Recombinant Staphylococcus epidermidis ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) in Staphylococcus epidermidis?

ATP synthase subunit c, encoded by the atpE gene in Staphylococcus epidermidis, is a critical component of the F0 domain of bacterial ATP synthase. This protein forms the c-ring structure within the membrane-embedded portion of the ATP synthase complex, which is essential for proton translocation and subsequent ATP synthesis. The atpE gene product is highly conserved across bacterial species, particularly within the Staphylococcus genus. Sequence alignment studies have revealed 100% identity in ATP synthase subunit C amino acid sequences across more than 1,000 strains of S. aureus and S. epidermidis, highlighting its evolutionary conservation and functional importance . This protein plays a crucial role in cellular bioenergetics and has been identified as a cellular target for certain antibiotic compounds, such as tomatidine and its derivatives, particularly in small colony variants (SCVs) of staphylococci.

Why is recombinant expression of S. epidermidis atpE important for research?

Recombinant expression of S. epidermidis atpE is essential for multiple research applications, including structural studies, functional characterization, and drug development efforts. By producing the protein in controlled laboratory conditions, researchers can obtain sufficient quantities of pure protein for detailed biochemical and biophysical analyses. This approach allows for the isolation of the protein away from its native complex, enabling the study of its specific properties without interference from other cellular components.

The recombinant expression also facilitates the introduction of specific mutations to study structure-function relationships, as demonstrated in studies where mutations in atpE were analyzed for their effects on antibiotic resistance and ATP synthesis capability . Additionally, recombinantly expressed atpE can be used in binding assays to screen for potential inhibitors, which is valuable for antibiotic development. The expression of atpE in surrogate hosts, such as demonstrated with other S. epidermidis proteins in S. carnosus, provides a platform to study protein interactions without interference from other staphylococcal surface proteins .

How does bacterial ATP synthase differ from mitochondrial ATP synthase?

Bacterial ATP synthase, including that of S. epidermidis, differs from mitochondrial ATP synthase in several key structural and functional aspects, making it a selective target for antibiotics. The c-subunit of bacterial ATP synthase typically contains a single hairpin domain with two transmembrane helices, whereas the mitochondrial counterpart often has additional structural elements. These structural differences create distinct binding pockets for inhibitors.

The bacterial ATP synthase also operates in a different membrane environment and may have distinct regulatory mechanisms. Furthermore, in bacteria like S. epidermidis, ATP synthase plays additional roles beyond ATP production, including maintenance of pH homeostasis, which is not a primary function in mitochondria . These differences provide opportunities for developing highly selective antibacterial agents that target bacterial ATP synthase without affecting host mitochondrial function.

What expression systems are suitable for recombinant production of S. epidermidis atpE?

Several expression systems have proven effective for the recombinant production of staphylococcal proteins, including atpE. Based on the experimental approaches described in the literature, the following systems are particularly suitable:

E. coli-based expression systems:
The BL21-DE3 strain of E. coli has been successfully used for the expression of staphylococcal proteins. This system allows for high-yield protein production when the gene of interest is cloned into vectors containing T7 promoters, such as the pET series. Expression can be induced using IPTG at concentrations of approximately 1 mM, with optimal results often achieved at lower temperatures (28°C) for 16 hours to prevent inclusion body formation .

Surrogate staphylococcal hosts:
For functional studies where the protein needs to be expressed in a more native-like cellular context, surrogate hosts such as Staphylococcus carnosus TM300 provide an excellent alternative. This approach is particularly valuable when studying membrane proteins like atpE or when investigating interactions with other staphylococcal components. Expression in S. carnosus can be induced using anhydrotetracycline (AHT) at concentrations of approximately 200 ng/ml .

Inducible systems in native S. epidermidis:
For studies requiring expression in the native organism, inducible promoter systems have been developed for S. epidermidis. The Pxyl/tet promoter system allows controlled expression of the target gene and has been used successfully for studying S. epidermidis proteins .

Selection of the optimal expression system should be based on the specific research goals, considering factors such as required protein yield, need for post-translational modifications, downstream applications, and whether the protein needs to be correctly inserted into membranes.

What purification strategies are most effective for recombinant S. epidermidis atpE?

