Recombinant Mycobacterium avium ATP synthase subunit beta (atpD)

What is the function of ATP synthase subunit beta (atpD) in Mycobacterium avium?

ATP synthase subunit beta (atpD) is a critical component of the F1F0-ATP synthase complex in M. avium, playing a central role in energy metabolism. This protein forms part of the catalytic core (F1) of the ATP synthase complex, which synthesizes ATP from ADP and inorganic phosphate using the proton gradient established across the bacterial membrane. In M. avium, this process is particularly important for survival in various environmental conditions, including during infection. Current research indicates that atpD functions within the ATP synthesis pathway connected to the TCA cycle, where acetaldehyde can be converted to acetyl-CoA, acetyl-phosphate, and pyruvate for ATP production . The expression and activity of atpD may be regulated differently under stress conditions such as biofilm formation or hypoxia, enabling the bacterium to adapt its energy metabolism to challenging environments.

How is atpD conserved across different Mycobacterium species?

The atpD gene is highly conserved across mycobacterial species due to its essential function in energy metabolism. While specific conservation data for atpD isn't directly mentioned in the provided resources, the fundamental role of ATP synthase in bacterial survival suggests strong evolutionary constraints. To study atpD conservation, researchers should employ comparative genomic analyses of various mycobacterial species, including M. avium, M. tuberculosis, and M. abscessus. Proteogenomic analyses, as demonstrated in studies that identified 2954 proteins in M. avium , provide valuable methodologies for comparing atpD sequences and expression levels across different mycobacterial species. When conducting such comparative studies, researchers should focus on:

• Sequence identity and similarity percentages

• Conservation of functional domains versus variable regions

• Potential species-specific adaptations in non-catalytic regions

• Correlations between sequence variations and ecological niches or pathogenicity

What methodologies can be used to study the structure-function relationship of M. avium atpD?

Investigating the structure-function relationship of M. avium atpD requires a multi-faceted approach:

• Structural determination methods:

  • X-ray crystallography of purified recombinant protein

  • Cryo-electron microscopy for visualizing the protein in the context of the ATP synthase complex

  • Homology modeling based on related ATP synthase structures

• Functional domain analysis:

  • Site-directed mutagenesis of predicted functional residues

  • Truncation studies to identify essential regions

  • Chimeric proteins with other mycobacterial atpD subunits

• Protein interaction studies:

  • Co-immunoprecipitation with other ATP synthase subunits

  • Surface plasmon resonance to measure binding kinetics

  • Cross-linking studies followed by mass spectrometry

Proteogenomic analyses have proven valuable for confirming the precise amino acid sequence of M. avium proteins, with studies identifying novel peptide sequences and correcting translation start sites for multiple proteins . These approaches are essential for accurate structural studies of atpD and understanding how its structure relates to ATP synthesis function in different environmental conditions.

What are the optimal conditions for expressing and purifying recombinant M. avium atpD?

Successful expression and purification of functional recombinant M. avium atpD requires careful optimization of multiple parameters:

Expression Systems:

• E. coli-based systems: BL21(DE3) strains with pET vectors containing T7 promoters

• Mycobacterial expression systems: M. smegmatis mc²155 for native-like post-translational modifications

Culture Conditions:

• For E. coli: LB or 2xYT media with appropriate antibiotics

• For mycobacterial hosts: Middlebrook 7H9 medium supplemented with 10% albumin-dextrose-catalase (ADC) and 0.05% Tween 80

Induction Parameters:

• IPTG concentration: 0.1-1.0 mM (optimize to minimize inclusion body formation)

• Temperature: 16-25°C during induction phase

• Duration: 4-16 hours (monitor by SDS-PAGE)

Purification Strategy:

• Affinity chromatography: His-tag or GST-tag

• Ion exchange chromatography: To remove contaminants

• Size exclusion chromatography: For final polishing and confirmation of oligomeric state

Buffer Optimization:

• pH range: 7.0-8.0

• Salt concentration: 150-300 mM NaCl

• Stabilizing agents: 10% glycerol, 1-5 mM DTT or β-mercaptoethanol

The methodologies used for growing M. avium under various conditions in the DosR regulon studies provide valuable insights for culturing conditions that might enhance recombinant protein expression and stability.

How can researchers validate the functionality of recombinant atpD protein?

