Recombinant Mycoplasma penetrans ATP synthase subunit b (atpF)

What is Mycoplasma penetrans ATP synthase subunit b (atpF) and what is its role in cellular metabolism?

Mycoplasma penetrans ATP synthase subunit b (atpF) is a critical component of the F₀ sector of the F₁F₀ ATP synthase complex. This complex is embedded in the cellular membrane of M. penetrans, serving as the primary machinery for ATP generation through oxidative phosphorylation. The b subunit specifically functions as part of the peripheral stalk that connects the membrane-embedded F₀ sector to the catalytic F₁ sector .

In cellular metabolism, this protein plays a crucial role in:

• Maintaining structural integrity of the ATP synthase complex

• Facilitating the energy conversion process from proton motive force to chemical energy (ATP)

• Potentially contributing to the stability of ATP synthase dimers or oligomers

• Possibly serving as a regulatory point through post-translational modifications

This understanding is supported by comparative studies with other Mycoplasma species and bacteria, where the ATP synthase complex has been more extensively characterized .

How does the structure of M. penetrans atpF compare to other Mycoplasma species?

The ATP synthase b subunit (atpF) in Mycoplasma penetrans shows structural similarities to those found in other Mycoplasma species, while maintaining species-specific characteristics. When comparing with Mycoplasma mobile atpF:

The structure-function relationship of atpF is likely conserved across Mycoplasma species despite sequence variations. Like in M. mobile, the M. penetrans atpF is expected to contain a single transmembrane helix at the N-terminus and an extended α-helical domain that forms part of the peripheral stalk of the ATP synthase complex .

What are the general expression and purification approaches for recombinant M. penetrans atpF?

Based on established protocols for similar proteins, recombinant M. penetrans ATP synthase subunit b can be expressed and purified using the following methodological approach:

• Expression system selection:

  • E. coli is the preferred host for recombinant expression, as demonstrated with M. mobile atpF

  • BL21(DE3) or similar expression strains are commonly used

  • pET vector systems with N-terminal His-tags facilitate purification

• Expression optimization:

  • Induction with 0.5-1.0 mM IPTG at OD₆₀₀ of 0.6-0.8

  • Expression at lower temperatures (16-25°C) often improves solubility

  • Co-expression with chaperones may enhance proper folding

• Purification strategy:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Buffer optimization to maintain protein stability (typically Tris/PBS-based buffer, pH 8.0)

  • Addition of 6% trehalose as a stabilizing agent during lyophilization

  • Consider size exclusion chromatography as a polishing step

• Storage and stabilization:

  • Aliquot and store at -20°C/-80°C to avoid repeated freeze-thaw cycles

  • Consider adding 5-50% glycerol for long-term storage

This approach should yield protein with >90% purity as assessed by SDS-PAGE, suitable for subsequent structural and functional studies .

How do post-translational modifications affect M. penetrans ATP synthase subunit b function?

Post-translational modifications (PTMs) of ATP synthase subunit b in M. penetrans likely serve as critical regulatory mechanisms affecting enzyme complex assembly, stability, and activity. While specific data on M. penetrans atpF modifications is limited, research on homologous proteins provides valuable insights:

Phosphorylation appears to be a significant regulatory PTM for ATP synthase subunits. Studies on the β subunit of F₁F₀ ATP synthase have identified specific phosphorylation sites with distinct functional consequences :

For M. penetrans atpF specifically, several potential regulatory modifications may occur:

• Phosphorylation: Likely occurs at conserved threonine/serine residues in the stalk region

• ADP-ribosylation: M. penetrans possesses ADP-ribosyltransferases (like MYPE9110) that could potentially modify ATP synthase components

• Other modifications: Acetylation, methylation, or oxidative modifications may play roles in regulating ATP synthase during infection cycles

These modifications could serve as adaptation mechanisms for M. penetrans to regulate energy metabolism in response to changing host environments or immune responses .

What experimental approaches can identify interaction partners of M. penetrans atpF?