Purifying recombinant S. epidermidis atpE requires specialized approaches due to its hydrophobic nature as a membrane protein. Based on successful purification strategies described for similar staphylococcal membrane proteins, a multi-step purification protocol is recommended:

Initial extraction and solubilization:

• Cell lysis is typically performed using sonication (30% amplitude, 15s on/off cycles) for approximately 3 minutes while maintaining samples on ice .

• Following centrifugation to remove cell debris, the membrane fraction can be isolated through ultracentrifugation.

• Membrane proteins require careful solubilization using mild detergents such as n-dodecyl-β-D-maltoside (DDM) or n-octyl-β-D-glucopyranoside (OG) at concentrations slightly above their critical micelle concentration.

Chromatographic purification sequence:

• Immobilized metal affinity chromatography (IMAC): Using Ni-NTA columns for His-tagged proteins with binding and washing buffers containing detergent to maintain protein solubility .

• Ion exchange chromatography (IEX): HiTrap Q FF columns can provide further purification and concentration of the target protein .

• Size exclusion chromatography (SEC): Final polishing step using columns such as Superdex 200 to separate the target protein from aggregates and other impurities .

Throughout the purification process, it's critical to maintain the protein in a properly solubilized state by including appropriate detergents in all buffers. For functional studies, detergent exchange to more suitable options for activity assays may be necessary in the final purification steps.

Quality assessment should be performed using SDS-PAGE after each purification step, and final protein identity can be confirmed using Western blotting with anti-His antibodies or mass spectrometry.

How can ATP synthase activity be measured in recombinant S. epidermidis atpE preparations?

Measuring ATP synthase activity of recombinant S. epidermidis atpE requires specialized assays that typically involve reconstitution of the purified protein into liposomes or the use of inverted membrane vesicles. Based on established protocols for bacterial ATP synthase activity measurements, the following approaches are recommended:

Inverted membrane vesicle assay:
This assay has been effectively used to measure ATP synthase activity in S. aureus, which shares 100% identity in ATP synthase subunit C with S. epidermidis . The procedure involves:

• Preparation of inverted membrane vesicles from bacterial cultures through French press or sonication techniques.

• Incubation of vesicles with ADP and inorganic phosphate in the presence of an artificial proton gradient.

• Measurement of ATP production using luminescence-based ATP detection kits or coupled enzyme assays with luciferase.

This method allows for the evaluation of ATP synthesis rates and can also be adapted to test the effects of potential inhibitors by measuring IC50 values. Research has shown that mutations in atpE can significantly impact ATP production capacity, with resistant mutants often showing reduced ATP synthesis capabilities compared to wild-type strains .

Reconstituted proteoliposome assay:
For more controlled studies with purified recombinant atpE:

• Reconstitution of purified atpE into liposomes with other essential ATP synthase subunits.

• Establishment of a proton gradient using techniques such as acid-base transitions or light-activated proton pumps co-reconstituted into the liposomes.

• Measurement of ATP synthesis using sensitive detection methods as described above.

These functional assays are essential for confirming that the recombinant protein maintains its native activity and for studying the effects of mutations or inhibitors on ATP synthase function.

How can site-directed mutagenesis of S. epidermidis atpE inform antibiotic resistance mechanisms?

Site-directed mutagenesis of S. epidermidis atpE provides valuable insights into antibiotic resistance mechanisms and structure-function relationships of ATP synthase. This approach has been particularly informative in understanding resistance to ATP synthase inhibitors and can guide rational drug design efforts.

Experimental approach for structure-function studies:

• Identification of resistance-associated mutations: Begin by analyzing the sequences of atpE from antibiotic-resistant strains that emerged either clinically or through in vitro selection experiments. Studies with tomatidine have successfully used in vitro-generated resistant S. aureus strains to identify key mutations in atpE associated with resistance .

• Rational design of mutagenesis targets: Based on structural models of ATP synthase subunit c, design mutations at conserved residues predicted to interact with inhibitors or contribute to critical functional domains.

• Site-directed mutagenesis protocol: Introduce specific mutations into the atpE gene using techniques such as QuikChange mutagenesis or overlap extension PCR. The mutated gene can then be expressed in appropriate systems as described in section 2.1.