Validating the functionality of recombinant M. avium atpD requires a combination of biochemical, biophysical, and genetic approaches:

Enzymatic Activity Assays:

• ATP Hydrolysis Assay:

  • Colorimetric measurement of inorganic phosphate release

  • Luminescence-based ATP consumption assays

  • Activity measurement under varying pH and temperature conditions

• ATP Synthesis Assay:

  • Reconstitution in liposomes with artificial proton gradients

  • Luciferase-based detection of ATP production

Structural Integrity Verification:

• Circular Dichroism Spectroscopy:

  • Secondary structure analysis

  • Thermal stability assessment

• Size Exclusion Chromatography:

  • Oligomeric state determination

  • Detection of proper complex formation with other subunits

Interaction Studies:

• Co-immunoprecipitation:

  • Verification of interactions with other ATP synthase subunits

  • Pull-down assays with tagged recombinant protein

• Surface Plasmon Resonance:

  • Binding kinetics with nucleotides (ATP, ADP)

  • Interaction with potential inhibitors

For mycobacterial-specific validation, the data-dependent and data-independent acquisition methods used in proteomic analysis of M. avium would help confirm protein identity and detect potential post-translational modifications that might affect function.

What approaches are recommended for studying atpD expression under different environmental conditions?

To comprehensively study atpD expression under various environmental conditions relevant to M. avium pathophysiology, researchers should employ the following methodologies:

Transcriptional Analysis:

• RT-qPCR: As demonstrated in research on the DosR regulon, where expression of various genes was measured under microaerophilic conditions and normalized to sigA expression

• RNA-sequencing: For genome-wide transcriptional profiling, similar to the approach used to study the transcriptional response to oxygen depletion

Protein Expression Analysis:

• Western Blotting: With specific antibodies against atpD

• Proteomics Approaches: Data-dependent and data-independent acquisition methods as described in proteogenomic investigations that identified thousands of M. avium proteins

• Mass Spectrometry-Based Quantification: Label-free or isotope-labeled quantification methods

Environmental Condition Models:

• Oxygen Limitation Models:

  • Microaerophilic conditions: Using standing T25 vented tissue culture flasks

  • Hypoxic conditions: Wayne model with tightly sealed tubes containing methylene blue as oxygen depletion indicator

• Biofilm Models: As referenced in metabolic pathway studies comparing biofilm and aerobic conditions

• Chemical Stress Models:

  • Nitric oxide stress: Using NO donors like DETA/NO (500 μM)

  • pH variation: Testing at different pH levels (pH 7.3 versus pH 5.7)

Data Analysis Approaches:

• Differential expression analysis comparing log2-fold changes across conditions

• Pathway analysis integrating expression data into metabolic network models

• Correlation analysis between atpD expression and bacterial survival or growth rates

How does atpD expression and function change under hypoxic conditions in M. avium?

While the provided research doesn't directly address atpD expression under hypoxic conditions, several insights can be gleaned from studies on M. avium adaptation to oxygen limitation:

M. avium grown under progressive microaerophilic conditions activates more than 4-fold a subset of 16 genes, with 13 dependent on the two-component system regulator DosRS . Given that ATP synthesis through oxidative phosphorylation requires oxygen as the final electron acceptor, atpD regulation likely plays a crucial role in hypoxic adaptation.

Research shows that "M. avium central metabolism leads to the production of acetoin under aerobic conditions and lactic acid mainly under anaerobic conditions" . This metabolic shift would necessitate adaptations in ATP production mechanisms, potentially involving changes in atpD expression or ATP synthase activity regulation through post-translational modifications.

The DosRS system plays a critical role in hypoxic adaptation, as "Loss of dosRS expression in M. avium led to a significant reduction in viability under hypoxia that was more marked at acidic than at neutral pH" . While atpD isn't explicitly identified among the DosR regulon genes, the energy metabolism adjustments required for hypoxic survival would logically involve ATP synthase regulation.

Recommended Research Approach:

• Compare atpD transcript and protein levels between aerobic, microaerophilic, and hypoxic conditions using RT-qPCR and proteomics

• Measure ATP synthase activity in membrane fractions isolated from bacteria grown under varying oxygen tensions

• Investigate potential post-translational modifications of atpD under hypoxia

• Create conditional atpD expression mutants to assess its importance for hypoxic survival

What role does atpD play in M. avium biofilm formation and maintenance?

Biofilm formation represents a significant adaptation strategy for M. avium that likely involves specific regulation of energy metabolism and ATP synthase activity:

Proteomic analysis reveals that "M. avium proteome in biofilm and aerobic conditions showed metabolic pathways implicated in fatty acid metabolism, and biosynthesis of amino acid and cofactors" . This metabolic reprogramming during biofilm formation would affect energy requirements and potentially ATP synthase regulation.

The research identified several proteins overproduced under biofilm conditions, including "MAV_0357 haloalkane dehalogenase, MAV_0039 putative acyl-CoA dehydrogenase, MAV_4265 aldehyde dehydrogenase (NAD) family protein, MAV_4069 KatE catalase HPII and MAV_0927 conserved hypothetical protein" . These changes reflect metabolic adaptations that would influence energy demands and ATP synthase activity.