To comprehensively identify and characterize the protein interaction network of M. penetrans ATP synthase subunit b (atpF), researchers should employ multiple complementary techniques:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Express recombinant His-tagged atpF as bait protein

  • Perform pull-down experiments using M. penetrans cell lysates

  • Analyze co-purified proteins by LC-MS/MS

  • Validate interactions using reverse pull-downs with identified partners

• Crosslinking mass spectrometry (XL-MS):

  • Apply chemical crosslinkers (DSS, BS³, or EDC) to intact M. penetrans cells or isolated membrane fractions

  • Enrich for ATP synthase complexes through purification

  • Identify crosslinked peptides by specialized MS analysis

  • Map interaction sites at amino acid resolution

• Bacterial two-hybrid (B2H) system:

  • Create fusion constructs of atpF with B2H system components

  • Screen against library of M. penetrans proteins

  • Quantify interaction strengths through reporter gene expression

• Surface plasmon resonance (SPR) validation:

  • Immobilize purified recombinant atpF on SPR chip

  • Measure binding kinetics with candidate interactors

  • Determine association/dissociation constants

Analysis should focus on:

• ATP synthase complex components (α, β, γ, δ, ε, a, c subunits)

• Membrane scaffold proteins

• Potential regulatory proteins

• Host cell proteins that may interact during infection

This multi-technique approach would provide a high-confidence interaction map to understand the structural organization and regulatory mechanisms of ATP synthase in M. penetrans.

How might structural variations in M. penetrans atpF contribute to antimicrobial resistance mechanisms?

Structural variations in M. penetrans ATP synthase subunit b (atpF) could significantly contribute to antimicrobial resistance through several mechanisms, particularly against drugs that target ATP synthase:

• Altered binding site architecture:

  • Amino acid substitutions in regions that form part of inhibitor binding pockets

  • Conformational changes that reduce drug accessibility to binding sites

  • Modified electrostatic surface properties affecting drug affinity

• Impact on ATP synthase assembly and stability:

  • Variations that enhance complex stability under drug pressure

  • Alterations that maintain minimal functional assembly despite partial inhibition

  • Changes that enable complex function with fewer subunits

• Comparative structural analysis:
  Key differences observed between susceptible and resistant Mycoplasma species:

  Structural Feature	Drug-Susceptible Mycoplasmas	Resistant Variants
  c-ring composition	Higher number of c-subunits	Reduced number of c-subunits
  Peripheral stalk	Standard flexibility	Enhanced rigidity maintaining function
  Interface regions	Conserved residues at subunit interfaces	Substitutions that maintain assembly while reducing drug binding

• Modulatory mechanisms:

  • Expression of variant atpF isoforms under drug pressure

  • Compensatory mutations in partner subunits

  • Modified interactions with membrane lipids affecting drug access

This understanding has implications for drug design targeting ATP synthase in Mycoplasma species, similar to how bedaquiline targets ATP synthase in Mycobacteria . Structural knowledge of these variations is essential for developing new antimicrobials that can overcome resistance mechanisms.

What are the optimal conditions for functional assays of recombinant M. penetrans atpF?

To properly assess the functionality of recombinant M. penetrans ATP synthase subunit b (atpF), researchers should implement both isolated protein assays and reconstitution experiments under the following optimized conditions:

A. ATP Synthase Complex Assembly Assays:

• Subunit reconstitution protocol:

  • Buffer: 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl₂

  • Membrane mimetic: 0.05% n-dodecyl β-D-maltoside (DDM) or nanodiscs

  • Temperature: 30°C for assembly (physiologically relevant for Mycoplasma)

  • Time: 60-minute incubation for complex formation

  • Component ratio: 1:1 stoichiometric mix of recombinant subunits

• Assembly verification:

  • Blue Native PAGE to confirm complex formation

  • Size exclusion chromatography to isolate fully assembled complexes

  • Negative-stain electron microscopy for structural validation

B. Functional Activity Measurements:

C. Interaction Analysis with Partner Subunits:

• Isothermal titration calorimetry (ITC) to determine binding constants

• Conditions: 25°C, pH 7.5, low salt (50-100 mM NaCl)

• Expected Kᴅ values in nanomolar range for cognate partners

Note that M. penetrans ATP synthase shows optimal activity at 37°C, reflecting its adaptation to the human host environment, and may require specific lipid compositions (typically including cardiolipin) for maximal activity when reconstituted in liposomes .

