Recombinant Staphylococcus aureus ATP synthase subunit c (atpE)

What is the molecular function of ATP synthase subunit c (AtpE) in Staphylococcus aureus?

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 .

How does ATP synthase function differ between prototypic S. aureus and small-colony variants (SCVs)?

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 .

What is the structural organization of the S. aureus AtpE subunit and the complete c-ring assembly?

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 .

What are the most effective approaches for expressing and purifying recombinant S. aureus AtpE protein?

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 .

How can researchers effectively measure ATP synthase activity in S. aureus membrane vesicles?

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.

What techniques are available for generating and screening AtpE mutations to study inhibitor resistance?

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 .

How does ATP synthase contribute to S. aureus biofilm persistence and immune evasion?

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 .

What is the relationship between ATP synthase function and S. aureus virulence in different infection models?

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.

How does ATP synthase activity influence antibiotic susceptibility profiles in S. aureus?

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:

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

• Shows altered ATP synthase dependence and function

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

• Compromised energy metabolism

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.

What structural features of tomatidine and its analogs are critical for selective inhibition of S. aureus ATP synthase?

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:

  • Tomatine (the 3β-glycosylated form of tomatidine) is devoid of inhibitory activity against S. aureus SCVs

  • This indicates that extensive modification of the 3β-hydroxyl group is detrimental to antibacterial effect

• FC04-100 Enantiomers:

  • The more potent enantiomer (FcM, MIC 0.06 μg/ml) shows an IC₅₀ of 18.9 ± 3.6 μg/ml

  • The less potent enantiomer (Fcm, MIC 2 μg/ml) shows a higher IC₅₀ of 47.0 ± 9.6 μg/ml

  • This demonstrates that subtle stereochemical differences significantly impact binding and inhibition

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.

How do mutations in AtpE affect binding of inhibitors and confer resistance?

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):

  • Located between subunits in the multimeric assembly

  • May affect assembly integrity or inter-subunit interactions

  • The mutation likely disrupts the optimal binding pocket for tomatidine

• Position 18 (Gly to Cys):

  • Also positioned between subunits

  • Introduction of a cysteine may allow formation of disulfide bonds

  • Potentially alters the conformational flexibility required for inhibitor binding

• Position 26 (Ser to Leu):

  • Located at the surface of the subunit in the internal portion of the assembly

  • The mutation from a polar serine to a hydrophobic leucine significantly changes the surface properties

  • Likely directly interferes with inhibitor binding due to its exposed position

• Position 47 (Phe to Leu):

  • Exposed in the external portion of the c-ring

  • Change from aromatic phenylalanine to aliphatic leucine alters potential π-stacking interactions

  • May affect the entry or positioning of inhibitors within the binding site

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

• Reduced competitiveness in infection models

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

How can ATP synthase inhibitors be optimized for therapeutic development against S. aureus infections?

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:

  • Optimize physicochemical properties to enhance penetration into biofilms

  • Target compounds disrupting the immune-modulating effects of ATP synthase

  • Consider the impact on biofilm architecture and dispersal

Structural Optimization:

• Essential Pharmacophore Preservation:

  • Maintain intact spiroaminoketal moiety and correct stereochemistry at position 3

  • Preserve key interaction points with binding site residues (positions 17, 18, 26, 47)

  • Optimize steroidal backbone while maintaining critical binding interactions

• Selectivity Enhancement:

  • Further increase selectivity index (SI) for bacterial over mammalian ATP synthase

  • Exploit structural differences between bacterial and mitochondrial c-subunits

  • Current tomatidine analogs already show excellent selectivity (SI >10⁵)

• 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:

  • Further develop the tomatidine-aminoglycoside synergistic approach

  • The combination generates heightened ROS and enhances aminoglycoside uptake

  • Effective against otherwise tomatidine-resistant prototypic S. aureus

• Anti-Virulence Approaches:

  • Combine ATP synthase inhibitors with compounds targeting other virulence factors

  • Address the immune-modulating effects of ATP synthase

  • Enhance host immune-mediated clearance

Translational Research:

• Appropriate Infection Models:

  • Validate candidates in relevant biofilm infection models

  • Test efficacy against both acute and persistent infections

  • Evaluate impact on host immune responses

• Resistance Monitoring:

  • Assess resistance development frequency in complex infection models

  • Characterize fitness costs of resistance mutations in vivo

  • Develop strategies to counter potential resistance mechanisms

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.