• Functional assessment of mutants: Compare ATP synthesis capabilities of wild-type and mutant proteins using the assays described in section 2.3. Research has demonstrated that mutations in atpE that confer resistance to inhibitors like tomatidine often result in reduced ATP synthesis capacity compared to wild-type enzymes .

The correlation between resistance levels and reduced ATP synthesis capability suggests that there is a functional trade-off between resistance and bioenergetic efficiency. This relationship is particularly pronounced in small colony variants (SCVs), where ATP synthesis is already compromised due to defects in the electron transport chain .

What is the role of S. epidermidis atpE in biofilm formation and persistence?

ATP synthase subunit c (atpE) plays a significant but often overlooked role in S. epidermidis biofilm formation and persistence through its central function in bacterial energy metabolism. While not directly involved in the structural aspects of biofilm formation, atpE's contribution to energy homeostasis profoundly influences this process.

Energy requirements during biofilm development:
Biofilm formation is an energy-intensive process that requires ATP for initial attachment, production of extracellular polymeric substances (EPS), and maintenance of cellular processes under the stressed conditions of biofilm life. S. epidermidis is a canonical opportunistic biofilm former, particularly notorious for forming biofilms on medical implants . The role of ATP synthase becomes crucial during:

• Initial adhesion phase: Surface attachment and the expression of adhesins like Embp (extracellular matrix-binding protein) require energy provided by ATP synthase .

• Maturation phase: The production of extracellular polymeric substances requires significant metabolic activity and energy expenditure.

• Persistence phase: Bacteria in established biofilms often enter a state of reduced metabolic activity, similar to small colony variants (SCVs), where ATP synthase function becomes especially important for survival with limited resources .

Metabolic adaptation in biofilms:
Studies have shown that extensive metabolic remodeling occurs in persistent bacteria such as SCVs and in biofilm-producing bacteria . Within biofilms, bacteria often face oxygen limitation and must adapt their energy generation strategies. ATP synthase activity becomes modulated to maintain essential cellular functions with minimal energy expenditure. This metabolic adaptation contributes significantly to antibiotic tolerance in biofilms, as many antibiotics target actively growing cells.

Therapeutic implications:
The critical role of ATP synthase in biofilm persistence makes it an attractive target for anti-biofilm strategies. Compounds targeting ATP synthase, such as tomatidine derivatives, have shown promise in combating persistent infections, including those involving biofilms . The high conservation of atpE across staphylococcal species (100% identity across >1,000 strains) makes it a potentially broad-spectrum target for anti-biofilm compounds .

How does S. epidermidis atpE compare with atpE from other clinically relevant staphylococcal species?

The ATP synthase subunit c (atpE) demonstrates remarkable conservation across staphylococcal species while maintaining distinct characteristics compared to more distantly related bacteria. This comparative analysis provides insights into evolutionary relationships and potential species-specific therapeutic targeting.

Sequence conservation among staphylococcal species:
Detailed sequence analysis has revealed 100% identity in ATP synthase subunit C amino acid sequences across more than 1,000 strains of S. aureus and S. epidermidis . This extraordinary level of conservation suggests:

• Strong evolutionary pressure to maintain the exact sequence of this protein, indicating its critical functional importance.

• Limited tolerance for mutations that might confer resistance to atpE-targeting antibiotics without severely compromising bacterial fitness.

• Potential broad-spectrum activity of atpE-targeting compounds against multiple staphylococcal species.

Functional conservation and specialization:
Despite sequence identity, the functional context of ATP synthase may differ between species:

• Role in pH homeostasis: In addition to ATP production, ATP synthase plays a role in pH homeostasis in Bacillales including Listeria monocytogenes . This function may be particularly important in the diverse ecological niches occupied by different staphylococcal species.

• Essential nature: Studies with Bacillus subtilis have shown that deletion of ATP synthase greatly affects growth . The essentiality of ATP synthase may vary between staphylococcal species depending on their metabolic flexibility and environmental adaptations.

• Response to inhibitors: Despite sequence identity, the susceptibility to ATP synthase inhibitors can vary between staphylococcal species and strains due to differences in membrane composition, efflux pump expression, or metabolic adaptations. Tomatidine and its analog FC04-100 show a narrow yet specific spectrum of activity against SCVs of Bacillales .