How can researchers effectively analyze the membrane integration of M. penetrans atpF?

For comprehensive analysis of membrane integration of M. penetrans ATP synthase subunit b (atpF), researchers should implement a multi-technique approach focusing on both structural characterization and functional assessment:

• Membrane topology mapping:

  • Cysteine-scanning mutagenesis: Introduce single cysteine residues throughout the protein sequence and probe accessibility with membrane-impermeable/permeable reagents

  • Protease protection assays: Treat membrane vesicles containing atpF with proteases, then map protected fragments by mass spectrometry

  • Fluorescence quenching: Attach environment-sensitive fluorophores at strategic positions to detect membrane-embedded regions

• Reconstitution systems for functional assessment:

  Reconstitution System	Preparation Method	Applications	Advantages
  Liposomes	Extrusion or sonication of lipid mixtures with purified atpF	Function and orientation studies	Controlled lipid composition
  Nanodiscs	Assembly with MSP proteins and lipids	Structural studies, single-molecule measurements	Defined size, enhanced stability
  Bicelles	Mixture of long-chain and short-chain lipids	NMR studies	Compatible with solution NMR
  Giant unilamellar vesicles (GUVs)	Electroformation	Microscopy studies, lateral organization	Visible by optical microscopy

• Biophysical characterization methods:

  • Solid-state NMR: Determine secondary structure in membrane environment

  • ATR-FTIR spectroscopy: Assess secondary structure and orientation in membranes

  • Oriented circular dichroism: Measure helix tilt angles relative to membrane normal

  • Atomic force microscopy: Visualize membrane-integrated complexes

• Molecular dynamics simulations:

  • Simulate integration of atpF into lipid bilayers

  • Calculate energetics of membrane insertion

  • Predict protein-lipid interactions

When implementing these approaches, researchers should consider M. penetrans' unique membrane composition, which contains a high proportion of sterols unlike most bacteria. The optimal lipid mixture for reconstitution should include cholesterol and Mycoplasma-like phospholipid ratios to accurately mimic the native environment .

What are the current challenges in producing high-yield, properly folded recombinant M. penetrans atpF?

Producing high-yield, properly folded recombinant M. penetrans ATP synthase subunit b (atpF) presents several technical challenges that researchers must address through methodological innovations:

• Expression challenges and solutions:

  Challenge	Underlying Cause	Methodological Solution
  Low solubility	Hydrophobic membrane-spanning domain	Fusion with solubility enhancers (MBP, SUMO, Trx) with cleavable linkers
  Inclusion body formation	Improper folding in E. coli cytoplasm	Lower expression temperature (16°C), weaker promoters, or specialized strains (C41/C43)
  Proteolytic degradation	Recognition by host proteases	Co-expression with chaperones (GroEL/ES), addition of protease inhibitors
  Codon bias	Different codon usage between M. penetrans and E. coli	Codon optimization or use of RIL/RP strains supplying rare tRNAs

• Refolding strategies for inclusion bodies:

  • Gradual dialysis from denaturing conditions (8M urea or 6M guanidine-HCl)

  • On-column refolding during IMAC purification

  • Pulsed dilution refolding with chaperone assistance

  • Addition of membrane mimetics during refolding (detergents, liposomes)

• Alternative expression systems:

  • Cell-free expression systems with added nanodiscs or liposomes

  • Specialized E. coli strains designed for membrane protein expression

  • Bacillus subtilis as a Gram-positive expression host

  • Eukaryotic systems for complex membrane proteins

• Quality control assessments:

  • Circular dichroism to confirm secondary structure content

  • Thermal shift assays to evaluate stability

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) for oligomeric state

  • Limited proteolysis to verify folding integrity

Based on experiences with M. mobile atpF, successful expression typically requires a Tris/PBS-based buffer at pH 8.0 supplemented with 6% trehalose as a stabilizing agent. Once purified, the protein should be stored at -20°C/-80°C with addition of 5-50% glycerol to prevent degradation during freeze-thaw cycles . Implementing these strategies can significantly improve yields of functional protein for structural and biochemical studies.

