The recombinant ATP synthase subunit c (atpE) from Staphylococcus aureus is a membrane-bound protein critical for bacterial ATP synthesis. Expressed in E. coli and fused with an N-terminal His tag for purification, this protein spans residues 1–70 (UniProt IDs: Q7A4E6 and A6QIV2) . Its primary role lies in proton translocation across the bacterial membrane, enabling ATP production via the F₀ sector of ATP synthase .
TO Resistance: Mutations (e.g., Phe47Leu) disrupt inhibitor binding near Glu54, critical for proton transfer .
Selectivity: FC04-100 inhibits bacterial ATP synthase >10⁵-fold more potently than mitochondrial enzymes, minimizing off-target effects .
Structural models of S. aureus subunit c (SWISS-MODEL, PDB 3ZO6/1WU0) reveal:
Binding Sites: Mutated residues (Ala17, Gly18, Ser26, Phe47) cluster near Glu54, suggesting interference with proton channeling or inhibitor binding .
Dodecameric Assembly: Leu26 and Leu47 substitutions in resistant mutants alter surface exposure, potentially disrupting oligomer stability or drug interaction .
KEGG: saj:SaurJH9_2144
ATP synthase subunit c (AtpE) in S. aureus forms a critical component of the F₀ domain of the bacterial ATP synthase complex. It assembles into a ring structure comprising multiple c subunits (dodecameric assembly) within the bacterial membrane. The primary function of AtpE is to participate in proton translocation across the membrane, which drives the rotation of the ATP synthase complex. This rotational energy is subsequently utilized by the F₁ domain to catalyze ATP synthesis from ADP and inorganic phosphate.
The glutamic acid residue at position 54 (Glu54) plays a crucial role in proton binding and transfer within the c-ring structure . This residue is located near several amino acid positions (positions 17, 18, 26, and 47) that, when mutated, can affect the binding of ATP synthase inhibitors such as tomatidine . The ATP synthase complex is central to S. aureus energy metabolism, maintaining the proton motive force and ensuring cellular homeostasis .
In prototypic (wild-type) S. aureus strains, ATP synthase functions efficiently, contributing to high ATP production levels and a strong membrane potential. This efficient energy metabolism contributes to the characteristic rapid growth and normal colony morphology of these strains .
In contrast, small-colony variants (SCVs) of S. aureus, particularly those with mutations in the electron transport chain (such as hemB mutations), exhibit significantly reduced ATP synthase activity. This results in:
Dramatically lower ATP production compared to wild-type strains
Altered membrane potential
Adoption of a slow-growth phenotype with distinct small colony morphology
Metabolic adaptations that favor persistence
These differences in ATP synthase activity between wild-type and SCV strains have direct implications for antibiotic susceptibility. For example, SCVs with their compromised ATP synthase function show hypersusceptibility to ATP synthase inhibitors like tomatidine (MIC of 0.06 μg/ml) but increased resistance to aminoglycosides like gentamicin . This differential susceptibility stems from the fact that the residual ATP synthase activity in SCVs represents a critical vulnerability that can be targeted by specific inhibitors .
The S. aureus AtpE subunit (ATP synthase subunit c) is a membrane-spanning protein that assembles into a ring structure. Based on structural models built using homology with known structures (PDB accession numbers 3ZO6 and 1WU0), several key features of the AtpE structure can be described :
The monomeric subunit contains several transmembrane helices that span the bacterial membrane
The c subunits assemble into a dodecameric ring (12 subunits) forming a central pore
The essential glutamic acid residue (Glu54) is critical for ion binding and proton translocation
Several amino acid positions (17, 18, 26, and 47) are located near this ion-binding site and serve as potential interaction sites for inhibitors
The spatial organization of these residues is significant:
Amino acid Leu26 (mutated from Ser26) is exposed at the surface of subunit c in the internal portion of the assembly
Leu47 (mutated from Phe47) appears exposed in the external portion
Ser17 and Cys18 are positioned between subunits, potentially affecting assembly integrity
These structural characteristics allow the c-ring to function in proton translocation while also providing binding sites for specific inhibitors like tomatidine that target this subunit .
