Recombinant Staphylococcus aureus ATP synthase subunit b (atpF)

What is the role of ATP synthase subunit b (atpF) in Staphylococcus aureus metabolism?

ATP synthase subunit b (atpF) is an integral membrane protein that forms part of the F₀ portion of the ATP synthase complex in S. aureus. This complex is a central metabolic enzyme driven by the proton motive force generated by the respiratory chain, functioning primarily to synthesize ATP . The b subunit specifically serves as part of the peripheral stalk that connects the membrane-embedded F₀ portion to the catalytic F₁ portion. In S. aureus, ATP synthase is crucial for energy production, homeostasis, and maintenance of the proton motive force across the bacterial membrane .

When investigating atpF function, it's important to consider its relationship with other ATP synthase components. Studies of ATP synthase mutants, particularly those affecting the catalytic core (α, β, and γ subunits encoded by atpA, atpD, and atpG respectively), demonstrate significant alterations in bacterial growth kinetics under both planktonic and biofilm conditions . While specific atpF mutant data is more limited, the interconnected nature of ATP synthase subunits suggests that disruption of atpF would similarly impact energy metabolism and downstream physiological processes.

For experimental approaches to study atpF function, researchers should consider comparative analyses between wild-type and mutant strains, measuring changes in ATP production, growth rates, and membrane potential. Complementation studies reintroducing the functional atpF gene can confirm phenotype specificity. Additionally, metabolomic approaches can reveal broader impacts on S. aureus metabolism when atpF function is compromised.

How does recombinant expression of S. aureus atpF differ from native expression?

When expressing recombinant atpF, researchers must address several technical considerations. As a membrane protein component, atpF requires a suitable expression system that facilitates proper membrane insertion. E. coli-based systems often require optimization of induction conditions, temperature, and membrane-targeting sequences. Alternative expression systems such as cell-free approaches may better preserve native conformation for structural studies. Purification typically requires careful solubilization using detergents that maintain protein stability without disrupting structural integrity.

Validation of correctly folded recombinant atpF can be performed through circular dichroism to assess secondary structure, binding assays to confirm interactions with other ATP synthase components, and functional reconstitution experiments. Researchers should always compare recombinant protein characteristics with those derived from native S. aureus membranes to ensure biological relevance of their findings.

What are the established protocols for purifying recombinant S. aureus atpF protein?

Purification of recombinant S. aureus atpF requires specialized approaches due to its membrane protein nature. The most effective protocol typically involves a multi-step process beginning with optimal expression conditions. Researchers should first transform an appropriate E. coli strain (such as C41(DE3) or C43(DE3), specifically designed for membrane protein expression) with a vector containing the atpF gene with an affinity tag (commonly His₆ or Strep-tag).

Following expression, bacterial cells should be disrupted by methods that preserve membrane protein integrity, such as gentle sonication or French press treatment. The membrane fraction is then isolated through differential centrifugation and subjected to solubilization using detergents. Critical considerations include detergent selection (typically DDM, LMNG, or C₁₂E₈) at concentrations above their critical micelle concentration (CMC), solubilization time, and temperature. The solubilized atpF can then be purified using affinity chromatography based on the incorporated tag.

For higher purity, especially for structural studies, size exclusion chromatography provides an essential additional purification step. Throughout the purification process, protein stability must be maintained by including appropriate detergent concentrations in all buffers. Functional integrity can be assessed through reconstitution experiments or binding assays with other ATP synthase components. Protein purity should be verified using SDS-PAGE, while Western blotting with anti-atpF antibodies confirms identity. Mass spectrometry provides final validation of the purified protein sequence and any post-translational modifications.

How does mutation of atpF impact S. aureus antibiotic resistance mechanisms?