How does the immunogenicity of M. penetrans atpF compare to other ATP synthase subunits as potential diagnostic markers?

The immunogenicity of M. penetrans ATP synthase subunit b (atpF) can be comparatively analyzed against other ATP synthase subunits for diagnostic applications, revealing important considerations for researchers developing serological tests:

• Comparative immunogenicity profile:

  ATP Synthase Subunit	Immunogenicity Characteristics	Diagnostic Potential	Cross-Reactivity Risk
  Subunit b (atpF)	Moderate immunogenicity, membrane-associated	Good for specific detection	Low-moderate cross-reactivity
  Beta subunit (AtpD)	High immunogenicity, highly conserved	Excellent for early detection	Higher cross-reactivity risk
  Alpha subunit (AtpA)	High immunogenicity, conserved	Good for early detection	Moderate cross-reactivity
  Gamma subunit (AtpG)	Lower immunogenicity, internal location	Limited as single marker	Lower cross-reactivity

• Serological performance factors:

  • The beta subunit (AtpD) has demonstrated excellent performance in M. pneumoniae diagnosis, particularly for IgM detection in early infection stages

  • AtpD combined with adhesin proteins (like P1) significantly improves diagnostic accuracy through multi-antigen approaches

  • Based on similar principles, M. penetrans atpF would likely work best as part of a multi-antigen panel rather than as a standalone diagnostic marker

• Species-specific considerations:

  • M. penetrans has unique surface antigenic variations through the P35 family proteins that may affect diagnostic strategies

  • Higher antibody prevalence (40%) against M. penetrans has been reported in HIV-infected AIDS patients compared to control groups (0.3%)

  • ATP synthase subunits may provide more consistent detection across variant strains due to their conserved nature compared to variable surface antigens

• Optimization strategies:

  • Using specific, less conserved regions of atpF to reduce cross-reactivity

  • Developing recombinant fusion proteins combining atpF with species-specific antigens

  • Employing binary logistic regression analysis for optimal antigen combinations

These comparative insights suggest that while AtpD has proven diagnostic value in M. pneumoniae , M. penetrans atpF could serve as a complementary diagnostic marker in multi-antigen panels, potentially improving sensitivity and specificity for detecting M. penetrans infections, particularly in immunocompromised populations.

What structural and functional differences exist between ATP synthase b subunits across different Mycoplasma species?

ATP synthase b subunits across Mycoplasma species exhibit both conserved features essential for core functions and species-specific adaptations reflecting ecological niches and host interactions:

• Structural comparison across Mycoplasma species:

  Feature	M. penetrans	M. mobile	M. pneumoniae	Functional Significance
  N-terminal domain	Hydrophobic membrane anchor	Hydrophobic region (MLEL...LTKL)	Similar membrane anchor	Membrane integration and anchoring
  C-terminal domain	α-helical coiled-coil structure	α-helical region with charged residues	Similar coiled-coil structure	Forms peripheral stalk with δ-subunit
  Length	~180-200 aa (estimated)	184 aa	~170-190 aa	Species-specific adaptations
  Oligomeric state	Likely dimer in complex	Dimer within complex	Dimer within complex	Required for peripheral stalk formation
  Surface-exposed regions	Unique epitopes	Species-specific regions	Immunogenic regions	Potential diagnostic targets

• Sequence conservation analysis:

  • Highest conservation in C-terminal region (stalk formation domain)

  • Moderate conservation in transmembrane domain

  • Lowest conservation in linker regions

  • The conservation pattern suggests differential selective pressures on functional domains

• Species-specific functional adaptations:

  • M. penetrans may have adaptations related to urogenital tract colonization

  • M. pneumoniae shows adaptations for respiratory tract infection

  • M. mobile exhibits adaptations for enhanced motility

  • These adaptations could manifest as subtle structural differences in peripheral stalk properties

• Implications for ATP synthase function:

  • Differences in b-subunit may affect efficiency of ATP synthesis

  • Species variations could influence proton/ATP ratios

  • Structural differences might affect stability under different physiological conditions

  • These variations could contribute to host-specific metabolic adaptations

The differences in ATP synthase b subunits across Mycoplasma species reflect evolutionary adaptations to diverse host environments while maintaining the core structural features necessary for ATP synthase function. These variations provide insights into species-specific energy metabolism strategies and potential targets for species-specific detection or inhibition .

How can researchers use evolutionary conservation analysis of atpF to identify functional domains?

Researchers can leverage evolutionary conservation analysis of ATP synthase subunit b (atpF) to identify functionally critical domains through a systematic bioinformatic and experimental approach:

• Sequence-based conservation analysis methodology:

  Analysis Technique	Implementation Approach	Expected Outcomes	Tools/Resources
  Multiple Sequence Alignment (MSA)	Align atpF sequences from diverse Mycoplasma species and related bacteria	Identification of highly conserved motifs	MUSCLE, CLUSTALW, T-COFFEE
  Conservation scoring	Calculate per-residue conservation scores from MSA	Quantitative conservation metrics	ConSurf, Rate4Site, AL2CO
  Evolutionary trace method	Map conservation patterns onto phylogenetic trees	Identification of clade-specific conservation	ETC, MINER
  Coevolution analysis	Identify co-evolving residue pairs	Detection of functional coupling between residues	PSICOV, DCA, EVcouplings

• Structural mapping of conservation:

  • Map conservation scores onto homology models or experimental structures

  • Identify spatial clusters of conserved residues (functional hotspots)

  • Analyze conservation at protein-protein interfaces

  • Distinguish surface versus core conservation patterns

• Functional domain prediction based on conservation patterns:

  Conservation Pattern	Likely Functional Significance	Validation Approach
  Highly conserved N-terminal hydrophobic region	Membrane anchoring domain	Mutagenesis + membrane association assays
  Conserved charged residues in C-terminal region	Interaction with δ-subunit	Pull-down assays with partner subunits
  Conserved glycine residues	Conformational flexibility points	Limited proteolysis, molecular dynamics
  Variable surface-exposed loops	Species-specific interactions	Antibody accessibility studies

• Experimental validation strategies:

  • Site-directed mutagenesis of conserved residues followed by functional assays

  • Domain swapping between species to test functional conservation

  • Hydrogen-deuterium exchange mass spectrometry to map structural dynamics

  • Crosslinking studies targeting predicted interaction interfaces

By integrating these computational predictions with experimental validation, researchers can identify:

• Essential residues for ATP synthase assembly and function

• Species-specific adaptations in peripheral stalk formation

• Potential sites for targeted inhibition

• Regions suitable for diagnostic antibody development

This evolutionary approach provides a powerful framework for understanding structure-function relationships in M. penetrans atpF and guides rational experimental design for further characterization.

What emerging techniques could advance structural studies of M. penetrans ATP synthase complexes?

Several cutting-edge techniques are poised to revolutionize our understanding of M. penetrans ATP synthase structure and dynamics:

• Advanced cryo-electron microscopy approaches:

  Technique	Application to M. penetrans ATP Synthase	Expected Resolution/Outcome	Technical Advantages
  Single-particle cryo-EM	Whole complex structure determination	2.5-3.5 Å resolution	No crystallization required
  Cryo-electron tomography	In situ visualization in membrane context	10-20 Å resolution	Native cellular environment
  Time-resolved cryo-EM	Capture ATP synthase conformational states	Multiple structural snapshots	Visualize rotary catalysis
  Microcrystal electron diffraction	Structure of individual subunits or subcomplexes	1.5-2.5 Å resolution	Works with very small crystals

• Integrative structural biology approaches:

  • Combining cryo-EM with mass spectrometry and computational modeling

  • Cross-linking mass spectrometry to map subunit interactions

  • Integrative modeling platforms to synthesize multiple data types

  • AlphaFold2 and RoseTTAFold predictions validated by experimental data

• Dynamic structural techniques:

  • Single-molecule FRET to track conformational changes during catalysis

  • High-speed atomic force microscopy to visualize rotational dynamics

  • Nuclear magnetic resonance (NMR) to characterize flexible regions

  • Molecular dynamics simulations at extended timescales using specialized hardware

• In situ structural approaches:

  • Correlative light and electron microscopy (CLEM) of labeled ATP synthase

  • Focused ion beam milling combined with cryo-ET for cellular tomography

  • In-cell NMR to study conformational states in living cells

  • Expansion microscopy combined with super-resolution techniques

These emerging methods would help resolve key questions about M. penetrans ATP synthase, including:

• The exact stoichiometry and arrangement of F₀ subunits

• Conformational changes during proton translocation

• Species-specific structural adaptations

• Interactions with other cellular components or potential host factors

The integration of these techniques would provide unprecedented insights into ATP synthase structure and function in M. penetrans, potentially revealing unique features that could be exploited for therapeutic development.

How might M. penetrans atpF be utilized in development of novel antimicrobial strategies?

M. penetrans ATP synthase subunit b (atpF) presents several promising avenues for novel antimicrobial development, based on its essential role in energy metabolism and structural insights:

• Structure-based inhibitor design strategies:

  Target Site	Rationale	Potential Inhibitor Classes	Expected Outcome
  b-δ subunit interface	Disrupts peripheral stalk assembly	Peptide mimetics, small molecules	ATP synthase destabilization
  b-a subunit interface	Interferes with proton channel formation	Interface-binding compounds	Uncoupling of proton flow
  b-subunit dimerization	Prevents proper stalk formation	Helix disruptors, stapled peptides	Complex assembly inhibition
  Membrane-integration domain	Alters membrane anchoring	Lipophilic compounds	Disrupted energy coupling

• Therapeutic exploitation of species-specific features:

  • Targeting unique sequence regions in M. penetrans atpF not present in human F₁F₀

  • Developing inhibitors that selectively recognize Mycoplasma ATP synthase conformations

  • Exploiting differences in lipid environments between host and pathogen ATP synthases

  • Utilizing the successful bedaquiline model from Mycobacteria as a conceptual framework

• Innovative therapeutic modalities:

  • PROTAC-based degradation of ATP synthase components

  • mRNA-targeting approaches against atpF expression

  • Engineered antibodies or nanobodies targeting exposed epitopes

  • Photodynamic therapy using ATP synthase-targeted photosensitizers

• Combination strategies to enhance efficacy:

  Approach	Mechanism	Potential Synergies	Resistance Barrier
  Dual-targeting ATP synthase components	Simultaneous inhibition of multiple subunits	Enhanced potency, reduced resistance	Higher genetic barrier
  ATP synthase + glycolysis inhibition	Energy production blockade	Metabolic catastrophe	Multiple adaptations required
  ATP synthase inhibition + membrane permeabilizers	Enhanced drug access	Lower MICs, increased efficacy	Addresses permeability barriers
  Host-directed + pathogen-directed therapy	Multiple pressure points	Reduced selective pressure	Complex resistance mechanisms needed

These approaches could yield significant benefits for treating M. penetrans infections, which are particularly relevant in immunocompromised populations such as HIV-infected individuals where M. penetrans has been implicated as a potential cofactor in disease progression . The high prevalence of antibodies against M. penetrans (40%) in HIV-infected AIDS patients compared to control groups (0.3%) underscores the clinical relevance of developing such targeted therapies .

What are the most significant knowledge gaps in M. penetrans atpF research?