Expressing and purifying functional recombinant S. aureus AtpE requires specialized techniques due to its hydrophobic nature and tendency to form insoluble aggregates. Based on research protocols, the following methodology has proven effective:
Expression System:
Use of E. coli C43(DE3) strain, which is specifically designed for membrane protein expression
Construction of expression vectors containing the atpE gene with an N-terminal His6-tag for purification
Incorporation of a TEV protease cleavage site between the tag and protein sequence for tag removal
Culture Conditions:
Growth in LB medium supplemented with appropriate antibiotics
Induction with low IPTG concentrations (0.1-0.5 mM) at lower temperatures (18-25°C) to promote proper folding
Extended expression periods (16-24 hours) to maximize yield
Membrane Isolation and Solubilization:
Cell disruption via French press or sonication in buffer containing protease inhibitors
Isolation of membrane fraction through differential centrifugation
Solubilization of membrane proteins using detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG)
Purification Steps:
Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin
Optional TEV protease treatment to remove the His-tag
Size exclusion chromatography for further purification and assessment of oligomeric state
The purified protein can be verified by SDS-PAGE, Western blotting, and mass spectrometry to confirm identity and purity. For functional studies, reconstitution into proteoliposomes may be necessary to evaluate proton translocation activity .
Measuring ATP synthase activity in S. aureus can be performed using inverted membrane vesicles, which provide a reliable system for assessing ATP production and inhibition. The following methodology has been established based on published research:
Preparation of Inverted Membrane Vesicles:
Cultivate S. aureus (prototypical strain or SCV) to mid-exponential phase
Harvest cells and wash with appropriate buffer
Disrupt cells via French press or sonication under controlled conditions
Remove unbroken cells and debris by low-speed centrifugation
Collect membrane vesicles by ultracentrifugation
Resuspend in assay buffer containing appropriate salts and pH
ATP Synthesis Assay:
Incubate membrane vesicles with ADP, inorganic phosphate, and an energy source (NADH or succinate)
Allow ATP synthesis to proceed for a defined period (typically 5-30 minutes)
Stop the reaction with a suitable method (e.g., addition of trichloroacetic acid)
Quantify ATP production using a luciferin-luciferase assay system
For inhibition studies, pre-incubate vesicles with potential inhibitors before initiating ATP synthesis
Control Experiments:
Include known ATP synthase inhibitors as positive controls (DCCD, CCCP, oligomycin), which should show IC₅₀ values consistent with literature (0.82-8.67 μg/ml)
Use unrelated antibiotics (e.g., levofloxacin, gentamicin) as negative controls
Test mitochondrial preparations in parallel to assess selectivity of inhibitors
This assay system has been successfully used to demonstrate that tomatidine and its analogs specifically inhibit ATP synthesis in S. aureus membrane vesicles, with IC₅₀ values correlating with their antimicrobial activity (MIC values) . The assay readily distinguishes between compounds that target ATP synthase and those with different mechanisms of action.
Several complementary approaches can be employed to generate and characterize AtpE mutations for inhibitor resistance studies:
Generation of Mutations:
Serial Passage Method:
Expose S. aureus cultures to sub-lethal concentrations of the inhibitor of interest
Gradually increase inhibitor concentration over multiple passages
Select colonies showing increased resistance
This approach has successfully identified spontaneous mutations in atpE conferring resistance to tomatidine
Site-Directed Mutagenesis:
Design primers to introduce specific mutations at positions of interest (e.g., positions 17, 18, 26, 47 in AtpE)
Create point mutations in the atpE gene using PCR-based methods
Clone mutated genes into appropriate vectors for expression
CRISPR-Cas9 Genome Editing:
Design guide RNAs targeting the atpE gene
Provide donor DNA containing desired mutations
Select edited cells and confirm mutations
Screening and Characterization Methods:
Susceptibility Testing:
Determine MIC values for inhibitors using broth microdilution
Compare wild-type and mutant strains to quantify resistance levels
ATP Synthesis Assays:
Prepare membrane vesicles from mutant strains
Measure ATP synthesis in presence of inhibitors
Determine IC₅₀ values to correlate with whole-cell susceptibility
Structural Analysis:
Generate molecular models of mutant proteins using homology modeling
Analyze potential impacts on inhibitor binding sites
Predict structure-activity relationships
Binding Studies:
Use isothermal titration calorimetry or surface plasmon resonance to directly measure inhibitor binding to wild-type and mutant proteins
Determine binding affinities and thermodynamic parameters
In Vivo Assessment:
Test virulence of resistant mutants in animal infection models
Assess fitness costs associated with resistance mutations
This multi-faceted approach has successfully identified key residues in AtpE that influence inhibitor binding and action. For example, mutations at positions 17 (A17S), 18 (G18C), 26 (S26L), and 47 (F47L) have been shown to confer resistance to tomatidine and its analogs, providing valuable insights into the inhibitor binding site and mechanism of action .