How does ATP synthase activity coordinate with other metabolic pathways in S. aureus adaptation to host environments?

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:

  • ATP synthase activity is intimately linked to the electron transport chain (ETC)

  • Mutations in NADH dehydrogenase (ndh2) affect membrane potential and subsequently alter ATP synthase function

  • This coordinated regulation allows metabolic flexibility in response to changing oxygen availability

• Adaptation to Anaerobiosis:

  • Under anaerobic conditions, S. aureus shifts to fermentative metabolism

  • ATP synthase activity is modulated to accommodate the reduced proton motive force

  • This adaptation enables survival in oxygen-limited infection sites such as deep abscesses or biofilms

Carbon Metabolism Regulatory Networks:

• CcpA-Mediated Regulation:

  • Catabolite control protein A (CcpA) mutations confer intermediate resistance to ATP synthase inhibitors

  • CcpA regulates carbon source utilization and influences ATP synthase expression

  • This regulatory connection ensures appropriate energy production based on available nutrients

• Acidification Requirements:

  • ATP synthase function is linked to intracellular acidification processes

  • This acidification is required for optimal activity of fermentative enzymes

  • The coordinated regulation ensures energy generation when respiration is compromised

Membrane Physiology Coordination:

• Surface Hydrophobicity Regulation:

  • ATP synthase activity influences cell surface hydrophobicity

  • Prototypic cells with high ATP production display low surface hydrophobicity

  • This may involve effects on membrane lipid composition and teichoic acid synthesis

• Proton Motive Force Maintenance:

  • ATP synthase helps maintain the proton motive force across the membrane

  • This affects numerous transport processes and cellular functions

  • Inhibition of ATP synthase causes reduction in membrane potential in both WT and SCV strains

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:

  • ATP synthase activity may influence the expression of antioxidant enzymes

  • This coordination helps manage oxidative stress during host-pathogen interactions

  • The balance affects bacterial survival during phagocyte encounters

Host-Adaptive Metabolic Shifts:

• Small Colony Variant Phenotype:

  • Reduced ATP synthase activity is a hallmark of the SCV phenotype

  • This metabolic adaptation promotes persistence in hostile host environments

  • The coordinated downregulation of multiple metabolic pathways enables long-term survival

• Biofilm-Specific Metabolism:

  • ATP synthase function differs between planktonic and biofilm growth states

  • This influences the characteristic antibiotic tolerance of biofilms

  • Coordination with other metabolic pathways shapes the immune-modulating properties of biofilms

Understanding these complex metabolic interactions provides insights into S. aureus adaptability and identifies potential combination therapeutic targets to disrupt these coordinated responses.

What are the molecular mechanisms by which AtpE inhibition leads to bacterial cell death in S. aureus?

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:

  • Direct inhibition of ATP synthesis reduces cellular ATP levels

  • This energy depletion affects numerous ATP-dependent cellular processes

  • Small colony variants (SCVs) are particularly vulnerable due to their already limited energy production capacity

• Membrane Potential Disruption:

  • Tomatidine causes significant reduction in membrane potential in both wild-type and SCV S. aureus

  • This disruption affects various membrane-dependent processes

  • The magnitude of this effect correlates with bactericidal activity

Secondary Consequences Leading to Cell Death:

• Reactive Oxygen Species (ROS) Generation:

  • ATP synthase inhibition by tomatidine triggers substantial ROS production in SCVs

  • The combination of tomatidine with aminoglycosides generates 2.5-fold more ROS in wild-type strains compared to aminoglycosides alone

  • These elevated ROS levels cause oxidative damage to DNA, proteins, and lipids

• Cell Envelope Integrity Compromise:

  • Disruption of ATP synthase affects cell wall and membrane biosynthesis

  • This leads to structural weaknesses and potential cell lysis

  • Cell lysis releases immunostimulatory components that enhance host inflammatory responses

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

How do differences in ATP synthase structure and function between S. aureus and other bacterial pathogens impact inhibitor development?

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:

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.