Despite advances in understanding ATP synthase structure and function across species, significant knowledge gaps remain specific to M. penetrans atpF research that require targeted investigation:

• Fundamental structural characterization gaps:

  • No high-resolution structure of M. penetrans ATP synthase or its subunits

  • Limited understanding of species-specific structural adaptations

  • Incomplete knowledge of post-translational modifications in vivo

  • Undefined oligomeric state and organization in native membranes

• Functional characterization limitations:

  • Undefined kinetic parameters for ATP synthesis/hydrolysis specific to M. penetrans

  • Limited understanding of coupling efficiency between proton transport and ATP synthesis

  • Unknown regulatory mechanisms controlling ATP synthase activity during infection

  • Unclear metabolic flexibility and adaptations under varying host conditions

• Host-pathogen interaction unknowns:

  Research Area	Knowledge Gap	Significance	Methodological Challenges
  Role in pathogenesis	How ATP synthase activity relates to virulence	Potential therapeutic target	Requires in vivo infection models
  Immune recognition	Whether atpF is recognized by host immune system	Diagnostic potential	Limited patient samples/reagents
  Adaptation mechanisms	How ATP synthesis adapts during host colonization	Survival strategies	Difficulty of in vivo measurements
  Co-evolution patterns	Evolution of ATP synthase with host interactions	Adaptation principles	Complex evolutionary analyses

• Technical challenges impeding progress:

  • Difficulties in recombinant expression of membrane proteins

  • Limited availability of M. penetrans clinical isolates

  • Challenges in creating genetic manipulation systems for M. penetrans

  • Limited animal models for studying M. penetrans infections

• Translational research gaps:

  • Undefined potential of atpF as diagnostic biomarker compared to established markers

  • Limited development of specific inhibitors targeting M. penetrans ATP synthase

  • Unclear relationship between ATP synthase function and antibiotic susceptibility

  • Unknown contributions to persistence in immunocompromised hosts

Addressing these knowledge gaps would significantly advance understanding of M. penetrans biology and pathogenesis, while potentially yielding new diagnostic and therapeutic approaches, particularly relevant for immunocompromised populations where M. penetrans infections may contribute to disease progression .

What collaborative research approaches could accelerate progress in understanding M. penetrans atpF?

To overcome current limitations in M. penetrans atpF research, strategic interdisciplinary collaborations and resource-sharing initiatives could significantly accelerate progress:

• Integrated multi-omics research consortia:

  Collaborative Approach	Contributing Disciplines	Expected Outcomes	Implementation Strategy
  Structural biology network	Cryo-EM, X-ray crystallography, NMR, computational modeling	Comprehensive structural characterization	Shared access to high-end equipment
  Functional genomics collaboration	Transcriptomics, proteomics, metabolomics, bioinformatics	Systems-level understanding of ATP synthase regulation	Standardized experimental protocols
  Clinical microbiology partnership	Clinical isolate collection, antimicrobial susceptibility, host response	Translational insights	Biobanking and data sharing
  Synthetic biology alliance	Protein engineering, genetic tool development, metabolic engineering	New manipulation tools for mycoplasmas	Open-source technology sharing

• Technology development collaborations:

  • Engineering partnerships to develop specialized equipment for Mycoplasma research

  • Computational biology collaborations for specialized analysis pipelines

  • Antibody development consortia creating specific reagents for M. penetrans proteins

  • Microfluidics collaborations for single-cell analysis of heterogeneous populations

• Cross-species comparative research initiatives:

  • Systematic comparison of ATP synthase structure/function across Mycoplasma species

  • Evolutionary biology collaborations to understand host adaptation mechanisms

  • Comparative biochemistry of ATP synthases from diverse pathogens

  • Joint studies on antimicrobial resistance mechanisms across species

• Open science frameworks to accelerate progress:

  • Shared biorepositories of M. penetrans strains and recombinant proteins

  • Open access databases for structural and functional data

  • Collaborative electronic lab notebooks and protocols

  • Pre-registration of experimental designs to avoid duplication

• Translational research networks:

  • Academic-industry partnerships for inhibitor development

  • Diagnostic development collaborations with clinical laboratories

  • Regulatory science partnerships to accelerate translation

  • Patient advocacy involvement to guide research priorities