane proteins like atpE, specialized E. coli strains (C41, C43) designed for membrane protein expression may improve yields

Optimization parameters:

• Temperature: Lower temperatures (16-20°C) often improve membrane protein folding

• IPTG concentration: 0.1-0.5 mM typically provides balanced expression

• Induction duration: 4-16 hours depending on expression temperature

• Media composition: Rich media supplemented with glucose can improve yields

Special considerations for atpE as a membrane protein:

• Fusion with solubility-enhancing tags (MBP, SUMO) may improve expression

• Codon optimization based on E. coli preference may enhance expression levels

• Co-expression with chaperones can assist proper folding

• For functional studies, expression in the membrane fraction rather than as inclusion bodies is preferable

As demonstrated with AtpD from M. pneumoniae, recombinant mycoplasma proteins can be successfully expressed in E. coli and purified in functional form . Similar approaches should be applicable to M. arthritidis atpE with appropriate optimization.

What purification approaches are most suitable for obtaining high-purity recombinant atpE protein?

Purifying membrane proteins like ATP synthase subunit c requires specialized approaches. Based on successful purification of other mycoplasma proteins , the following strategy is recommended:

Initial extraction:

• Careful selection of detergents is critical for solubilizing atpE from membranes

• Screening of multiple detergents (DDM, LDAO, OG, Triton X-100) to identify optimal solubilization conditions

• Inclusion of protease inhibitors to prevent degradation during extraction

Chromatographic purification:

• Immobilized metal affinity chromatography (IMAC):

  • Utilizing His-tag affinity for initial capture

  • Optimized imidazole gradient to minimize non-specific binding

  • Maintaining detergent above critical micelle concentration throughout

• Ion exchange chromatography:

  • Secondary purification step as used for AtpD

  • Selection of cation or anion exchange based on theoretical pI of atpE

• Size exclusion chromatography:

  • Final polishing step to remove aggregates

  • Assessment of oligomeric state

  • Buffer exchange to optimal storage conditions

Quality control:

• SDS-PAGE to assess purity

• Western blotting with anti-His antibodies to confirm identity

• Mass spectrometry for definitive identification

This multi-step purification approach, similar to that used successfully for AtpD from M. pneumoniae , should yield high-purity recombinant atpE suitable for functional and structural studies.

What are the major challenges in working with recombinant membrane proteins like atpE and how can they be addressed?

Recombinant membrane proteins like atpE present several unique challenges that must be addressed for successful outcomes:

Challenge 1: Low expression levels

• Solution: Screen multiple expression strains and conditions

• Optimize codon usage and reduce expression temperature

• Consider fusion with solubility-enhancing tags

• Evaluate the impact of different media compositions and induction parameters

Challenge 2: Protein misfolding and aggregation

• Solution: Express at reduced temperatures (16-20°C)

• Co-express with molecular chaperones

• Use specialized strains developed for membrane protein expression

• Employ slow induction methods (auto-induction media)

Challenge 3: Maintaining native structure during solubilization

• Solution: Systematic screening of detergents and detergent mixtures

• Include appropriate lipids during solubilization

• Consider alternative solubilization systems (nanodiscs, amphipols)

• Optimize buffer conditions (pH, ionic strength, additives)

Challenge 4: Protein instability after purification

• Solution: Minimize purification duration

• Include stabilizing agents (glycerol, specific lipids)

• Determine optimal storage conditions

• Prepare single-use aliquots to avoid freeze-thaw cycles

Challenge 5: Functional assessment

• Solution: Develop specific activity assays for atpE

• Consider reconstitution into liposomes for functional studies

• Use complementary biophysical techniques to verify structural integrity

• Compare with native protein when possible

The successful expression and purification of other mycoplasma proteins as described in previous research suggests that these challenges can be overcome with systematic optimization.

What methods can be used to determine the structural features of recombinant Mycoplasma arthritidis atpE?

Determining the structural features of recombinant atpE requires a combination of computational and experimental approaches:

Computational methods:

• Sequence analysis:

  • Multiple sequence alignment with atpE from other species

  • Identification of conserved functional residues

  • Secondary structure prediction (transmembrane helices, orientation)

• Structure prediction:

  • Homology modeling based on known c-subunit structures

  • Ab initio modeling using tools like AlphaFold

  • Molecular dynamics simulations to study conformational dynamics

Experimental methods:

• Spectroscopic techniques:

  • Circular dichroism (CD) to assess secondary structure content

  • Fluorescence spectroscopy to probe tertiary structure

  • FTIR spectroscopy to analyze secondary structure in membrane environment

• Advanced structural biology approaches:

  • X-ray crystallography (challenging but possible with lipidic cubic phase)

  • Cryo-electron microscopy for structures of the assembled c-ring

  • Solid-state NMR for structural details in membrane environment

• Biochemical approaches:

  • Cross-linking studies to identify interaction interfaces

  • Limited proteolysis to probe structural domains

  • Hydrogen-deuterium exchange mass spectrometry to assess structure dynamics

• Functional mapping:

  • Site-directed mutagenesis of predicted functional residues

  • Proton translocation assays after reconstitution

  • Inhibitor binding studies to identify functional sites

These approaches would provide complementary information about the structure of atpE, especially in the context of M. arthritidis' non-glycolytic energy metabolism .