Taxonomic and therapeutic implications:
The high conservation of atpE within the Staphylococcus genus but divergence from other bacterial groups and human mitochondrial ATP synthase has significant implications for drug development. This pattern of conservation allows for the design of inhibitors with high selectivity indices (>10^5-fold for compounds like FC04-100) , minimizing off-target effects on human cells while potentially addressing multiple staphylococcal species with a single therapeutic agent.

What are the current challenges in expressing and studying recombinant S. epidermidis atpE?

Despite significant progress in understanding ATP synthase biology, several technical and conceptual challenges remain in the expression and study of recombinant S. epidermidis atpE:

Technical challenges in recombinant expression:

• Membrane protein solubility: As a highly hydrophobic membrane protein, atpE presents challenges in expression, solubilization, and maintaining proper folding during purification. Current detergent-based approaches may not optimally preserve the native conformation and interactions of the protein.

• Functional reconstitution: Studying atpE function requires reconstitution of complex multisubunit assemblies, as the c-subunit alone is not functional without other ATP synthase components. Creating artificial systems that accurately mimic the native environment remains technically demanding.

• Post-translational modifications: Potential species-specific post-translational modifications may not be correctly introduced in heterologous expression systems, potentially affecting protein function or interaction studies.

Conceptual and biological challenges:

• Strain-level heterogeneity: S. epidermidis exhibits high strain-level heterogeneity , which may extend to subtle variations in ATP synthase regulation or assembly despite sequence conservation of atpE itself. Understanding these strain-specific differences requires comprehensive comparative studies.

• Biofilm-specific adaptations: ATP synthase function may be differently regulated in biofilm environments compared to planktonic growth, but studying protein expression and function within intact biofilms presents methodological difficulties.

• Host-pathogen interactions: The potential role of ATP synthase components in host-pathogen interactions during colonization or infection remains understudied, partly due to technical limitations in modeling these complex interactions in vitro.

Emerging solutions:
Advanced technologies like cryo-electron microscopy, nanodiscs for membrane protein stabilization, and microfluidic systems for biofilm studies offer promising approaches to address these challenges. Additionally, CRISPR-based genome editing techniques have opened new possibilities for studying atpE function in its native context through precise genetic manipulation of S. epidermidis.

How might S. epidermidis atpE be targeted for novel antimicrobial development?

S. epidermidis atpE represents a promising target for novel antimicrobial development due to its essential function, high conservation, and demonstrated vulnerability to selective inhibitors. Several strategic approaches show particular promise:

Rational drug design approaches:

• Structure-based design: Using molecular modeling and crystal structures of bacterial ATP synthase c-subunits to design compounds that specifically interact with conserved binding pockets. Studies with tomatidine derivatives have validated this approach by demonstrating selective inhibition of bacterial ATP synthase with minimal effects on mitochondrial ATP production .

• Natural product derivatives: Modification of natural compounds known to interact with ATP synthase, such as tomatidine, to enhance their specificity and pharmacological properties. The tomatidine derivative FC04-100 has shown promising activity against small colony variants of staphylococci with an exceptional selectivity index of >10^5-fold compared to human mitochondrial ATP synthase .

• Peptidomimetics: Design of peptides that mimic natural antimicrobial peptides known to target bacterial membranes and membrane proteins, with specific modifications to enhance interaction with atpE.

Potential advantages of atpE-targeting antimicrobials:

• Activity against persistent infections: Compounds targeting ATP synthase have shown activity against metabolically altered bacteria such as small colony variants, which are often implicated in persistent infections .

• Reduced resistance development: The 100% conservation of atpE across >1,000 staphylococcal strains suggests limited tolerance for mutations without significant fitness costs . This constraint may reduce the frequency of resistance development compared to other antibiotic targets.

• Biofilm activity: As bacteria in biofilms undergo metabolic remodeling, ATP synthase becomes particularly important for survival in this state . Targeting atpE may therefore provide enhanced activity against biofilm-associated infections, which are particularly problematic with S. epidermidis on medical devices .

Development considerations:
Successful development of atpE-targeting antimicrobials will require careful consideration of pharmacokinetic properties, particularly membrane permeability and stability. Additionally, combination therapies with conventional antibiotics may enhance efficacy against heterogeneous bacterial populations and reduce resistance development.