The study notes that "acetaldehyde is the product of chloroalkane pathway which reversibly can be converted into an acetyl-CoA, acetyl-phosphate and pyruvate, and then processed in the TCA cycle for ATP synthesis" . This suggests active ATP synthesis pathways during biofilm conditions, involving atpD functionality.

Experimental Approaches to Study atpD's Role in Biofilm Formation:

• Compare atpD expression between planktonic and biofilm growth phases using RT-qPCR and proteomics

• Create atpD conditional expression mutants to assess impact on biofilm initiation and maturation

• Perform ATP measurements in biofilm versus planktonic cells at different developmental stages

• Evaluate the effects of ATP synthase inhibitors on biofilm formation and stability

• Visualize atpD localization within biofilm structures using fluorescent protein fusions or immunofluorescence

How can proteogenomic approaches enhance our understanding of atpD variants in M. avium strains with different virulence profiles?

Proteogenomic approaches offer powerful tools for characterizing atpD variations across M. avium strains and correlating these with functional differences:

Recent proteogenomic analysis of M. avium employed "both data-dependent and data-independent acquisition methods" and conducted investigations "using (i) a protein database for Mycobacterium tuberculosis, (ii) an M. avium genome six-frame–translated database, and (iii) a variant protein database of M. avium" . This comprehensive approach identified 2954 proteins and 1301 novel peptide sequences, and corrected translation start sites for 15 proteins .

When applied specifically to atpD, similar proteogenomic strategies could:

Identify Novel atpD Variants:

• Search mass spectrometry data against genome six-frame translations to detect novel protein forms

• Identify single amino acid variants through database searches against variant databases

• Correlate variants with strain virulence properties

Characterize Post-Translational Modifications:

• Identify phosphorylation, acetylation, or other regulatory modifications

• Quantify modification stoichiometry under different conditions

• Determine how modifications affect ATP synthase function

Study Strain-Specific Variations:

• Compare atpD sequences across clinical and environmental isolates

• Correlate genetic variations with functional differences in ATP production

• Identify potential virulence-associated variants

The creation of a spectral library, as described in the proteogenomic study which included "29,033 peptide precursors supported by 0.4 million fragment ions" , provides a valuable resource for consistently identifying and quantifying atpD peptides across different studies and laboratories.

How does atpD contribute to M. avium virulence and pathogenicity?

The contribution of atpD to M. avium virulence can be inferred from its essential role in bacterial energy metabolism and the bacterium's adaptation to host environments:

During infection, "M. avium is likely exposed to a variety of stressors, including hypoxic conditions inside activated macrophages and in the avascular necrotic regions of granulomas" . ATP synthase activity, mediated by atpD, would be critical for generating energy under these challenging conditions.

The metabolic flexibility of M. avium, where "acetaldehyde is the product of chloroalkane pathway which reversibly can be converted into an acetyl-CoA, acetyl-phosphate and pyruvate, and then processed in the TCA cycle for ATP synthesis" , likely contributes to its ability to survive in diverse host environments. This metabolic adaptability requires functional ATP synthase with properly regulated atpD.

Research on genetic variations between M. avium strains has identified "unique genetic features" that could be used to "trace the putative transmission route via their host" . If such variations occur in metabolic genes including atpD, they might influence virulence through altered energy metabolism efficiency.

Methodological Approaches to Study atpD's Role in Virulence:

• Create atpD conditional expression mutants and assess survival in macrophage infection models

• Compare atpD sequence and expression between clinical isolates with different virulence profiles

• Evaluate ATP production capacity of different strains during macrophage infection

• Test the effects of ATP synthase inhibitors on M. avium growth and virulence in infection models

What is the potential of atpD as a target for anti-mycobacterial drug development?

ATP synthase represents a validated drug target in mycobacteria, as demonstrated by bedaquiline (which targets the c subunit of ATP synthase) against M. tuberculosis. The potential of atpD as a specific target can be evaluated based on several factors:

Target Validation Considerations:

• Essentiality: ATP synthase is essential for energy metabolism, particularly in the context of the "TCA cycle for ATP synthesis" mentioned in metabolic studies

• Differential regulation: M. avium adapts to various stress conditions through metabolic reprogramming , suggesting that targeting ATP synthase might be effective against bacteria in different physiological states

• Drug accessibility: As a membrane-associated protein complex, ATP synthase may be accessible to inhibitors without requiring intracellular penetration

Experimental Approaches for Drug Development:

• Structural characterization of M. avium atpD to identify potential binding sites

• High-throughput screening of compound libraries against recombinant atpD

• Evaluation of existing ATP synthase inhibitors against M. avium

• Assessment of synergy between atpD inhibitors and current anti-mycobacterial drugs

• Selectivity analysis comparing M. avium atpD with human ATP synthase to minimize toxicity

Research demonstrated that "Loss of dosRS expression in M. avium led to a significant reduction in viability under hypoxia" , suggesting that targeting stress response mechanisms might enhance the efficacy of ATP synthase inhibitors, particularly against persistent bacteria in hypoxic niches.