S. aureus ATP synthase plays a critical role in biofilm persistence and immune evasion through several interconnected mechanisms:
Modulation of Host Immune Responses:
ATP synthase, particularly the alpha subunit (AtpA), significantly influences how host immune cells respond to S. aureus biofilms. Wild-type S. aureus biofilms with functional ATP synthase skew leukocytes toward an anti-inflammatory state, which promotes bacterial persistence . This immune modulation involves:
Suppression of proinflammatory cytokine production (IL-12p70, TNF-α, IL-6) by myeloid-derived suppressor cells (MDSCs) and macrophages
Induction of anti-inflammatory cytokine IL-10
Inhibition of effective bacterial clearance by phagocytes
Cell Lysis-Dependent Immune Signaling:
ATP synthase mutants (ΔatpA) elicit heightened proinflammatory responses compared to wild-type biofilms. This effect is dependent on bacterial cell lysis, as inhibition of lysis eliminates the augmented cytokine production . This suggests that ATP synthase activity regulates the release of immunostimulatory components from S. aureus cells.
Resistance to Immune-Mediated Clearance:
Functional ATP synthase renders S. aureus more resistant to macrophage bactericidal activity. Experiments have shown that ΔatpA mutants are more susceptible to killing by mouse macrophages compared to wild-type bacteria .
Biofilm Structure and Persistence:
ATP synthase function appears to influence biofilm architecture and structural integrity, which in turn affects accessibility to immune cells and antibiotics. This contributes to the characteristic persistence of biofilm infections, particularly in the context of medical device-associated infections .
The significance of these findings is highlighted in mouse models of prosthetic joint infection, where ATP synthase mutants (ΔatpA) demonstrate impaired ability to establish persistent infections compared to wild-type S. aureus, correlating with enhanced proinflammatory responses and improved bacterial clearance .
ATP synthase function has significant implications for S. aureus virulence across different infection scenarios:
Prosthetic Joint Infection Model:
In a mouse model of prosthetic joint infection, ATP synthase alpha subunit mutants (ΔatpA) showed impaired ability to establish persistent infections. This reduced virulence was associated with:
Heightened proinflammatory cytokine production by host immune cells
Improved biofilm clearance
Diminished tissue colonization compared to wild-type S. aureus
Skin and Soft Tissue Infections:
Studies have identified that the gamma subunit of ATP synthase (AtpG) is required for S. aureus virulence in skin and soft tissue infection models. This has been attributed to:
The role of ATP synthase in intracellular acidification
Support of fermentative enzyme activity when respiration is compromised
Maintenance of energy homeostasis under the varying conditions encountered during infection
Impact of Resistance Mutations:
S. aureus strains carrying mutations in ATP synthase genes that confer resistance to inhibitors such as tomatidine or its combination with aminoglycosides demonstrate significantly altered virulence profiles:
Strains with atpE mutations show impaired ability to colonize tissues in vivo
Mutations in related genes (such as ccpA and ndh2) that contribute to ATP synthase inhibitor resistance also affect bacterial fitness in infection models
Metabolic Adaptation During Infection:
The ability of S. aureus to modulate ATP synthase activity contributes to its metabolic flexibility, allowing adaptation to diverse host environments:
Under anaerobic conditions, S. aureus adopts a fermentative slow-growth phenotype resembling SCVs, altering its susceptibility to ATP synthase inhibitors
This metabolic adaptation enables persistence in oxygen-limited infection sites
ATP synthase function influences membrane potential and ROS production, which are critical determinants of bacterial survival during host-pathogen interactions
These findings collectively demonstrate that ATP synthase function is intimately connected to S. aureus virulence through effects on energy metabolism, immune modulation, and adaptive responses to host environments.
