Recombinant Mycoplasma penetrans ATP synthase subunit c (atpE)

What is the structure and function of Mycoplasma penetrans ATP synthase subunit c?

ATP synthase subunit c (atpE) in M. penetrans, like in other prokaryotes, forms a ring structure in the F0 portion of the ATP synthase complex embedded in the cell membrane. This c-ring plays a crucial role in the rotary mechanism of ATP synthesis by facilitating proton translocation across the membrane. The c-subunit contains a highly conserved carboxyl residue (typically glutamate) that is essential for proton binding during the catalytic cycle . Similar to other bacterial species, the M. penetrans c-subunit likely contains approximately 70-80 amino acids arranged in a hairpin-like structure with two transmembrane α-helices connected by a polar loop . While the specific structure of M. penetrans ATP synthase subunit c has not been fully characterized, comparative analysis with related mycoplasma species suggests significant structural conservation with some species-specific variations.

What are the recommended methods for cloning the atpE gene from Mycoplasma penetrans?

For successful cloning of the M. penetrans atpE gene, researchers should consider the following methodological approach:

• DNA extraction: Use a specialized mycoplasma DNA extraction kit to obtain high-quality genomic DNA, as M. penetrans has a low G+C content and lacks a cell wall.

• PCR amplification: Design primers based on the published M. penetrans genome sequence, with consideration for the following:

  • Include appropriate restriction sites for subsequent cloning

  • Consider codon optimization if expressing in E. coli

  • Include a 6xHis-tag or other affinity tag for purification purposes

• Vector selection: Choose an expression vector with a strong promoter suitable for membrane protein expression, such as pET or pBAD series vectors.

• Transformation: Use competent E. coli strains optimized for membrane protein expression, such as C41(DE3) or C43(DE3) .

This approach should be modified based on specific experimental requirements and the challenging nature of membrane protein expression.

What expression systems are most effective for producing recombinant Mycoplasma ATP synthase subunit c?

Based on comparative studies with other bacterial ATP synthase subunits, the following expression systems have proven effective and can be adapted for M. penetrans atpE:

For optimal results, expression should be carried out at lower temperatures (18-25°C) with reduced inducer concentrations to minimize toxicity and protein aggregation . The addition of mild detergents like n-dodecyl-β-D-maltoside (DDM) during cell lysis can aid in solubilizing the membrane-embedded c-subunit.

What purification strategies are recommended for recombinant ATP synthase subunit c?

A multi-step purification strategy is recommended:

• Cell lysis: Use a combination of enzymatic (lysozyme) and mechanical (sonication) methods in a buffer containing protease inhibitors and appropriate detergents.

• Membrane fraction isolation: Perform differential centrifugation to isolate the membrane fraction (typically 20,000×g for cell debris removal, followed by 100,000×g for membrane collection).

• Solubilization: Use mild detergents like DDM or n-octyl-β-D-glucopyranoside (OG) at 1-2% concentration.

• Affinity chromatography: If His-tagged, use Ni-NTA columns with imidazole gradient elution (50-300 mM).

• Size exclusion chromatography: For higher purity, especially for structural studies, perform SEC using Superdex 200 columns.

The purity can be assessed using SDS-PAGE and Western blotting with antibodies specific to the target protein or the affinity tag .

What functional assays can be used to evaluate the activity of recombinant M. penetrans ATP synthase subunit c?

Researchers can employ several complementary functional assays:

• Reconstitution into liposomes: Incorporate purified recombinant c-subunit into liposomes with other ATP synthase components to measure ATP synthesis/hydrolysis activity.

• Proton translocation assays: Use pH-sensitive fluorescent dyes (ACMA or pyranine) to monitor proton movement across reconstituted proteoliposomes.

• Binding assays with known inhibitors: Assess interaction with specific ATP synthase inhibitors like oligomycin or TMC207 using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) .

• Complementation studies: Test functional activity by complementing ATP synthase-deficient bacterial strains with the recombinant M. penetrans atpE gene.