What methodological approaches are recommended for studying atpD expression and sequence variations in clinical isolates?

Studying atpD in clinical isolates requires robust methodologies applicable to samples with potentially limited bacterial numbers:

Molecular Methods:

• RT-qPCR: For targeted quantification of atpD expression, normalized to reference genes like sigA as demonstrated in DosR regulon studies

• Digital droplet PCR: For absolute quantification from samples with low bacterial loads

• Targeted amplicon sequencing: For atpD sequence analysis across multiple isolates

Proteomic Methods:

• Selected Reaction Monitoring (SRM): For targeted quantification of atpD peptides

• Data-independent acquisition: As used in comprehensive proteogenomic analysis that identified thousands of M. avium proteins

• Parallel Reaction Monitoring (PRM): For highly sensitive detection of atpD peptides and potential variants

Genetic Characterization Methods:

• Variable Number of Tandem Repeat (VNTR) analysis: For typing isolates as demonstrated in genomic studies

• Whole genome sequencing: To identify genetic variations in atpD and associated genes

• Comparative genomic analysis: To correlate atpD variations with other genetic markers

For clinical isolate comparison, researchers could adopt approaches used in studies comparing "MAH isolates from pigs, humans and the environment" , which involved:

• Collection and culturing of isolates from different sources

• Genetic characterization using molecular typing methods

• Comparative analysis of target gene sequences and expression levels

• Correlation of genetic variations with phenotypic differences in growth, virulence, or drug susceptibility

What are the major technical challenges in studying recombinant M. avium atpD and how can they be overcome?

Studying recombinant M. avium atpD presents several technical challenges that researchers must address:

Expression Challenges:

• Problem: Mycobacterial protein codon usage may cause poor expression in E. coli

• Solution: Codon optimization of the atpD gene sequence for the expression host

• Problem: Membrane association may cause inclusion body formation

• Solution: Use solubility tags (MBP, SUMO) and lower induction temperatures (16-18°C)

Purification Challenges:

• Problem: Maintaining the native structure during purification

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

• Problem: Co-purification of host ATP synthase components

• Solution: Employ stringent washing steps and additional purification techniques like ion exchange chromatography

Functional Assay Challenges:

• Problem: Assessing activity outside the complete ATP synthase complex

• Solution: Develop reconstitution methods with other ATP synthase components

• Problem: Distinguishing recombinant atpD activity from host ATP synthase

• Solution: Use specific inhibitors of host ATP synthase or create ATP synthase-deficient host strains

Structural Analysis Challenges:

• Problem: Obtaining sufficient quantities of pure protein for structural studies

• Solution: Scale up expression or use structural prediction tools in combination with limited experimental data

• Problem: Crystallization difficulties

• Solution: Screen multiple constructs with varying terminal regions

The proteogenomic approaches described in the literature, which have successfully identified and characterized thousands of M. avium proteins , provide valuable methodologies for overcoming some of these challenges.

How can contradictory data on atpD expression in different studies be reconciled and interpreted?

When facing contradictory data on atpD expression across different studies, researchers should consider several factors that might explain the discrepancies:

Methodological Differences:

• Different normalization strategies in RT-qPCR (reference gene selection)

• Variations in protein extraction efficiency affecting proteomic detection

• Differences in sensitivity between targeted and global expression analysis methods

Experimental Condition Variations:

• Subtle differences in growth media composition

• Variations in oxygen tension or pH in nominally similar conditions

• Different time points of sample collection during growth phases

Strain-Specific Factors:

• Genetic variations between M. avium strains used in different studies

• Adaptations to laboratory growth conditions over time

• Potential contamination with other mycobacterial species

Resolution Strategies:

• Standardize methods across laboratories:

  • Adopt consistent protocols for bacterial culture

  • Use the same reference genes for expression normalization (e.g., sigA)

  • Employ similar proteomic workflows

• Perform comprehensive comparative studies:

  • Study multiple strains under identical conditions

  • Include biological replicates to assess variability

  • Employ multiple complementary techniques (e.g., RT-qPCR and proteomics)

• Validate findings with functional assays:

  • Correlate expression data with ATP synthesis activity measurements

  • Assess growth phenotypes under relevant conditions

  • Evaluate virulence correlates in standardized models

The comprehensive approaches used in recent M. avium studies, including RNA-sequencing for transcriptional profiling and proteogenomic analysis identifying thousands of proteins , provide methodological templates for resolving contradictory data through multi-omics approaches.