Recombinant Streptococcus pyogenes serotype M5 ATP synthase subunit alpha (atpA), partial

What is ATP synthase in Streptococcus pyogenes, and how does it differ from human ATP synthase?

ATP synthase in S. pyogenes is a multi-subunit enzyme complex that catalyzes ATP synthesis using energy from the proton gradient across the bacterial membrane. It consists of two domains: F₁ (containing the catalytic core with α, β, γ, δ, and ε subunits) and F₀ (comprising the membrane proton channel with a, b, and c subunits) .

Key differences from human (mitochondrial) ATP synthase:

• Bacterial ATP synthases are located in the plasma membrane, while human ATP synthase is in the inner mitochondrial membrane

• Different inhibitor sensitivity profiles

• Variations in subunit composition and molecular weight

• Bacterial ATP synthase often functions in reverse during anaerobic growth to maintain membrane potential

Research considerations: When designing inhibitor studies or structural analyses, these differences are crucial for developing antimicrobial targets that don't affect human ATP synthase.

What expression systems are most effective for producing recombinant S. pyogenes ATP synthase subunit alpha?

For recombinant expression of S. pyogenes ATP synthase subunit alpha, several systems have proven effective:

Methodology notes:

• For E. coli expression, BL21(DE3) or Rosetta strains often provide optimal results

• Codon optimization is critical due to GC content differences

• Growth at lower temperatures (16-25°C) after induction helps reduce inclusion body formation

• Consider fusion partners (MBP, SUMO) to enhance solubility if inclusion bodies are problematic

These strategies have been successfully employed for other S. pyogenes proteins (including nucleases and ADP-ribosyltransferases) , suggesting their applicability to ATP synthase subunits.

What purification challenges are specific to recombinant ATP synthase subunits?

Purifying recombinant S. pyogenes ATP synthase alpha subunit presents several specific challenges:

• Solubility issues: ATP synthase subunits often form inclusion bodies or aggregate during expression

  • Solution: Screen solubilization buffers with different detergents (0.5-1% Triton X-100, CHAPS, or DDM)

  • Denaturation/refolding protocols may be necessary

• Maintaining native conformation: The alpha subunit's function depends on proper folding

  • Include ATP or ADP (1-5 mM) in purification buffers to stabilize conformation

  • Avoid harsh elution conditions when using affinity chromatography

• Co-purification of contaminants: Bacterial chaperones often co-purify with ATP synthase subunits

  • Employ multi-step purification (ion exchange followed by size exclusion chromatography)

  • Use high salt washes (300-500 mM NaCl) during initial affinity purification steps

• Verification of purity: ATP synthase subunit alpha has a similar molecular weight to common contaminants

  • SDS-PAGE should show >90% purity (as reported for other S. pyogenes recombinant proteins)

  • Western blotting with anti-His or specific antibodies confirms identity

The recombinant protein should be stored in Tris-based buffer with 50% glycerol at -20°C for extended storage, with aliquoting recommended to avoid freeze-thaw cycles .

How can researchers verify the functional integrity of purified recombinant ATP synthase subunit alpha?

Verifying functional integrity requires multiple approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure

  • Thermal shift assays to assess protein stability

  • Limited proteolysis to evaluate proper folding

• Nucleotide binding analysis:

  • Fluorescence-based assays using MANT-ATP or TNP-ATP

  • Isothermal titration calorimetry (ITC) to measure binding affinities

  • Surface plasmon resonance (SPR) for binding kinetics

• ATPase activity assessment:

  • Colorimetric phosphate release assays (e.g., malachite green assay)

  • Coupled enzyme assays (with pyruvate kinase and lactate dehydrogenase)

  • Luciferase-based ATP detection methods

• Complex formation evaluation:

  • Size-exclusion chromatography to assess oligomerization state

  • Native PAGE to detect complex formation with other ATP synthase subunits

  • Chemical crosslinking followed by mass spectrometry

When analyzing ATP synthase activity, researchers should establish appropriate positive controls (such as commercially available F₁ ATP synthase) and negative controls (heat-inactivated enzyme) .

How does ATP synthase contribute to S. pyogenes survival under different environmental conditions?