The ATP synthase complex in S. aureus plays a surprisingly important role in antibiotic resistance, particularly against cationic antimicrobial peptides like polymyxins. Research has demonstrated that mutations in ATP synthase subunits, including atpA, atpB, atpG, and atpH, significantly reduce S. aureus resistance to polymyxin B, with MIC reductions of up to 8-fold . While specific studies on atpF mutations are more limited, the functional integration of all ATP synthase subunits suggests atpF likely contributes to similar resistance mechanisms.

The mechanistic relationship between ATP synthase function and antimicrobial resistance appears to involve multiple pathways. ATP depletion resulting from compromised ATP synthase function may impair energy-dependent efflux systems that typically expel antibiotics. Additionally, alterations in membrane potential due to ATP synthase dysfunction could increase permeability to cationic antimicrobial peptides. Supporting this connection, pharmacological inhibition of ATP synthase with oligomycin A increases polymyxin B-mediated killing of S. aureus .

Researchers investigating atpF's role in antibiotic resistance should design experiments comparing wild-type and atpF mutant strains across multiple antibiotic classes. Important parameters to measure include MIC values, kill curve kinetics, membrane potential using fluorescent probes like DiSC3(5), and ATP levels. Cross-resistance profiles would help determine whether atpF mutation specifically affects certain antibiotic classes or creates broader susceptibility. Complementation studies reintroducing functional atpF would confirm phenotype specificity.

What structural features of atpF contribute to ATP synthase assembly and function in S. aureus?

The b subunit (atpF) of ATP synthase serves critical structural roles that facilitate proper complex assembly and function. In bacterial ATP synthases, the b subunit typically forms a peripheral stalk extending from the membrane-embedded F₀ portion to the catalytic F₁ portion, maintaining proper spatial orientation between these domains during the rotational catalytic mechanism. Specific structural features include a transmembrane anchor, an extended alpha-helical domain, and interaction sites with other subunits.

In S. aureus, atpF likely contains specialized structural elements adapted to the Gram-positive bacterial membrane environment. While detailed structural information specific to S. aureus atpF remains limited, comparative analysis with better-characterized bacterial homologs suggests importance of the C-terminal domain for interactions with the δ and α subunits of the F₁ portion. The transmembrane domain likely contains specific residues that facilitate assembly within the F₀ complex and potentially contribute to proton translocation efficiency.

For researchers investigating atpF structure-function relationships, several experimental approaches are recommended. Site-directed mutagenesis targeting conserved residues can identify critical functional domains. Crosslinking studies between atpF and other ATP synthase subunits can map interaction interfaces. Advanced structural techniques such as cryo-electron microscopy of the assembled ATP synthase complex or X-ray crystallography of individual domains can provide high-resolution structural information. Molecular dynamics simulations based on homology models can predict conformational changes during the catalytic cycle.

How does atpF contribute to S. aureus biofilm formation and host immune evasion?

The relationship between ATP synthase function and S. aureus biofilm dynamics represents an emerging area of research with significant clinical implications. Studies of the ATP synthase alpha subunit (atpA) mutant have revealed altered biofilm architecture with more diffuse structure that permits greater leukocyte infiltration . This suggests the ATP synthase complex, likely including the b subunit (atpF), influences biofilm structural integrity and consequent immune evasion capabilities.

ATP synthase dysfunction in S. aureus significantly alters host immune responses to biofilm infection. The atpA mutant elicits heightened proinflammatory cytokine production from leukocytes both in vitro and in vivo, with increased levels of IL-12p70, TNF-α, and IL-6 . This inflammatory shift coincides with improved biofilm clearance in a mouse model of prosthetic joint infection. Mechanistically, ATP synthase disruption appears to increase bacterial susceptibility to immune clearance through multiple pathways, including increased cell lysis that releases immunostimulatory components, altered toxin production, and changes in biofilm structure that enhance leukocyte access.