What insights can recombinant S. epidermidis atpE provide for understanding bacterial bioenergetics in diverse environments?

Recombinant S. epidermidis atpE serves as a valuable model for understanding broader principles of bacterial bioenergetics across diverse environmental conditions, particularly in the context of host-associated lifestyles and antimicrobial resistance:

Adaptation to microenvironmental niches:
S. epidermidis colonizes diverse microenvironments on human skin with varying oxygen availability, pH, and nutrient conditions . Recombinant atpE studies can reveal how ATP synthase function adapts to these diverse conditions:

• Oxygen limitation: In skin crevices and deeper layers of biofilms where oxygen is limited, ATP synthase function becomes particularly crucial for energy conservation. Studies with recombinant atpE can elucidate adaptations to microaerobic and anaerobic conditions relevant to infection settings.

• pH adaptation: ATP synthase plays a role in pH homeostasis in Bacillales . Recombinant systems allow controlled studies of how atpE function responds to the variable pH environments encountered on skin and during infection.

• Nutrient limitation: During colonization of nutrient-poor environments such as implant surfaces, ATP synthesis efficiency becomes critical for survival. Recombinant systems enable precise measurement of ATP synthase efficiency under controlled nutrient conditions.

Bioenergetic basis of antibiotic tolerance:
Recombinant atpE studies provide insights into how energy metabolism relates to antibiotic tolerance:

• SCV phenotype: Small colony variants (SCVs) with altered respiratory chains show distinct susceptibility profiles to ATP synthase inhibitors . Recombinant atpE studies can clarify how ATP synthase compensates for electron transport chain defects in these persistent variants.

• Resistance trade-offs: Mutations in atpE that confer resistance to inhibitors typically result in reduced ATP synthesis capacity . Quantitative studies with recombinant systems can precisely determine these energetic trade-offs and their implications for bacterial fitness.

Comparative bioenergetics across bacterial taxa:
The remarkable conservation of atpE across staphylococcal species provides an opportunity for comparative studies:

• Species-specific adaptations: Despite sequence identity, differences in the regulatory context of ATP synthase between species may reveal adaptations to specific ecological niches.

• Evolutionary constraints: The 100% conservation across >1,000 strains suggests strong evolutionary pressure, which can be further explored through comparative studies with more distantly related taxa to identify the key features under selection.

These insights from recombinant atpE studies contribute to our fundamental understanding of bacterial bioenergetics while also informing practical applications in antimicrobial development and biotechnology.

What are common pitfalls in recombinant S. epidermidis atpE expression and how can they be addressed?

Recombinant expression of membrane proteins like S. epidermidis atpE presents several common challenges that researchers should anticipate and address proactively:

Expression and toxicity issues:

• Toxicity to expression host: Overexpression of membrane proteins often leads to toxicity in the host organism. This can be mitigated by using tightly controlled inducible systems with careful optimization of induction conditions. Lower induction temperatures (28°C instead of 37°C) and reduced inducer concentrations often improve viable protein yields .

• Inclusion body formation: Membrane proteins frequently form inclusion bodies when overexpressed. This can be addressed by:

  • Using specialized E. coli strains designed for membrane protein expression (C41/C43)

  • Adding fusion partners such as MBP (maltose-binding protein) or SUMO to enhance solubility

  • Optimizing growth temperature and inducer concentration

  • Considering expression in a native-like host such as S. carnosus TM300

• Codon bias: Differences in codon usage between S. epidermidis and expression hosts can hinder translation. This can be addressed by:

  • Codon optimization of the gene sequence for the expression host

  • Using expression hosts with supplemental tRNAs for rare codons

Purification challenges:

• Inefficient solubilization: Incomplete extraction from membranes is common with hydrophobic proteins like atpE. Systematic screening of detergents including DDM, OG, LDAO, and newly developed detergents like GDN can identify optimal solubilization conditions.

• Aggregation during purification: Protein aggregation during purification can be minimized by:

  • Maintaining detergent above critical micelle concentration in all buffers

  • Including stabilizing agents such as glycerol (10-20%)

  • Keeping samples cold throughout purification

  • Adding lipids to stabilize the protein-detergent complex

• Low purity or yield: This can be addressed through optimization of the multi-step purification strategy, potentially incorporating additional chromatography steps like hydroxyapatite chromatography or using alternative affinity tags.