ATP synthase plays crucial roles in S. pyogenes adaptation to changing environmental conditions:

• pH homeostasis:

  • ATP synthase helps maintain intracellular pH during acid stress

  • In low pH environments, ATP synthase can function in reverse to expel protons

  • During stationary phase, ATP synthase works in concert with arginine deiminase pathway to balance acid production

• Nutritional stress response:

  • When glucose is depleted, ATP synthase expression is altered

  • ATP synthase contributes to survival in long-term stationary phase cultures

  • Strains isolated from long-term stationary phase cultures show mutations affecting ATP synthase regulation

• Oxidative stress management:

  • F₀F₁-ATP synthase (atpB-H) is downregulated in S. pyogenes in the presence of arginine

  • This regulation is linked to oxidative stress response mechanisms

• Biofilm formation:

  • ATP synthase expression changes during biofilm development

  • Energy production via ATP synthase supports extracellular polysaccharide synthesis

In vitro experiments demonstrated that ATP synthase is critical for maintaining S. pyogenes viability during transition to stationary phase, with mutant strains showing reduced survival .

What evidence exists that ATP synthase subunits could serve as vaccine candidates against S. pyogenes?

Several lines of evidence suggest ATP synthase subunits might be valuable vaccine candidates:

• Surface accessibility:

  • Proteomic analysis identified ATP synthase F₀F₁ subunit alpha among cell surface-associated proteins in S. pyogenes

  • This unexpected surface localization makes it potentially accessible to antibodies

• Conservation across strains:

  • ATP synthase subunits are highly conserved across different S. pyogenes serotypes

  • Genome analysis of multiple strains showed >97% sequence identity for ATP synthase genes

  • This conservation suggests a vaccine could provide broad protection

• Immunogenicity:

  • Patients recovering from S. pyogenes infections develop antibodies against numerous cell surface proteins

  • Convalescent sera from patients with invasive disease recognize surface-exposed proteins including ATP synthase components

  • ATP synthase subunits are expressed during human infection, as demonstrated by seroconversion

• Potential for protective immunity:

  • Other bacterial ATP synthase components have shown promise as vaccine antigens

  • Similar approaches with other highly conserved metabolic enzymes have demonstrated protection in animal models

How does genetic variation in atpA affect enzymatic function across different S. pyogenes strains?

Genetic variation in atpA across S. pyogenes strains has several functional implications:

• Sequence conservation and variation:

  • Core catalytic regions show high conservation (>95% amino acid identity)

  • Most variations occur in non-catalytic regions

  • Recombination events contribute to genetic diversity in S. pyogenes

• Functional impacts:

  • Single nucleotide polymorphisms (SNPs) may alter:

    • Catalytic efficiency

    • Thermal stability

    • pH sensitivity

    • Interaction with other subunits

  • Strains adapted to different tissue niches show subtle variations in ATP synthase genes

• Expression level differences:

  • Promoter region variations affect transcription levels

  • Post-transcriptional regulation varies between strains

  • Some strains show altered regulation during infection stages

• Evolutionary selection:

  • ATP synthase genes are subject to purifying selection

  • Mutations affecting core function are typically eliminated

  • Variations in regulatory regions persist more frequently

A comparative study of S. pyogenes strains showed that while coding sequences are highly conserved, promoter regions and intergenic spaces show more variation, suggesting that expression regulation rather than protein structure is the primary target of evolutionary adaptation .

What controls are necessary when studying recombinant S. pyogenes ATP synthase activity?

Robust experimental design for ATP synthase activity studies requires careful consideration of controls:

Essential Controls:

Additional validation approaches:

• Mutant variants: Create known catalytic site mutants (H225A, H262A) to serve as negative controls

• Subunit interactions: Test isolated alpha subunit vs. alpha in complex with beta/other subunits

• Time-course measurements: Establish linearity of activity over experimental timeframe

• Concentration dependencies: Verify Michaelis-Menten kinetics with varying substrate concentrations

When using spectrophotometric assays, incorporate controls for background absorbance and non-enzymatic hydrolysis of ATP, particularly at extreme pH values or temperatures .

What techniques are available for studying ATP synthase-protein interactions in S. pyogenes?

Several complementary techniques can elucidate ATP synthase-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against atpA to pull down interaction partners

  • Employ epitope-tagged recombinant atpA (His, FLAG, or HA tags)

  • Analyze by mass spectrometry to identify binding partners

  • Verify with reciprocal Co-IP experiments

• Surface Plasmon Resonance (SPR):

  • Immobilize purified atpA on sensor chip

  • Measure real-time binding kinetics with potential interacting proteins

  • Determine association/dissociation rates and binding affinities

  • Can detect weak and transient interactions

• Crosslinking coupled with mass spectrometry:

  • Use chemical crosslinkers (e.g., DSS, formaldehyde) to capture interactions

  • Apply in vivo crosslinking to capture physiologically relevant interactions

  • Identify crosslinked peptides by mass spectrometry

  • Map interaction interfaces at amino acid resolution

• Bacterial two-hybrid systems:

  • Adapt bacterial two-hybrid systems for studying S. pyogenes protein interactions

  • Use as screening tool to identify novel interaction partners

  • Verify with other methods

• Fluorescence-based methods:

  • Fluorescence resonance energy transfer (FRET)

  • Bioluminescence resonance energy transfer (BRET)

  • Fluorescence correlation spectroscopy (FCS)

  • Can be applied in vitro or in live cells

• Structural biology approaches:

  • X-ray crystallography of complexes

  • Cryo-electron microscopy for larger assemblies

  • NMR for mapping interaction surfaces

Studies using these approaches have identified interactions between ATP synthase subunits and various regulatory proteins in S. pyogenes, including potential links to virulence regulation networks .

How can researchers design effective gene deletion studies to understand ATP synthase function in S. pyogenes?

Designing effective gene deletion studies for ATP synthase requires specialized approaches:

• Selection of target genes:

  • Complete atpA deletion may be lethal – consider conditional knockouts

  • Target regulatory genes controlling ATP synthase expression

  • Create partial deletions or point mutations in critical domains

  • Consider deleting other ATP synthase subunits that may be less essential

• Methodological approaches:

  • Use efficient one-step methods for gene deletion via homologous recombination

  • Consider CRISPR-Cas9 systems adapted for S. pyogenes

  • For essential genes, employ inducible expression systems

  • Create merodiploid strains with a second copy under inducible control

• Verification strategies:

  • PCR verification with primers outside the flanking regions

  • RT-qPCR to confirm transcriptional changes

  • Western blotting to verify protein deletion

  • Whole-genome sequencing to confirm clean deletion and detect potential compensatory mutations

• Phenotypic characterization:

  • Growth curves in different media and stress conditions

  • ATP production measurement

  • pH homeostasis assessment

  • Membrane potential analysis

  • Virulence in infection models

  • Competition assays with wild-type strains

• Controls and complementation:

  • Include wild-type and single recombinant controls

  • Create complemented strains to verify phenotypes result from target gene deletion

  • Use trans-complementation with inducible promoters

  • Create point mutants to identify critical residues

Recent successful gene deletion strategies in S. pyogenes employ colE1-type plasmids as suicide vectors, allowing generation of non-polar mutants in just 3 days, with 73-93% success rates for various target genes .

How does ATP synthase contribute to S. pyogenes adaptation during infection?

ATP synthase plays multifaceted roles during S. pyogenes infection:

• Metabolic adaptation:

  • Adjusts energy production according to host microenvironments

  • Responds to fluctuating nutrient availability in different tissues

  • Coordinates with other metabolic pathways during infection phases

• pH tolerance:

  • Helps maintain intracellular pH during exposure to acidic phagolysosomes

  • Works with arginine deiminase pathway to control acid production

  • Contributes to survival in various host niches with different pH levels

• Stress response integration:

  • ATP synthase expression is regulated by stress-responsive transcription factors

  • Downregulation of ATP synthase (atpB-H) occurs under specific stress conditions

  • Coordinates with virulence factor expression through central regulatory networks

• Biofilm formation:

  • Contributes to energy requirements during biofilm development

  • Expression changes coordinate with adhesin production

  • May influence extracellular matrix composition

• Immune evasion connections:

  • Unexpected surface exposure may contribute to immune interactions

  • Regulated as part of complex virulence networks

  • May interact with host factors directly or indirectly

Transcriptomic analysis revealed that F₀F₁-type ATP synthase genes (atpB-H) are downregulated in the presence of arginine, which correlates with upregulation of virulence factors like streptolysin S and streptolysin O, suggesting coordinated regulation between metabolism and virulence .

What structural features distinguish bacterial ATP synthase alpha subunit from other species?

The ATP synthase alpha subunit from S. pyogenes contains several distinctive structural features:

• Domain organization:

  • N-terminal beta-barrel domain (residues ~1-95)

  • Central nucleotide-binding domain (residues ~96-380)

  • C-terminal alpha-helical domain (residues ~381-510)

• Key functional regions:

  • Phosphate-binding loop (P-loop) containing the conserved sequence GXGXGKT/S

  • DELSEED region (or bacterial equivalent) that interacts with the gamma subunit

  • Nucleotide-binding pocket with specific metal coordination sites

• Species-specific features:

  • Surface-exposed epitopes unique to S. pyogenes

  • Specialized interfaces for interaction with other bacterial subunits

  • Surface residues that may contribute to membrane association

Comparative structural analysis between bacterial and human ATP synthase alpha subunits reveals differences in:

These structural differences provide potential targets for selective inhibition and are important considerations for both antimicrobial development and immunological studies .