How can the functionality of recombinant atpE be assessed in experimental settings?

Assessing the functionality of recombinant atpE requires specialized approaches that address its role in the ATP synthase complex:

Reconstitution systems:

• Proteoliposome preparation:

  • Incorporation of purified atpE into liposomes

  • Co-reconstitution with other ATP synthase components when available

  • Optimization of lipid composition to support functionality

• Functional measurements:

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • Membrane potential measurements using potentiometric dyes

  • ATP synthesis/hydrolysis coupling when reconstituted with F1 components

Interaction studies:

• Binding assays with partner proteins:

  • Pull-down assays with other ATP synthase components

  • Surface plasmon resonance to quantify binding affinities

  • Isothermal titration calorimetry for thermodynamic parameters

• Assembly assessment:

  • Blue native PAGE to assess c-ring formation

  • Analytical ultracentrifugation to determine oligomeric state

  • Electron microscopy to visualize assembled complexes

Inhibitor studies:

• Binding assays with known ATP synthase inhibitors:

  • Oligomycin binding studies (c-subunit is a target)

  • Fluorescence competition assays to characterize binding sites

  • Inhibitor-resistant mutant analysis

• Comparative inhibition profiles:

  • Comparison with inhibition patterns of other mycoplasma ATP synthases

  • Evaluation of species-specific inhibitor sensitivity

  • Structure-activity relationship studies with inhibitor variants

These functional assessments would provide insights into whether M. arthritidis atpE has unique properties related to the organism's non-glycolytic energy metabolism .

How might the properties of ATP synthase subunit c in Mycoplasma arthritidis differ from those in glycolytic mycoplasma species?

Given that M. arthritidis is non-glycolytic and relies on arginine catabolism for energy production , its ATP synthase components, including atpE, might show several adaptations compared to glycolytic mycoplasmas:

Structural adaptations:

• c-ring composition:

  • Potentially different stoichiometry (number of c subunits)

  • Modified c-subunit interfaces to optimize ring stability

  • Adaptations at the interface with other ATP synthase components

• Proton-binding site:

  • Potentially altered proton-binding residue environment

  • Modified pKa of the catalytic carboxylate residue

  • Adaptations for different membrane potential conditions

Functional differences:

• Energetic parameters:

  • Potentially different H⁺/ATP ratios

  • Altered ATP synthesis/hydrolysis equilibrium

  • Modified regulation by proton motive force magnitude

• Operational pH range:

  • Adaptation to pH gradients generated by arginine catabolism versus glycolysis

  • Different optimal pH for activity

  • Modified pH-dependent regulatory mechanisms

Regulatory properties:

• Interaction with metabolic enzymes:

  • Potential physical or functional coupling with arginine catabolism enzymes

  • Different regulatory mechanisms linking metabolic state to ATP synthase activity

  • Unique inhibitory/activatory mechanisms

• Response to environmental conditions:

  • Adaptation to arginine availability fluctuations

  • Modified response to oxygen levels

  • Different temperature-dependent properties

Comparative biochemical and structural studies between atpE from M. arthritidis and glycolytic mycoplasmas would help elucidate these potential differences and their functional significance in the context of different energy metabolism strategies.

How can recombinant Mycoplasma arthritidis atpE be used to study host-pathogen interactions?

Recombinant atpE can serve as a valuable tool for investigating various aspects of host-pathogen interactions in M. arthritidis infections:

Immunological studies:

• Antigenicity assessment:

  • Development of ELISA assays to detect anti-atpE antibodies in infected animals

  • Evaluation of atpE immunogenicity during infection

  • Comparison with other mycoplasma ATP synthase components like AtpD, which has demonstrated immunogenicity in M. pneumoniae infections

• Cytokine response profiling:

  • Measurement of cytokine production by immune cells exposed to recombinant atpE

  • Comparison with immune responses to other M. arthritidis antigens like MAM

  • Correlation with disease progression in arthritis models

Diagnostic applications:

• Serological test development:

  • Similar to applications demonstrated for M. pneumoniae AtpD

  • Evaluation as a diagnostic biomarker for M. arthritidis infection

  • Development of multiplex assays with other antigens for improved sensitivity

• Experimental infection monitoring:

  • Tracking antibody responses to atpE during experimental arthritis

  • Correlation with bacterial load and disease severity

  • Comparison with responses to MAM superantigen

Pathogenesis mechanisms:

• Potential role beyond energy metabolism:

  • Investigation of possible moonlighting functions in host interactions

  • Assessment of potential role in adhesion or immune evasion

  • Comparative studies with MAAs (M. arthritidis adhesins) mentioned in the literature

• Vaccine candidate evaluation:

  • Assessment of protective potential in animal models

  • Epitope mapping for subunit vaccine design

  • Evaluation of adjuvant formulations for optimal immune response

These applications would complement studies with other M. arthritidis virulence factors like MAM superantigen and contribute to a comprehensive understanding of mycoplasma pathogenesis.