Verification of proper folding:
A critical but often overlooked challenge is verifying that the recombinant protein maintains its native conformation. This can be assessed through:

Addressing these challenges requires systematic optimization and often an iterative approach, but the resulting protocols can provide valuable insights for other membrane protein studies beyond atpE.

How can researchers optimize ATP synthase activity assays for screening inhibitors against S. epidermidis atpE?

Developing robust ATP synthase activity assays optimized for inhibitor screening requires careful consideration of assay design, controls, and data analysis. The following methodological approach has proven effective for similar screening efforts:

Assay platform optimization:

• Membrane vesicle preparation:

  • Inverted membrane vesicles provide a relatively simple system that maintains the native lipid environment of atpE. These can be prepared through French press or sonication techniques from S. epidermidis or recombinant expression systems .

  • The vesicle quality can be assessed by measuring the proton gradient formation using pH-sensitive fluorescent dyes.

• Purified enzyme reconstitution:

  • For higher-throughput screening, purified and reconstituted ATP synthase can provide a more defined system.

  • Reconstitution into liposomes with defined lipid composition allows for investigation of lipid-dependent effects on inhibitor binding.

  • ATP synthase activity can be coupled to fluorescent or luminescent readouts for microplate-based screening.

Assay parameters for optimal sensitivity:

• Buffer composition:

  • pH optimization is critical as ATP synthase activity is highly pH-dependent

  • Ionic strength affects both enzyme activity and inhibitor binding

  • Addition of membrane stabilizers like BSA can improve assay robustness

• Substrate concentrations:

  • ADP and Pi concentrations should be optimized to operate at 0.5-1× Km values to ensure sensitivity to competitive inhibitors

  • ATP/ADP ratio modulation can help identify compounds affecting specific steps of the catalytic cycle

• Signal detection:

  • Luciferase-based ATP detection provides high sensitivity but may be subject to interference from test compounds

  • Coupled enzyme assays using phosphoenolpyruvate/pyruvate kinase/lactate dehydrogenase systems offer continuous monitoring but with more potential for off-target effects

  • Consider using orthogonal assays to confirm hits

Data analysis and validation approaches:

• Dose-response assessment:

  • Full dose-response curves rather than single-point measurements provide more reliable inhibitor characterization

  • IC50 determination should include appropriate controls such as oligomycin as a reference ATP synthase inhibitor

  • Studies with tomatidine derivatives have demonstrated the value of this approach, revealing steep transitions from partial to complete inhibition of ATP synthesis

• Counter-screening strategies:

  • Mitochondrial ATP synthase counter-screening is essential to determine selectivity indices

  • Compounds like FC04-100 have shown remarkable selectivity (>10^5-fold) between bacterial and mitochondrial ATP synthases

  • Additional counter-screens against common assay interference mechanisms (aggregators, redox cyclers, luciferase inhibitors) are recommended

• Validation with resistant mutants:

  • Testing compounds against atpE mutants known to confer resistance to other ATP synthase inhibitors provides validation of on-target activity

  • This approach has successfully validated tomatidine as an ATP synthase inhibitor using resistant mutants with defined mutations in atpE

Optimization of these parameters will yield a robust screening platform capable of identifying selective inhibitors of S. epidermidis ATP synthase with potential therapeutic applications.

How might cryo-EM studies of S. epidermidis ATP synthase contribute to drug discovery efforts?

Cryo-electron microscopy (cryo-EM) studies of S. epidermidis ATP synthase represent a frontier in structural biology with profound implications for antimicrobial drug discovery. This approach offers several advantages over traditional structural techniques and can significantly accelerate the development of selective ATP synthase inhibitors:

Structural insights into inhibitor binding:
Cryo-EM can provide high-resolution structures of ATP synthase in complex with inhibitors, revealing critical molecular interactions. For S. epidermidis atpE, such studies could:

• Define binding pockets: Identify precise binding sites of known inhibitors like tomatidine derivatives in the context of the entire ATP synthase complex . This would extend beyond what can be inferred from resistance mutations alone.