What advanced biophysical methods are most effective for studying ATP synthase dynamics?

Several cutting-edge biophysical methods provide insights into ATP synthase dynamics:

• Single-molecule techniques:

  • Single-molecule FRET to monitor conformational changes

  • Optical tweezers to measure rotational force generation

  • Magnetic tweezers for torque measurements

  • High-speed atomic force microscopy to visualize rotational dynamics

• Advanced spectroscopic methods:

  • Hydrogen/deuterium exchange mass spectrometry (HDX-MS) to map conformational dynamics

  • Electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling

  • Solid-state NMR to study membrane-embedded regions

  • Time-resolved fluorescence spectroscopy to measure conformational transitions

• Structural methods with dynamic information:

  • Time-resolved cryo-electron microscopy

  • X-ray free-electron laser (XFEL) crystallography

  • Molecular dynamics simulations based on structural data

  • Normal mode analysis of structural ensembles

• Functional assays with temporal resolution:

  • Real-time ATP synthesis/hydrolysis measurements

  • Simultaneous proton translocation and ATP synthesis monitoring

  • Patch-clamp electrophysiology to measure proton currents

  • pH-sensitive fluorescent probes to monitor local pH changes

These methods have revealed that ATP synthase functions as a rotary motor, with the gamma subunit rotating within the alpha/beta hexamer during catalysis. The bacterial ATP synthase completes this rotation in discrete 120° steps, with each step coupled to ATP synthesis or hydrolysis .

How can researchers address the challenges of studying membrane-associated ATP synthase complexes?

Studying membrane-associated ATP synthase presents unique challenges requiring specialized approaches:

• Membrane mimetic systems:

  • Detergent micelles (DDM, CHAPS, OG) for initial extraction

  • Nanodiscs with defined lipid composition for controlled environment

  • Liposome reconstitution for functional studies

  • Amphipols for maintaining native-like environment without detergents

• Advanced isolation techniques:

  • Styrene-maleic acid lipid particles (SMALPs) to extract membrane proteins with native lipids

  • Digitonin extraction for preserving supercomplexes

  • Native electrophoresis (BN-PAGE, CN-PAGE) for intact complex analysis

  • Density gradient ultracentrifugation to separate intact complexes

• Functional reconstitution strategies:

  • Proteoliposome creation with controlled lipid composition

  • Co-reconstitution with proton pumps to generate proton gradients

  • Incorporation of pH or voltage sensors

  • Surface-tethered membranes for single-molecule studies

• Structural analysis of membrane complexes:

  • Cryo-electron microscopy for near-atomic resolution

  • Electron crystallography for 2D crystals

  • Solid-state NMR for atomic-level dynamics

  • X-ray crystallography with lipidic cubic phase

• Overcoming expression challenges:

  • Cell-free expression systems with supplied lipids or detergents

  • Specialized host strains for membrane protein expression

  • Fusion with membrane protein expression enhancers

  • Codon optimization and controlled expression rate

Studies using CN-PAGE with mild detergents have successfully analyzed ATP synthase complexes from bacteria, showing that assembly occurs from separate modules: the c-ring, F₁, and the stator arm. This approach preserves important protein-protein interactions that are disrupted by harsher conditions .

What experimental approaches can identify inhibitors specific to S. pyogenes ATP synthase?

Several specialized approaches can identify selective inhibitors:

• High-throughput screening platforms:

  • ATP hydrolysis assays using purified enzyme

  • Whole-cell ATP production assays

  • Growth inhibition screening with counter-screening against human cell lines

  • Membrane potential-sensitive fluorescent dyes to detect ATP synthase inhibition

• Structure-based drug design:

  • Homology modeling of S. pyogenes ATP synthase

  • Molecular docking against unique binding pockets

  • Fragment-based screening approaches

  • Structure-activity relationship studies of lead compounds

• Target-based approaches:

  • Photoaffinity labeling to identify binding sites

  • Thermal shift assays to detect stabilizing compounds

  • Surface plasmon resonance for binding kinetics

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

• Physiological validation:

  • Membrane potential measurement

  • Intracellular ATP quantification

  • Proton translocation assays

  • Effects on virulence factor expression

• Selectivity assessment:

  • Counter-screening against human ATP synthase

  • Cytotoxicity evaluation in mammalian cells

  • Mitochondrial function assays

  • Cardiac cell contractility assessment

These approaches have successfully identified selective inhibitors of other bacterial ATP synthases, suggesting similar strategies would be effective for S. pyogenes. The goal is to identify compounds that exploit structural differences between bacterial and human enzymes to achieve selective toxicity .