What is the relationship between ATP synthase function and virulence in Mycoplasma arthritidis?

The relationship between ATP synthase function and virulence in M. arthritidis is complex and multifaceted:

Energy production for virulence:

• Metabolic support for pathogenesis:

  • ATP production to power virulence factor expression and secretion

  • Energy supply for colonization and persistence

  • Sustaining bacterial viability during host immune response

• Adaptation to host environment:

  • Utilization of arginine as energy source in host tissues

  • Potential metabolic flexibility during different infection stages

  • Energy production under varying nutrient availability conditions

Potential direct roles in virulence:

• Surface exposure and host interactions:

  • Potential exposure of ATP synthase components on bacterial surface

  • Possible moonlighting functions beyond energy production

  • Comparison with roles of other ATP synthase components in mycoplasmas

• Immune modulation:

  • Potential immunomodulatory effects similar to other bacterial ATP synthases

  • Interaction with host immune receptors

  • Comparison with the immunomodulatory effects of MAM superantigen

Genetic evidence:

• Transposon mutagenesis studies:

  • The development of a transposon library in M. arthritidis could reveal whether atpE or other ATP synthase components are essential during infection

  • Comparison with the dispensability of MAM for arthritis induction

  • Correlation between ATP synthase function and in vivo fitness

• Comparative genomics:

  • Evaluation of ATP synthase gene conservation across virulent and avirulent strains

  • Assessment of genetic variation in atpE sequence

  • Identification of potential virulence-associated polymorphisms

Further research using recombinant atpE and genetic approaches would help elucidate the specific contributions of ATP synthase to M. arthritidis virulence.

How does the arginine catabolism pathway interact with ATP synthase in Mycoplasma arthritidis?

As a non-glycolytic organism, M. arthritidis relies on arginine catabolism for energy production , suggesting important functional interactions between this pathway and ATP synthase:

Metabolic coupling:

• Energy transfer mechanisms:

  • Arginine catabolism to ornithine generates ATP via substrate-level phosphorylation

  • This process also produces ammonia, potentially creating a proton gradient

  • ATP synthase likely utilizes this proton gradient for additional ATP synthesis

• Stoichiometric considerations:

  Parameter	Value/Characteristic
  ATP yield from arginine pathway	1 ATP per arginine molecule via substrate-level phosphorylation
  Potential additional ATP from ATP synthase	Dependent on proton gradient generated and c-ring stoichiometry
  Net energy yield	Likely higher than from arginine catabolism alone
  Efficiency compared to glycolysis	Potentially lower ATP yield but utilizing different nutrients

Regulatory integration:

• Coordinated expression:

  • Likely co-regulation of ATP synthase and arginine catabolism genes

  • Potential shared transcriptional regulators

  • Response to common environmental signals (arginine availability, pH, energy status)

• Functional regulation:

  • ATP levels potentially regulating arginine catabolism enzymes

  • Proton gradient affecting both pathways

  • Potential feedback mechanisms linking ATP production to arginine utilization

Spatial organization:

• Membrane architecture:

  • Potential co-localization of ATP synthase with arginine catabolism components

  • Possible formation of metabolic microdomains in the membrane

  • Optimization of proton gradient utilization through spatial arrangement

• Protein-protein interactions:

  • Possible direct interactions between components of both pathways

  • Formation of supramolecular complexes for enhanced efficiency

  • Specialized structural adaptations at interaction interfaces

Understanding these interactions would provide insights into how M. arthritidis has adapted its energy metabolism to support its pathogenic lifestyle, particularly in comparison to glycolytic mycoplasmas.

How could genetic manipulation techniques be applied to study atpE function in Mycoplasma arthritidis?