• Structural integrity assessment: Use circular dichroism (CD) spectroscopy to confirm proper folding of the recombinant protein.

These assays should be validated using appropriate positive and negative controls, including ATP synthase components from well-characterized species.

How can researchers investigate the role of M. penetrans ATP synthase in host-pathogen interactions?

To investigate the role of M. penetrans ATP synthase in host-pathogen interactions, researchers should consider these methodological approaches:

• Generate atpE knockdown or knockout strains using CRISPR-Cas systems adapted for mycoplasmas or transposon mutagenesis.

• Develop specific antibodies against the c-subunit for immunolocalization studies during host cell infection.

• Perform adhesion and invasion assays comparing wild-type and atpE-modified strains using human cell lines relevant to M. penetrans infection (urogenital epithelial cells).

• Assess immune response to recombinant ATP synthase subunit c using serum from patients with confirmed M. penetrans infection, similar to approaches used for M. pneumoniae ATP synthase beta subunit (AtpD) .

• Investigate potential surface exposure of ATP synthase components during infection using cell-surface biotinylation and proteomics approaches.

This multi-faceted approach can provide insights into whether ATP synthase components play roles beyond energy metabolism, possibly contributing to adhesion or immune modulation similar to what has been observed with P35 lipoproteins in M. penetrans .

What are the challenges and solutions for structural studies of recombinant M. penetrans ATP synthase subunit c?

Structural studies of membrane proteins like ATP synthase subunit c present several challenges:

For successful structural determination, researchers should consider:

• X-ray crystallography with specialized crystallization methods for membrane proteins

• Cryo-electron microscopy for the intact c-ring structure

• Solid-state NMR for specific structural elements

• Molecular dynamics simulations based on homology models

These approaches have yielded structural insights for c-subunits from other bacteria and could be adapted for M. penetrans .

What are common issues in recombinant expression of M. penetrans ATP synthase subunit c and how can they be resolved?

Researchers frequently encounter several challenges when expressing membrane proteins like ATP synthase subunit c:

• Toxicity to expression host:

  • Solution: Use tightly regulated expression systems (pBAD, Tet-inducible)

  • Reduce expression temperature to 16-20°C

  • Decrease inducer concentration significantly (0.01-0.1 mM IPTG)

• Inclusion body formation:

  • Solution: Co-express with chaperones (GroEL/ES, DnaK/J)

  • Use solubility-enhancing fusion partners (SUMO, thioredoxin)

  • Optimize cell lysis conditions to prevent aggregation

• Poor membrane integration:

  • Solution: Consider using E. coli strains with enhanced membrane protein expression capabilities

  • Test different detergents for efficient extraction

  • Optimize expression constructs to include proper signal sequences

• Low yield:

  • Solution: Scale-up cultivation (bioreactors rather than shake flasks)

  • Consider codon optimization for expression host

  • Use enriched media formulations (terrific broth or auto-induction media)

Each issue requires systematic optimization based on protein-specific characteristics and experimental objectives .

How can researchers distinguish between native and recombinant M. penetrans ATP synthase subunit c in analytical assays?

To distinguish between native and recombinant proteins in analytical assays, researchers should implement these methodological approaches:

• Epitope tagging: Include distinguishable tags (His, FLAG, etc.) on recombinant proteins that can be detected with specific antibodies.

• Mass spectrometry analysis: Utilize MS to identify peptide mass fingerprints unique to recombinant versus native protein, particularly if modifications were introduced.

• Immunological detection: Develop antibodies that specifically recognize recombinant ATP synthase subunit c but not the native form, targeting unique epitopes created by the expression system.

• Size differentiation: Design recombinant constructs with slightly altered molecular weights that can be resolved by high-resolution SDS-PAGE or native PAGE.

• Functional assays with inhibitors: Engineer inhibitor resistance or sensitivity into recombinant proteins to distinguish their activity from native proteins.

These approaches enable reliable distinction between native and recombinant proteins in complex samples or co-expression systems .