• Capture conformational states: Unlike crystallography, cryo-EM can capture multiple conformational states, providing insights into how inhibitors might interfere with the rotary mechanism of ATP synthase.

• Visualize lipid interactions: Cryo-EM can reveal specific lipid-protein interactions that may influence inhibitor binding, offering new targets for drug design.

Comparative structural biology:
The 100% sequence identity between S. epidermidis and S. aureus ATP synthase subunit c makes comparative structural studies particularly informative:

• Species-specific features: Despite sequence identity in atpE, differences in other subunits of the ATP synthase complex may reveal species-specific structural features that could be exploited for selective targeting.

• Resistance mechanisms: Structural comparison between wild-type and resistant mutant ATP synthases can provide detailed mechanistic understanding of how mutations confer resistance while typically reducing enzymatic efficiency .

Structure-based drug design applications:
Cryo-EM structures can directly inform rational drug design efforts:

• Fragment-based approaches: High-resolution structures enable fragment-based drug discovery, where small molecular fragments are identified as weak binders and then elaborated into more potent compounds.

• In silico screening: Cryo-EM structures provide templates for computational docking studies to screen virtual libraries for potential ATP synthase inhibitors.

• Structure-activity relationship (SAR) studies: Correlating structural data with the activity of compound series like tomatidine derivatives can guide medicinal chemistry optimization of lead compounds .

Technical considerations:
Successful cryo-EM studies of S. epidermidis ATP synthase will require overcoming technical challenges including:

• Stable expression and purification of the intact ATP synthase complex

• Optimization of detergent or nanodisc systems to maintain native-like membrane environment

• Development of strategies to capture functionally relevant conformational states

The resulting structures would provide unprecedented insights into the molecular machinery of S. epidermidis energy production and establish a foundation for developing next-generation antibiotics targeting this essential bacterial process.

What role might S. epidermidis atpE play in developing combination therapies for biofilm-associated infections?

ATP synthase subunit c (atpE) represents a promising component in developing effective combination therapies for biofilm-associated infections, particularly those involving medical devices where S. epidermidis is a primary concern . The strategic targeting of this protein could address several challenges in biofilm eradication:

Synergistic targeting of metabolically diverse subpopulations:
Biofilms contain bacterial subpopulations with diverse metabolic states, contributing to treatment recalcitrance. ATP synthase inhibitors can target these heterogeneous populations in several ways:

• Persister cell elimination: Persister cells and small colony variants (SCVs) within biofilms rely heavily on ATP synthase for survival, making them particularly vulnerable to atpE inhibitors. Research has shown that tomatidine derivatives have enhanced activity against SCVs of staphylococci .

• Metabolic potentiation: ATP synthase inhibitors can disrupt energy metabolism, potentially increasing bacterial susceptibility to other antibiotics that require active cellular processes for efficacy, such as cell wall synthesis inhibitors.

• Biofilm dispersal enhancement: Energy depletion through ATP synthase inhibition may trigger stress responses that promote biofilm dispersal, rendering bacteria more susceptible to conventional antibiotics.

Design principles for combination therapies:
Effective combination regimens targeting atpE would consider:

• Mechanistic complementarity: Pairing ATP synthase inhibitors with antibiotics targeting other essential processes (cell wall synthesis, protein synthesis, DNA replication) to create multiple simultaneous pressures on the bacterial population.

• Penetration enhancement: Combining ATP synthase inhibitors with compounds that enhance biofilm matrix penetration, such as matrix-degrading enzymes or surfactants.

• Resistance prevention: ATP synthase mutations conferring resistance typically result in reduced ATP production capacity , creating an evolutionary constraint that may be exploited in combination therapy design to minimize resistance development.

Novel delivery approaches:
The effectiveness of atpE-targeting combination therapies could be enhanced through innovative delivery strategies:

• Surface-functionalized implants: Medical devices coated with controlled-release formulations of ATP synthase inhibitors in combination with other antimicrobials could prevent initial biofilm formation.

• Nanoparticle delivery systems: Encapsulation in nanoparticles could enhance penetration into established biofilms and provide sustained release of ATP synthase inhibitors and companion drugs.

• Biofilm-responsive drug release: Smart delivery systems that respond to biofilm-specific signals could provide targeted release of ATP synthase inhibitors specifically at sites of biofilm formation.