How can recombinant ATP synthase subunits contribute to vaccine development strategies?

Recombinant ATP synthase subunits offer several advantages for vaccine development:

• Antigen preparation approaches:

  • High-purity recombinant protein production

  • Engineered constructs focusing on immunogenic epitopes

  • Fusion with carrier proteins or adjuvants

  • Multivalent designs incorporating multiple antigens

• Immunological considerations:

  • Surface epitope mapping to identify accessible regions

  • B-cell and T-cell epitope prediction

  • Cross-reactivity assessment with human homologs

  • Evaluation in multiple serotype challenge models

• Delivery platforms:

  • Protein-based subunit vaccines

  • DNA vaccines encoding optimized sequences

  • Viral vector delivery systems

  • mRNA-based approaches

  • Outer membrane vesicles displaying ATP synthase components

• Combination strategies:

  • ATP synthase components combined with established antigens (e.g., M protein derivatives)

  • Multi-epitope constructs targeting different virulence factors

  • Prime-boost regimens with different delivery platforms

Research has shown that other surface-exposed proteins in S. pyogenes can elicit protective immune responses. Proteomic analysis identified ATP synthase F₀F₁ subunit alpha among the surface-associated proteins in S. pyogenes , suggesting it may be accessible to antibodies. The high conservation of ATP synthase across different S. pyogenes strains makes it potentially valuable for broad-spectrum protection .

What are the most promising future research directions for understanding ATP synthase's role in S. pyogenes pathogenesis?

Several emerging research directions show particular promise:

• Systems biology approaches:

  • Integrating transcriptomics, proteomics, and metabolomics data

  • Network analysis of ATP synthase interactions with virulence regulation

  • Flux balance analysis of energy metabolism during infection

  • Multi-omics analysis across infection stages

• In vivo dynamics:

  • Real-time imaging of ATP production during infection

  • Single-cell analysis of ATP synthase expression

  • Tissue-specific metabolic adaptations

  • Host-pathogen metabolic interactions

• Regulatory mechanisms:

  • Small RNA regulation of ATP synthase expression

  • Post-translational modifications affecting activity

  • Protein-protein interactions modulating function

  • Environmental sensing and signal transduction

• Unexpected functions:

  • Potential moonlighting roles of ATP synthase components

  • Surface-exposed functions unrelated to ATP synthesis

  • Interactions with host immune factors

  • Involvement in biofilm matrix organization

• Therapeutic applications:

  • Attenuated strains through ATP synthase modulation

  • Metabolic inhibitors as antivirulence compounds

  • Combination therapies targeting energy production

  • Host-directed therapies affecting bacterial energy requirements

These approaches may reveal how S. pyogenes coordinates energy production with virulence expression during infection. Recent findings showing coordinated regulation between ATP synthase and virulence factors under arginine-rich conditions highlight the complex integration of metabolism and pathogenesis .

How can genetic variation in ATP synthase be leveraged to understand S. pyogenes evolution and adaptation?

Genetic variation in ATP synthase provides valuable insights into evolutionary processes:

• Comparative genomics approaches:

  • Analysis of ATP synthase sequences across diverse clinical isolates

  • Correlation with geographical distribution and disease manifestations

  • Identification of selection signatures

  • Mapping recombination events affecting ATP synthase genes

• Experimental evolution:

  • Laboratory evolution under different energy stresses

  • Selection for altered ATP synthase function

  • Tracking compensatory mutations

  • Fitness landscape mapping for ATP synthase variants

• Structure-function correlations:

  • Mapping natural variants onto structural models

  • Identifying functionally important residues under purifying selection

  • Characterizing the effects of natural variants on enzyme function

  • Determining structural constraints on evolution

• Host adaptation signatures:

  • Comparing ATP synthase sequences from different host-adapted strains

  • Identifying adaptive mutations for specific host environments

  • Correlating genetic variants with tissue tropism

  • Experimental validation of adaptive hypotheses

• Clinical correlations:

  • Association of ATP synthase variants with disease severity

  • Correlation with antibiotic resistance profiles

  • Identification of hypervirulent lineage markers

  • Development of molecular typing schemes