What are the critical factors to consider when designing site-directed mutagenesis studies of M. penetrans ATP synthase subunit c?

For effective site-directed mutagenesis studies of ATP synthase subunit c, researchers should consider:

• Target residue selection:

  • Focus on the conserved ion-binding site (likely Glu61 or equivalent)

  • Target residues at the interface between adjacent c-subunits

  • Investigate species-specific residues that differ from other mycoplasmas

• Mutation strategy:

  • Conservative mutations: Asp for Glu to maintain charge but alter geometry

  • Non-conservative mutations: Ala or Gly substitutions to eliminate side chain function

  • Introduction of reporter residues (Cys) for labeling studies

• Functional assessment:

  • Growth complementation assays in ATP synthase-deficient strains

  • ATP synthesis/hydrolysis activity measurements

  • Proton translocation efficiency

• Structural integrity verification:

  • Circular dichroism to confirm secondary structure maintenance

  • Thermal stability assays to detect destabilizing effects

  • Oligomerization status assessment

Based on studies in mycobacteria, mutations in key residues of ATP synthase subunit c (Asp28→Gly, Asp28→Ala, Leu59→Val, Glu61→Asp, Ala63→Pro, and Ile66→Met) can significantly affect function and drug binding . Similar approaches could yield valuable insights into M. penetrans ATP synthase structure-function relationships.

How can recombinant M. penetrans ATP synthase subunit c be used in serological assays for detecting M. penetrans infections?

Recombinant ATP synthase subunit c can be utilized in serological diagnostic applications following these methodological guidelines:

• ELISA development:

  • Optimize coating conditions for recombinant proteins (typically 1-5 μg/ml in carbonate buffer, pH 9.6)

  • Determine optimal blocking conditions to minimize background

  • Validate with known positive and negative serum samples

  • Establish cutoff values based on ROC curve analysis

• Multiplexing with other antigens:

  • Combine with other M. penetrans antigens like P35 lipoproteins for improved sensitivity

  • Design a panel that distinguishes M. penetrans from other mycoplasma species

  • Consider bead-based multiplex assays for simultaneous detection of multiple antibodies

• Performance assessment:

  • Determine sensitivity, specificity, positive and negative predictive values

  • Compare performance with existing commercial assays

  • Establish cross-reactivity profiles with other mycoplasma species

This approach builds on successful serological assays developed for M. pneumoniae using recombinant ATP synthase beta subunit (AtpD), which demonstrated excellent discrimination between infected patients and healthy subjects .

What is known about the potential of M. penetrans ATP synthase as a therapeutic target?

ATP synthase represents a promising therapeutic target based on several key considerations:

• Essential function: ATP synthase is critical for energy metabolism in mycoplasmas, which have limited metabolic capabilities due to their reduced genome.

• Structural uniqueness: Bacterial ATP synthases differ structurally from human counterparts, potentially allowing for selective targeting.

• Drug development precedent: TMC207 (Bedaquiline) successfully targets mycobacterial ATP synthase subunit c, demonstrating the viability of this approach .

• Accessibility: In mycoplasmas, which lack a cell wall, membrane proteins may be more accessible to drugs.

Research strategies should include:

• High-throughput screening for small molecule inhibitors specific to M. penetrans ATP synthase

• Structure-based drug design targeting the ion-binding site or subunit interfaces

• Repurposing existing ATP synthase inhibitors with modifications for increased specificity

The identified mutations in mycobacterial ATP synthase that confer resistance to TMC207 (at positions Asp28, Leu59, Glu61, Ala63, and Ile66) highlight potential binding sites that could be targeted in M. penetrans as well .

How does M. penetrans ATP synthase compare with ATP synthases from other human pathogens as a potential vaccine candidate?

When evaluating M. penetrans ATP synthase as a vaccine candidate compared to other pathogens, consider:

For vaccine development, researchers should:

• Evaluate the immune response to recombinant ATP synthase subunit c in animal models

• Assess protective efficacy against challenge with M. penetrans

• Identify specific epitopes that are immunodominant and accessible

• Investigate potential for cross-protection against other mycoplasma species

The successful use of M. pneumoniae ATP synthase beta subunit (AtpD) in serological diagnosis suggests that ATP synthase components can be recognized by the immune system during infection, supporting their potential as vaccine candidates .