ATP synthase activity significantly impacts antibiotic susceptibility in S. aureus through several mechanisms:
Differential Susceptibility Between Growth Phenotypes:
The table below summarizes how ATP synthase inhibitors and reference compounds affect different S. aureus strains:
Compound | S. aureus Δhemb (SCV) | Prototypic S. aureus (Newbould) | ||
---|---|---|---|---|
MIC (μg/ml) | IC₅₀ (μg/ml) | MIC (μg/ml) | IC₅₀ (μg/ml) | |
Tomatidine (TO) | 0.06 | 18.5 ± 1.9 | >128 | 95.5 ± 13.7 |
FcM | 0.06 | 18.9 ± 3.6 | 2 | 40.2 ± 13.8 |
Fcm | 2 | 47.0 ± 9.6 | 8 | 111.0 ± 11.3 |
FC02-190 | 8 | 85.1 ± 7.0 | >128 | >1,024 |
This data reveals that:
SCVs (Δhemb) with reduced ATP synthase function are hypersusceptible to ATP synthase inhibitors like tomatidine
Prototypic strains with normal ATP synthase activity are inherently resistant to tomatidine alone
IC₅₀ values for ATP synthase inhibition correlate with whole-cell MIC values, confirming the mechanism of action
Synergistic Effects with Aminoglycosides:
ATP synthase inhibition by tomatidine creates synergistic effects with aminoglycosides against prototypic S. aureus strains:
Tomatidine reduces membrane potential, enhancing aminoglycoside uptake
The combination generates increased levels of reactive oxygen species (ROS)
This synergistic effect results in effective killing of normally tomatidine-resistant wild-type S. aureus
Adaptation to Anaerobic Conditions:
Under anaerobic conditions, prototypic S. aureus:
Adopts a fermentative slow-growth phenotype resembling SCVs
Becomes susceptible to tomatidine alone
Resistance Development and Fitness Costs:
Resistance to ATP synthase inhibitors develops through:
Mutations in the ATP synthase subunit c (atpE) gene leading to altered binding sites
Mutations in regulatory genes like ccpA (catabolite control protein A) causing intermediate resistance
Mutations in ndh2 (NADH dehydrogenase) affecting membrane potential and drug uptake
Importantly, these resistance mutations carry significant fitness costs:
Impaired virulence in animal infection models
Altered membrane properties
These findings demonstrate that ATP synthase activity is a critical determinant of antibiotic susceptibility in S. aureus, with potential therapeutic implications for targeting persistent infections.
Structure-activity relationship studies of tomatidine and its analogs have revealed several crucial molecular features required for selective inhibition of S. aureus ATP synthase:
Essential Structural Elements:
Modifications Affecting Activity:
Glycosylation at 3β-Position:
FC04-100 Enantiomers:
Binding Site Interactions:
Molecular modeling and resistance studies suggest that tomatidine and its analogs likely interact with specific amino acids in AtpE:
Residues at positions 17, 18, 26, and 47 appear critical for inhibitor binding
These residues are located near the essential ion-binding site (Glu54)
Mutations at these positions confer resistance, suggesting direct interaction with the inhibitors
Selectivity Features:
A key advantage of tomatidine-based inhibitors is their selectivity for bacterial ATP synthase over mammalian mitochondrial ATP synthase:
No inhibitory activity toward ATP production by mitochondria (IC₅₀ >1,024 μg/ml)
Estimated selectivity index (SI) of >10⁵ for bacterial ATP synthase over mitochondria
This contrasts with non-selective inhibitors like DCCD and oligomycin (SI ≤1)
These structure-activity insights provide a foundation for rational design of improved ATP synthase inhibitors with enhanced potency and selectivity profiles.