Researchers investigating atpF's specific contribution to biofilm dynamics should employ comparative analyses between wild-type and atpF-mutant strains. Critical measurements include biofilm biomass quantification, confocal microscopy to assess structural characteristics, extracellular DNA quantification, and leukocyte penetration assays. Immune response parameters should include cytokine production profiles from cocultured leukocytes and in vivo biofilm clearance rates. RNA-seq and proteomics approaches comparing wild-type and atpF mutant biofilms can identify downstream pathways affected by atpF disruption.

What expression systems optimize yield and functionality of recombinant S. aureus atpF?

Optimizing expression of functional recombinant S. aureus atpF requires careful selection of expression systems and conditions due to its membrane protein nature. Several expression platforms offer distinct advantages and limitations for atpF production. E. coli remains the most accessible system, with specialized strains like C41(DE3), C43(DE3), and Lemo21(DE3) designed specifically for membrane protein expression. These strains help mitigate toxicity often associated with membrane protein overexpression.

For E. coli-based expression, vector selection significantly impacts success. Vectors with tunable promoters (such as the arabinose-inducible pBAD system) allow precise control over expression levels. Including fusion partners like MBP or SUMO can enhance solubility, while specialized tags like Mistic or YidC may improve membrane targeting. Expression conditions require systematic optimization, with lower temperatures (16-25°C), reduced inducer concentrations, and extended expression times generally favoring proper folding over inclusion body formation.

Alternative expression systems offer advantages for specific research objectives. Bacillus subtilis provides a Gram-positive expression environment potentially more suitable for S. aureus membrane proteins. Cell-free expression systems eliminate cellular toxicity concerns and allow direct incorporation of detergents or lipids during synthesis. For highest yields of functional protein, insect cell expression using baculovirus vectors offers a eukaryotic membrane environment with sophisticated folding machinery. Each system requires optimization of expression constructs and conditions, with protein functionality verified through binding assays or reconstitution experiments.

The following table summarizes key expression systems for recombinant S. aureus atpF:

What analytical techniques best characterize structural interactions between atpF and other ATP synthase subunits?

Characterizing the structural interactions between atpF and other ATP synthase subunits requires complementary analytical approaches that address different aspects of protein-protein interactions. Several techniques have proven particularly valuable for investigating membrane protein complexes like ATP synthase.

Crosslinking coupled with mass spectrometry provides a powerful approach for mapping interaction interfaces between atpF and partner subunits. Chemical crosslinkers with different spacer lengths (BS³, DSS, or photo-activatable crosslinkers) can covalently link proteins in close proximity. Following digestion, crosslinked peptides are identified by mass spectrometry, revealing interaction sites at amino acid resolution. This approach works particularly well for dynamic interactions that might be lost during traditional purification.

Förster Resonance Energy Transfer (FRET) enables detection of protein-protein interactions in native-like environments. By labeling atpF and potential partner subunits with compatible fluorophore pairs, interaction distances can be measured with nanometer precision. This technique is particularly valuable for monitoring dynamic interactions during the ATP synthase catalytic cycle. Similarly, Bioluminescence Resonance Energy Transfer (BRET) offers advantages for cellular systems by eliminating the need for external illumination.

For high-resolution structural characterization, cryo-electron microscopy has revolutionized our understanding of large membrane protein complexes like ATP synthase. This technique can resolve the entire ATP synthase complex at near-atomic resolution, clearly showing atpF positioning and interactions. For focused investigation of specific interaction domains, X-ray crystallography of co-crystallized fragments can provide atomic-level detail of binding interfaces, though crystallization of membrane proteins remains challenging.

Complementary biophysical methods include Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC) to determine binding affinities and thermodynamic parameters of atpF interactions, while analytical ultracentrifugation and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) help characterize complex stoichiometry and assembly states.

How can researchers effectively design atpF mutants to investigate specific structure-function relationships?

Site-directed mutagenesis should target specific structural elements with distinct mutation types. Conservative substitutions (maintaining similar physicochemical properties) help determine the importance of specific chemical groups, while non-conservative substitutions can reveal more dramatic functional changes. Alanine scanning, systematically replacing residues with alanine, provides a neutral approach to assess individual amino acid contributions. For transmembrane domains, leucine scanning may be more appropriate given the hydrophobic environment.