The creation of a transposon library in M. arthritidis mentioned in search result indicates that genetic manipulation of this organism is feasible. Several genetic approaches could be applied to study atpE function:

Gene disruption strategies:

• Transposon mutagenesis:

  • Random insertional mutagenesis to disrupt atpE

  • Similar to approaches used for MAM gene disruption

  • Phenotypic characterization of mutants

• Targeted gene modification:

  • Development of CRISPR-Cas systems adapted for mycoplasmas

  • Creation of conditional knockdown systems if complete knockout is lethal

  • Site-directed mutagenesis of key functional residues

Complementation approaches:

• Expression systems:

  • Reintroduction of wild-type atpE in mutant strains

  • Expression of variant forms to study structure-function relationships

  • Comparison with complementation approaches used for MAM studies

• Reporter fusions:

  • Creation of atpE-reporter fusions for expression and localization studies

  • Promoter-reporter constructs to study regulation

  • Tagged versions for in vivo tracking

Experimental applications:

• Phenotypic characterization:

  Parameter	Wild-type	atpE mutant	Complemented strain
  Growth rate	Baseline	Potentially reduced	Restored
  Energy metabolism	Normal arginine utilization	Potentially altered	Restored
  Virulence	Full virulence	Potentially attenuated	Restored
  Membrane potential	Normal	Potentially compromised	Restored

• In vivo studies:

  • Virulence assessment in arthritis models

  • Persistence in host tissues

  • Immune response elicitation

  • Comparison with MAM mutant phenotypes

These genetic approaches would complement biochemical studies with recombinant atpE, providing a comprehensive understanding of its role in M. arthritidis physiology and pathogenesis.

What new therapeutic strategies might emerge from studying ATP synthase in Mycoplasma arthritidis?

Research on M. arthritidis ATP synthase, including the atpE component, could lead to several novel therapeutic approaches:

Target-based drug development:

• Specific inhibitor design:

  • Identification of unique structural features in M. arthritidis ATP synthase

  • Structure-based design of selective inhibitors

  • High-throughput screening using recombinant components

• Exploitation of non-glycolytic metabolism:

  • Development of combination therapies targeting both ATP synthase and arginine metabolism

  • Exploitation of the limited metabolic flexibility of M. arthritidis

  • Comparative analysis with inhibitors of glycolytic mycoplasmas

Immunological approaches:

• Vaccine development:

  • Evaluation of ATP synthase components as vaccine antigens

  • Design of multi-epitope vaccines including ATP synthase-derived peptides

  • Comparison with immune responses to natural infection

• Immunomodulation strategies:

  • Targeting the inflammatory response in mycoplasma arthritis

  • Comparison with approaches targeting MAM superantigen

  • Development of therapies that preserve beneficial immune responses

Potential therapeutic advantages:

• Specificity considerations:

  Target feature	Therapeutic potential	Potential advantages
  Unique structural features of mycoplasma ATP synthase	Selective inhibition	Reduced impact on host ATP synthase
  Non-glycolytic energy metabolism	Metabolic vulnerability	Targeting pathway absent in host
  Arginine-ATP synthase coupling	Synergistic inhibition	Enhanced efficacy through dual targeting

• Resistance management:

  • Essential nature of ATP synthase may limit resistance development

  • Multiple targeting sites within the complex

  • Potential for combination therapies with existing antibiotics

These therapeutic approaches would build on the understanding of M. arthritidis as a model for inflammatory arthritis and could potentially extend to other mycoplasma infections.

How could systems biology approaches integrate ATP synthase function into the broader metabolic network of Mycoplasma arthritidis?

Systems biology offers powerful approaches to understand the integration of ATP synthase in the minimal but specialized metabolism of M. arthritidis:

Metabolic modeling:

• Genome-scale metabolic reconstruction:

  • Integration of the 635 predicted coding regions from the M. arthritidis genome

  • Detailed mapping of arginine catabolism and ATP synthase function

  • Flux balance analysis to predict energy production capabilities

• Comparative metabolic analysis:

  • Comparison with metabolic models of glycolytic mycoplasmas

  • Identification of unique metabolic capabilities and constraints

  • Prediction of essential metabolic reactions and potential drug targets

Multi-omics integration:

• Integrated data analysis:

  Omic level	Contribution to understanding
  Genomics	Gene content, organization of energy metabolism genes
  Transcriptomics	Expression patterns, co-regulation of energy pathways
  Proteomics	Protein abundance, post-translational modifications
  Metabolomics	Metabolic fluxes, energy intermediates, redox state
  Fluxomics	Quantitative analysis of arginine metabolism and ATP production

• Regulation network reconstruction:

  • Identification of transcriptional regulators controlling energy metabolism

  • Mapping of signaling pathways affecting ATP synthase function

  • Integration with stress response networks

Host-pathogen systems biology:

• Interaction mapping:

  • Metabolic interactions between host and pathogen

  • Energy competition or complementation during infection

  • Effects of host immune response on bacterial metabolism

• Dynamic modeling:

  • Temporal changes in energy metabolism during infection

  • Adaptation to changing host environments

  • Prediction of metabolic vulnerabilities during different infection stages

These systems approaches would place ATP synthase function in the broader context of M. arthritidis biology and host interaction, potentially revealing emergent properties and new intervention points not apparent from reductionist approaches.