Mutations in the AtpE subunit of S. aureus ATP synthase confer resistance to inhibitors through several mechanisms that affect binding interactions and protein function:
Key Resistance-Conferring Mutations:
Four specific amino acid positions in AtpE have been identified as critical sites for resistance development:
Position 17 (Ala to Ser):
Position 18 (Gly to Cys):
Position 26 (Ser to Leu):
Position 47 (Phe to Leu):
Structural Basis for Resistance:
Molecular modeling studies of the AtpE c-ring have revealed that:
Each of these mutations is in proximity to the essential ion-binding site (Glu54)
The mutations likely prevent inhibitor binding or interaction with the functional center of the protein
Alternatively, they may allow proton transfer to continue even when the inhibitor is bound
Functional Consequences:
Resistance mutations in AtpE lead to:
High-level resistance to tomatidine and its analogs (MIC increases from 0.06 μg/ml to >128 μg/ml)
Altered ATP synthase function, often with fitness costs
Stepwise Resistance Development:
Resistance to ATP synthase inhibitors typically develops in a stepwise fashion:
Initial mutations in regulatory genes (e.g., ccpA) conferring intermediate resistance
Subsequent mutations in atpE providing high-level resistance
This multistep process may explain the relatively low frequency of resistance development observed in laboratory settings
Understanding these resistance mechanisms provides valuable insights for drug development by:
Identifying critical inhibitor binding determinants
Revealing potential strategies to overcome resistance
Guiding structure-based design of next-generation inhibitors with reduced potential for resistance development
Optimizing ATP synthase inhibitors for therapeutic development against S. aureus infections requires a multi-faceted approach addressing potency, selectivity, pharmacokinetics, and resistance potential:
Target Population Optimization:
Dual-Action Compounds:
Design inhibitors effective against both SCVs and prototypic S. aureus
Incorporate structural features enabling synergy with conventional antibiotics
Example: FC04-100 (FcM) shows improved activity against prototypic S. aureus (MIC 2 μg/ml) compared to tomatidine (MIC >128 μg/ml) while maintaining potency against SCVs
Biofilm Penetration:
Structural Optimization:
Essential Pharmacophore Preservation:
Selectivity Enhancement:
Resistance Barrier Improvement:
Design compounds that interact with multiple conserved residues
Develop inhibitors targeting alternative sites within the ATP synthase complex
Create molecules requiring multiple mutations for resistance development
Pharmacokinetic Optimization:
Solubility Enhancement:
Address the inherent hydrophobicity of steroidal compounds
Incorporate solubilizing groups without compromising activity
Develop appropriate formulations for different routes of administration
Tissue Distribution:
Optimize compounds for reaching infection sites (bone, joint, biofilm)
Consider the impact of protein binding on effective concentration
Develop targeted delivery systems for biofilm-associated infections
Combination Strategies:
Synergistic Combinations:
Anti-Virulence Approaches:
Translational Research:
Appropriate Infection Models:
Resistance Monitoring:
Through these optimization strategies, ATP synthase inhibitors can be developed into effective therapeutic agents against both persistent and acute S. aureus infections, addressing a critical need in antimicrobial therapy.
S. aureus ATP synthase functions as a central metabolic hub that coordinates with multiple pathways to enable bacterial adaptation to diverse host environments:
Integration with Respiratory Metabolism:
Electron Transport Chain Coordination:
Adaptation to Anaerobiosis:
Carbon Metabolism Regulatory Networks:
CcpA-Mediated Regulation:
Acidification Requirements:
Membrane Physiology Coordination:
Surface Hydrophobicity Regulation:
Proton Motive Force Maintenance:
Redox Balance and Oxidative Stress:
ROS Generation and Management:
ATP synthase inhibition by tomatidine causes significant ROS production in SCVs
The combination of tomatidine with gentamicin generates 2.5-fold more ROS in wild-type S. aureus compared to gentamicin alone
This suggests coordination between ATP synthase, electron transport, and ROS management systems
Antioxidant Defense Coordination:
Host-Adaptive Metabolic Shifts:
Small Colony Variant Phenotype:
Biofilm-Specific Metabolism:
Understanding these complex metabolic interactions provides insights into S. aureus adaptability and identifies potential combination therapeutic targets to disrupt these coordinated responses.