Beyond single amino acid substitutions, more extensive mutational approaches provide complementary insights. Domain swapping between S. aureus atpF and homologs from other species can identify species-specific functional regions. Deletion analysis systematically removing protein segments helps map essential domains for assembly or function. Insertion of reporter groups like cysteine residues enables subsequent labeling for fluorescence or crosslinking studies.

The following table outlines strategic approaches to atpF mutagenesis:

Mutant characterization should employ multiple complementary assays, including ATP synthesis activity, growth complementation in ATP synthase-deficient strains, complex assembly analysis, and binding studies with partner subunits. Combining functional data with structural information creates a comprehensive understanding of structure-function relationships.

How might atpF be exploited as a potential antimicrobial target in multidrug-resistant S. aureus?

ATP synthase represents an underexplored but promising antimicrobial target in S. aureus, with the b subunit (atpF) offering specific targeting opportunities. Evidence supporting ATP synthase as a viable target includes the finding that ATP synthase inactivation renders S. aureus hypersusceptible to polymyxins and other antibiotics like gentamicin . Additionally, pharmacological inhibition of ATP synthase with oligomycin A increases polymyxin B-mediated killing of S. aureus . These findings suggest that ATP synthase inhibitors could function as antibiotic potentiators even if not directly bactericidal alone.

The atpF subunit specifically offers strategic advantages as a drug target. As part of the peripheral stalk, atpF contains regions exposed to the periplasmic space that may be more accessible to inhibitors compared to deeply membrane-embedded subunits. Structural differences between bacterial and human ATP synthase b subunits provide potential selectivity windows for antimicrobial development. Small molecules or peptides that disrupt atpF interactions with other subunits could destabilize the entire ATP synthase complex, reducing ATP production and enhancing antibiotic susceptibility.

Research approaches for atpF-targeted drug discovery should include high-throughput screening of compound libraries using ATP synthase activity assays, fragment-based drug discovery targeting specific atpF domains, and structure-guided design based on atpF interaction interfaces. Promising compounds should be evaluated for direct antimicrobial activity, antibiotic potentiation effects, resistance development frequency, and cytotoxicity to human cells. In vivo efficacy studies should assess both standalone activity and combination effects with existing antibiotics in relevant infection models.

What methodological advances would improve our understanding of atpF's role in S. aureus metabolic adaptation?

Understanding atpF's role in S. aureus metabolic adaptation requires advanced methodological approaches that integrate genetic, biochemical, and systems biology techniques. Real-time monitoring of ATP synthase function in living cells represents a critical methodological advance. This could be achieved through development of genetically encoded ATP sensors specifically designed for S. aureus, allowing visualization of ATP dynamics during metabolic shifts. Similarly, fluorescent probes for membrane potential would enable correlation between ATP synthase activity and proton motive force under different environmental conditions.

Controlled expression systems for atpF would significantly enhance functional studies. Development of inducible atpF expression constructs with titratable promoters would allow precise control over atpF levels, enabling investigation of dose-dependent effects on ATP production, metabolism, and virulence. CRISPR interference (CRISPRi) approaches offer complementary benefits by allowing tunable repression of atpF expression without permanent genetic modification.

Metabolic flux analysis specifically designed for S. aureus ATP synthase investigations would provide comprehensive insights into how atpF alterations affect global metabolism. This approach uses isotopically labeled substrates (¹³C, ¹⁵N) to track metabolite flow through various pathways under different conditions. Combining this with transcriptomic and proteomic analyses of wild-type versus atpF mutant strains under various stressors (oxygen limitation, pH shifts, antimicrobial exposure) would reveal how atpF contributes to metabolic adaptation mechanisms.

The following table outlines methodological advances for investigating atpF in metabolic adaptation: