Recombinant Staphylococcus carnosus ATP synthase subunit c (atpE)

What is the function of ATP synthase subunit c in S. carnosus?

ATP synthase subunit c, encoded by the atpE gene, forms the critical rotor component of the F0 domain in the F1F0-ATP synthase complex. This rotor turns when a proton flux enters the ATP synthase, generating the energy necessary for ATP synthesis. The c-subunit forms a ring structure that rotates as protons pass through the membrane domain, coupling proton movement to the conformational changes required for ATP production in the F1 catalytic domain. This mechanism is fundamental to energy production across bacterial species including S. carnosus, similar to the well-characterized process in S. aureus .

What are the optimal vector systems for expressing recombinant atpE in S. carnosus?

For recombinant expression of atpE in S. carnosus, several proven vector systems can be employed. The pSPPmABPXM expression system has demonstrated high efficiency for heterologous protein expression in S. carnosus . This vector contains origins of replication compatible with both E. coli and Staphylococcus species, facilitating initial cloning steps in E. coli followed by transformation into S. carnosus. For atpE expression specifically, inserting the gene sequence using BamHI and XhoI restriction sites in the pSPPmABPXM plasmid would be advantageous, as this approach has successfully expressed other recombinant proteins . Chloramphenicol (10 μg/ml) should be used for selection in S. carnosus transformants. Alternative systems including pSE-derived vectors could also be considered, though they have shown lower efficiency for surface display in comparative studies with S. carnosus .

What methods are most effective for transforming S. carnosus with recombinant atpE constructs?

The most effective transformation method for S. carnosus involves protoplast preparation followed by polyethylene glycol (PEG)-mediated DNA uptake. The protocol adapted from Götz's method has proven particularly successful . The procedure involves:

• Growing S. carnosus TM300 to mid-logarithmic phase (A578 ≈ 0.5-0.7)

• Harvesting cells and resuspending in hypertonic buffer containing lysostaphin

• Enzymatic digestion of the cell wall to generate protoplasts

• Gentle washing and stabilization of protoplasts in osmotically balanced media

• Mixing protoplasts with 1-5 μg of plasmid DNA in the presence of PEG

• Recovery in regeneration medium for 2-3 hours

• Plating on selection media containing chloramphenicol (10 μg/ml)

This method typically yields transformation efficiencies of 103-104 transformants per μg DNA . Electroporation has been attempted but generally shows lower efficiency for S. carnosus compared to the protoplast method.

How can researchers verify successful expression of recombinant atpE in S. carnosus?

Verification of recombinant atpE expression in S. carnosus should employ multiple complementary techniques:

• Western Blot Analysis: Prepare cell lysates from overnight cultures of transformed S. carnosus, separate proteins by SDS-PAGE, and transfer to membranes for immunodetection. Use antibodies specific to atpE or to fusion tags incorporated in the construct. This method confirms proper protein synthesis and expected molecular weight .

• Surface Display Verification: If atpE is designed for surface display, employ an immunofluorescence assay using whole cells. Incubate intact recombinant bacteria with specific antibodies followed by fluorescently-labeled secondary antibodies, then analyze using flow cytometry or fluorescence microscopy .

• Functional Assays: Measure ATP synthesis activity using luciferin-luciferase assays to quantify ATP production. Compare ATP synthesis rates between wild-type and recombinant strains to assess functional expression.

• Extraction and Analysis of Cell Fractions: Separate cell wall, membrane, and cytoplasmic fractions using differential centrifugation after enzymatic treatment with lysostaphin. This approach, as demonstrated with other S. carnosus recombinant proteins, helps determine the subcellular localization of expressed atpE .

A combination of these techniques provides comprehensive validation of recombinant atpE expression, localization, and functionality.

What structural models best explain the interaction between atpE and antimicrobial compounds?

Structural modeling of S. carnosus atpE interactions with antimicrobial compounds can be approached through homology modeling based on known structures of bacterial ATP synthase c-subunits. Currently, no crystal structure specifically for S. carnosus atpE exists, but models can be constructed using the high sequence similarity to S. aureus atpE.

Key findings from resistance studies indicate that specific residues in atpE are critical for antimicrobial binding. For example, mutations A17S and S26L in S. aureus atpE confer resistance to tomatidine, suggesting these positions form part of the binding pocket . Similarly, mutations at positions A28, G61, A63, and I66 in mycobacterial atpE affect bedaquiline binding .

A proposed structural model would include:

• The two transmembrane helices forming a hairpin structure

• A binding pocket formed at the interface between adjacent c-subunits in the c-ring

• Key residues that interact with the antimicrobial compound through hydrogen bonding and hydrophobic interactions

This model explains the high selectivity of compounds like tomatidine derivatives for bacterial versus mitochondrial ATP synthases, with a selectivity index estimated to be >105-fold for the compound FC04-100 .

How can recombinant S. carnosus atpE be used to screen for novel antimicrobial compounds?

Recombinant S. carnosus expressing wild-type or mutant forms of atpE can serve as a powerful platform for screening novel antimicrobial compounds. A comprehensive screening approach would include:

• Whole-Cell Viability Assays: Express wild-type atpE in S. carnosus and evaluate antimicrobial compound efficacy using growth inhibition assays. Calculate minimum inhibitory concentrations (MICs) for each compound.

• Resistance Profiling: Generate a panel of S. carnosus strains expressing atpE with known resistance mutations (e.g., A17S, S26L equivalents from S. aureus). Test compounds against this panel to identify cross-resistance patterns and predict binding mechanisms .

• ATP Synthesis Inhibition Assay: Measure ATP production in inverted membrane vesicles prepared from recombinant S. carnosus in the presence of test compounds. A correlation between antibiotic potency and ATP synthase inhibition would confirm the target mechanism, as demonstrated with tomatidine derivatives .

• Structure-Activity Relationship Analysis: Systematically modify chemical structures of lead compounds and test against wild-type and mutant atpE, as was done with tomatidine to develop FC04-100, which showed improved activity against both small-colony variants and prototypical strains .

• Selectivity Assessment: Compare inhibition of bacterial ATP synthase versus mitochondrial ATP synthase to calculate selectivity indices, which is critical for developing safe antimicrobials.

This multi-faceted approach enables identification of compounds that specifically target bacterial ATP synthase with limited cross-resistance and high selectivity over mammalian homologs.

What resistance mechanisms develop against ATP synthase inhibitors in Staphylococcus species?

Resistance to ATP synthase inhibitors in Staphylococcus species primarily develops through specific mutations in the atpE gene. Based on studies with S. aureus, several distinct resistance mechanisms have been characterized:

• Target Site Mutations: Point mutations in atpE that alter the binding site for inhibitors represent the primary mechanism. Identified mutations include:

  • A17S substitution in the first transmembrane helix of atpE, observed in high-level tomatidine-resistant S. aureus isolates

  • S26L substitution, also conferring high-level tomatidine resistance through a different nucleotide substitution (C77T)

  • Similar to bedaquiline resistance in mycobacteria where A28V, A28P, G61A, A63P, and I66M mutations confer 10-128-fold increases in MIC

• Metabolic Adaptation: Secondary mutations in metabolic regulators, such as the G149V substitution in the catabolite control protein A (CcpA) gene found in S. aureus, can contribute to resistance by altering bacterial metabolism to compensate for reduced ATP synthase function .

• Efflux Systems: While not directly documented for ATP synthase inhibitors in Staphylococcus, homologous systems to the MmpS5-MmpL5 efflux system (upregulated in bedaquiline-resistant mycobacteria) may play a role in reduced susceptibility .

Interestingly, resistance development patterns differ between in vitro selection and clinical settings, suggesting fitness costs associated with some resistance mutations under physiological conditions . Compounds like FC04-100, a tomatidine derivative, show advantages in limiting high-level resistance development, particularly in prototypic strains .

What genomic approaches can identify mutations associated with atpE inhibitor resistance?

Advanced genomic approaches for identifying mutations associated with atpE inhibitor resistance include:

• Whole-Genome Sequencing (WGS) of Resistant Isolates: Generate inhibitor-resistant S. carnosus through serial passages with increasing inhibitor concentrations, then sequence the entire genome of resistant isolates and compare to the parent strain. This approach successfully identified mutations in atpE (G49T leading to A17S) and ccpA (G149V) in tomatidine-resistant S. aureus .

• Targeted Deep Sequencing: Focus sequencing efforts on the atpE gene and known resistance-associated genes to achieve higher coverage and detect low-frequency mutations that might be missed by WGS.

• Transcriptomics Analysis: Compare gene expression profiles between susceptible and resistant strains using RNA-Seq to identify compensatory mechanisms and pathways activated in response to atpE inhibition.

• Functional Validation Through Gene Overexpression: Confirm the role of identified mutations by overexpressing mutant atpE genes in susceptible strains, as demonstrated with S. aureus where overexpression of atpE containing resistance mutations conferred resistance to tomatidine .

• Cross-Species Validation: Introduce identified mutations into homologous atpE genes in different bacterial species to confirm the conserved mechanism of resistance, similar to the introduction of S. aureus resistance mutations into Bacillus subtilis atpE .

These genomic approaches should be complemented with biochemical and structural studies to fully characterize the resistance mechanisms and inform the development of inhibitors less prone to resistance.

How can researchers design ATP synthase inhibitors that minimize resistance development?