The bactericidal activity of ATP synthase inhibitors targeting AtpE involves multiple interrelated mechanisms that collectively lead to S. aureus cell death:
Primary Effects of AtpE Inhibition:
Energy Depletion:
Membrane Potential Disruption:
Secondary Consequences Leading to Cell Death:
Reactive Oxygen Species (ROS) Generation:
Cell Envelope Integrity Compromise:
Phenotype-Specific Killing Mechanisms:
Cellular Stress Responses and Death Pathways:
Metabolic Futile Cycles:
ATP synthase inhibition may lead to futile cycling of protons across the membrane
This further depletes cellular energy reserves
The process accelerates metabolic collapse and cell death
Bacterial Programmed Cell Death:
Evidence suggests that persistent ATP synthase inhibition may trigger bacterial programmed cell death pathways
This involves coordinated degradation of cellular components
The process may be accelerated by ROS-mediated damage
Understanding these bactericidal mechanisms is crucial for:
Optimization of ATP synthase inhibitors
Development of combination therapies that enhance killing
Designing strategies to address persistent S. aureus infections that are refractory to conventional antibiotics
The structural and functional characteristics of ATP synthase vary significantly across bacterial species, creating both challenges and opportunities for selective inhibitor development:
Structural Divergence Among Bacterial ATP Synthases:
Sequence Variation in AtpE:
Significant sequence differences exist in the c subunit (AtpE) across bacterial species
These variations create distinct binding pockets and interaction sites
For example, tomatidine effectively inhibits S. aureus ATP synthase but shows limited activity against E. coli (MIC >128 μg/ml, IC₅₀ 264.73 ± 30.8 μg/ml)
C-ring Stoichiometry Differences:
The number of c subunits forming the ring varies between species (8-15 subunits)
This affects the geometry and accessibility of potential binding sites
These structural differences can be exploited for species-selective inhibition
Functional Adaptations in Different Pathogens:
Respiratory Flexibility:
S. aureus possesses remarkable respiratory flexibility, adapting to varying oxygen levels
Some pathogens are obligate aerobes or anaerobes with different ATP synthase dependencies
These differences influence the vulnerability to ATP synthase inhibition across species
Metabolic Integration:
The degree of integration between ATP synthase and other metabolic pathways varies
In S. aureus, ATP synthase inhibition affects multiple aspects of bacterial physiology
Other species may have alternative energy generation pathways reducing dependency on ATP synthase
Species-Specific Inhibitor Development:
Selective Targeting:
Bedaquiline, a diarylquinoline that inhibits Mycobacterium tuberculosis ATP synthase, shows no inhibitory activity against S. aureus ATP synthase (IC₅₀ >1,028 μg/ml)
Conversely, tomatidine selectively targets S. aureus but not mycobacterial ATP synthase
This demonstrates that bacterial ATP synthases are sufficiently distinct to respond to specific inhibitors
Structural Basis for Selectivity:
Amino acid sequence analysis reveals significant differences between bacterial species
These differences explain the observed selectivity of inhibitors
Structure-based design can exploit these distinctions to develop pathogen-specific therapeutics
Comparative Inhibitor Efficacy Table:
Inhibitor | Target Pathogen | S. aureus (IC₅₀) | M. tuberculosis (IC₅₀) | E. coli (IC₅₀) | Human Mitochondria (IC₅₀) |
---|---|---|---|---|---|
Tomatidine | S. aureus SCV | 18.5 ± 1.9 μg/ml | No significant inhibition | 264.73 ± 30.8 μg/ml | >1,024 μg/ml |
Bedaquiline | M. tuberculosis | >1,028 μg/ml | 0.01-0.1 μg/ml | No significant inhibition | No significant inhibition |
DCCD | Non-selective | 1-10 μg/ml | 1-10 μg/ml | 1-10 μg/ml | 1-10 μg/ml |
Implications for Therapeutic Development:
Narrow-Spectrum Antibiotics:
The structural diversity of bacterial ATP synthases enables development of narrow-spectrum antibiotics
This approach reduces impact on beneficial microbiota
Targeted therapy may reduce selection pressure for resistance in non-target species
Combination Therapy Design:
Understanding species-specific ATP synthase characteristics allows rational design of combination therapies
Synergistic effects (like tomatidine-aminoglycoside) may vary between pathogens
Optimized combinations can be tailored to specific infection types
Multi-Species Inhibitor Development:
Identifying conserved elements across ATP synthases of multiple pathogens
Designing broad-spectrum ATP synthase inhibitors for polymicrobial infections
Balancing spectrum of activity with selectivity over mammalian ATP synthase
The significant structural and functional differences between bacterial ATP synthases provide a strong foundation for developing highly selective antimicrobial agents with reduced off-target effects and potential for customized therapeutic approaches.