What advanced imaging techniques are most suitable for studying the localization of ATP synthase in M. penetrans?

For investigating ATP synthase localization in the small (0.2-0.8 μm) and wall-less M. penetrans cells, the following advanced imaging approaches are recommended:

• Super-resolution microscopy:

  • STORM or PALM techniques can achieve 20-30 nm resolution

  • Methodology: Use photoactivatable fluorophores conjugated to anti-ATP synthase antibodies

  • Requires careful sample preparation to preserve membrane integrity

• Cryo-electron tomography:

  • Provides 3D visualization of intact cells at molecular resolution

  • Methodology: Flash-freeze whole M. penetrans cells without fixation, collect tilt series

  • Can be combined with immunogold labeling for specific identification

• Correlative light and electron microscopy (CLEM):

  • Combines fluorescence localization with ultrastructural context

  • Methodology: Label cells with fluorescent antibodies, then process for EM

  • Particularly useful for examining ATP synthase in relation to the specialized tip structure of M. penetrans

• Expansion microscopy:

  • Physically expands specimens while maintaining relative spatial relationships

  • Methodology: Embed samples in expandable polymer, followed by immunolabeling

  • Can achieve effective super-resolution with standard confocal microscopes

These techniques can help determine whether ATP synthase localizes to specific membrane domains in M. penetrans and potentially reveal unexpected non-canonical locations .

How can researchers investigate potential non-canonical functions of ATP synthase subunit c in M. penetrans?

To explore non-canonical functions of ATP synthase subunit c, researchers should implement these methodological approaches:

• Protein-protein interaction studies:

  • BioID or proximity labeling to identify proteins in close proximity to ATP synthase in vivo

  • Co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening using the hydrophilic domains of the c-subunit

• Conditional knockdown/knockout systems:

  • Create conditional expression systems to study phenotypes beyond growth defects

  • Analyze effects on membrane properties, host cell interactions, and stress responses

  • Use time-course studies to distinguish direct from indirect effects

• Host response studies:

  • Examine host cell responses to purified recombinant c-subunit

  • Investigate potential immunomodulatory effects

  • Assess whether c-subunit affects host cell signaling pathways

• Localization during infection:

  • Study dynamic localization during different stages of host cell interaction

  • Use live-cell imaging with minimally disruptive tags

  • Correlate localization changes with functional readouts

This approach could reveal whether ATP synthase subunit c in M. penetrans has evolved additional functions beyond its role in energy metabolism, similar to the dual roles observed for some surface lipoproteins .

What computational approaches are valuable for predicting structure-function relationships in M. penetrans ATP synthase subunit c?

Computational approaches offer powerful tools for studying proteins that are challenging to work with experimentally:

• Homology modeling:

  • Build structural models based on c-subunits from related species

  • Validate models using molecular dynamics simulations in membrane environments

  • Assess conservation of key functional residues

• Molecular dynamics simulations:

  • Simulate behavior of c-ring in lipid bilayers

  • Investigate conformational changes during proton translocation

  • Model interactions with other ATP synthase components

• Evolutionary analysis:

  • Identify conserved versus variable regions through multiple sequence alignments

  • Calculate selection pressures on different domains

  • Detect potential co-evolving residues that maintain functional interactions

• Protein-ligand docking:

  • Predict binding sites for potential inhibitors

  • Virtual screening of compound libraries

  • Structure-based drug design

• Systems biology modeling:

  • Integrate ATP synthase function into whole-cell metabolic models of M. penetrans

  • Predict systemic effects of ATP synthase modifications

  • Model energetic consequences of ATP synthase inhibition

These computational approaches can guide experimental design and help interpret experimental results, particularly for membrane proteins that pose significant technical challenges .