Designing ATP synthase inhibitors with reduced resistance potential requires multifaceted approaches based on structural, functional, and evolutionary insights:

• Target Conserved Residues: Design inhibitors that interact with highly conserved residues in atpE that cannot tolerate mutations without significant fitness costs. Computational analysis of sequence conservation across bacterial species can identify these critical residues.

• Multi-Site Binding: Develop compounds that simultaneously interact with multiple sites on the ATP synthase, requiring multiple mutations for resistance to develop. The tomatidine derivative FC04-100 demonstrates this principle, showing reduced resistance development compared to the parent compound .

• Dual-Target Inhibitors: Design molecules that inhibit both ATP synthase and a second essential bacterial target, requiring simultaneous mutations in two targets for resistance to emerge.

• Structure-Based Design: Utilize homology models of S. carnosus atpE based on crystal structures from related species to design inhibitors that maximize interactions with the target while maintaining selectivity over human ATP synthase.

• Resistance Mutation Prediction: Employ computational approaches to predict potential resistance mutations and preemptively design inhibitors that maintain activity against these predicted mutants.

• Pharmacokinetic Optimization: Design inhibitors with optimal pharmacokinetic properties to achieve high concentrations at the infection site, exceeding the mutant prevention concentration (MPC) to suppress the emergence of resistant subpopulations.

Implementation of these strategies requires integration of structural biology, medicinal chemistry, microbial genetics, and computational modeling to develop robust ATP synthase inhibitors less prone to resistance development.

What are the optimal conditions for expressing recombinant atpE in S. carnosus for structural studies?

Optimizing expression conditions for structural studies of recombinant atpE requires careful consideration of growth parameters and expression systems:

• Vector Selection: The pSPPmABPXM vector system has demonstrated high-level expression of recombinant proteins in S. carnosus . For structural studies, consider incorporating a C-terminal His6-tag for purification while maintaining protein functionality.

• Growth Conditions:

  • Medium: Basic broth medium supplemented with 1% glucose and appropriate antibiotics (chloramphenicol at 10 μg/ml)

  • Temperature: Growth at 30°C rather than 37°C often improves proper folding of membrane proteins

  • Induction: If using an inducible promoter, optimize inducer concentration and induction timing (typically at mid-log phase, A578 ≈ 0.5-0.7)

  • Duration: Extended expression times (16-24 hours) at lower temperatures may improve yield of properly folded protein

• Membrane Extraction Protocol:

  • Harvest cells and wash in phosphate buffer

  • Cell wall removal using lysostaphin (0.5 mg/ml) in isotonic buffer

  • Membrane fraction isolation through differential centrifugation

  • Solubilization of membranes using mild detergents like n-dodecyl β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG)

• Protein Stabilization:

  • Include specific lipids like cardiolipin during purification to maintain native-like environment

  • Add ATP or non-hydrolyzable ATP analogs to stabilize the protein complex

  • Optimize buffer conditions (pH 6.5-7.5, 100-300 mM NaCl) for protein stability

These conditions should be empirically optimized for S. carnosus specifically, as parameters may differ from those established for other Staphylococcus species .

What methods are most effective for purifying functional atpE from recombinant S. carnosus?

Purification of functional atpE from recombinant S. carnosus for enzymatic and structural studies requires specialized techniques for membrane protein isolation:

• Cell Disruption and Membrane Preparation:

  • Enzymatic cell wall digestion with lysostaphin (0.5 mg/ml) to create protoplasts

  • Gentle lysis of protoplasts using osmotic shock or French press

  • Isolation of membrane fraction by ultracentrifugation (100,000 × g for 1 hour)

  • Washing membranes to remove peripheral proteins

• Detergent Solubilization:

  • Screen detergents for optimal solubilization efficiency and protein stability

  • Recommended detergents: n-dodecyl β-D-maltoside (DDM) at 1% for initial solubilization

  • Solubilize at 4°C with gentle agitation for 1-2 hours

  • Remove insoluble material by ultracentrifugation (100,000 × g for 30 minutes)

• Affinity Chromatography:

  • For His-tagged constructs, use Ni-NTA or TALON resin

  • Apply solubilized membrane fraction to equilibrated resin

  • Wash extensively with buffer containing low imidazole (20-30 mM) and reduced detergent (0.05% DDM)

  • Elute with higher imidazole concentrations (250-300 mM)

• Size Exclusion Chromatography:

  • Further purify by gel filtration to isolate properly assembled c-ring complexes

  • Superdex 200 or similar matrix in buffer containing 0.05% DDM, 150 mM NaCl, 20 mM Tris-HCl pH 7.5

• Functional Validation:

  • Assess protein folding by circular dichroism spectroscopy

  • Verify ATP synthase activity using reconstituted proteoliposomes and ATP production assays

  • Confirm inhibitor binding through thermal shift assays or isothermal titration calorimetry

This purification scheme should yield homogeneous, functional atpE protein suitable for enzymatic assays, antibody production, or structural studies .