Recombinant Staphylococcus aureus ATP synthase subunit alpha (atpA), partial

What is the role of ATP synthase subunit alpha (atpA) in Staphylococcus aureus?

ATP synthase subunit alpha (atpA) is a critical component of the F1F0-ATP synthase complex in S. aureus, responsible for ATP production during oxidative phosphorylation. The utilization of ATP within bacterial cells plays a fundamental role in cellular processes essential for the regulation of host-pathogen dynamics and bacterial survival during infection. In S. aureus specifically, atpA contributes to energy metabolism pathways that are enriched when the bacterium is located inside human cells, including nutrient acquisition and biosynthesis of amino acids .

How is the atpA gene organized in the genome of Staphylococcus aureus?

The atpA gene in S. aureus is part of a conserved operon structure, similar to other bacterial species. While the search results don't provide the exact genomic organization in S. aureus, comparative studies with other organisms suggest that atpA is typically found in an operon containing multiple ATP synthase subunit genes. For instance, in the case of Chlamydomonas reinhardtii (a model organism for studying chloroplast gene expression), the atpA gene is clustered with other genes including psbI, cemA, and atpH, forming a tetracistronic transcript . In S. aureus, the atpA gene likely has a similar arrangement with other ATP synthase subunits, facilitating coordinated expression of all components required for ATP synthesis.

What are common methods for expressing recombinant S. aureus atpA protein?

Table 1: Common Expression Systems for Recombinant S. aureus atpA

For recombinant expression of S. aureus atpA, E. coli-based expression systems remain the most widely used platform due to their efficiency and ease of genetic manipulation. The methodology typically involves:

• PCR amplification of the atpA gene from S. aureus genomic DNA

• Cloning into an expression vector containing appropriate tags (His, GST, etc.)

• Transformation into an E. coli expression strain (BL21, Rosetta, etc.)

• Optimization of expression conditions (temperature, IPTG concentration, media)

• Purification using affinity chromatography based on the chosen tag

When expressing membrane-associated proteins like ATP synthase components, it is often beneficial to express only the soluble portions or to optimize solubilization conditions using detergents during purification.

How can activity-based protein profiling be used to study atpA function in S. aureus?

Activity-based protein profiling (ABPP) using ATP probes has emerged as a powerful technique to study ATP-interacting proteins in S. aureus during host-pathogen interactions. The methodology involves:

• Culture preparation: Establish bacterial cultures in appropriate conditions (either free-living or during infection of host cells)

• Probe labeling: Apply desthiobiotin-ATP probe to selectively label active ATP-binding proteins

• Enrichment: Isolate labeled proteins using streptavidin-based affinity purification

• Proteomic analysis: Identify and quantify enriched proteins using mass spectrometry

This chemoproteomic technique offers significant advantages for studying atpA function as it focuses on enzymatic activity rather than mere protein presence. In S. aureus studies, this approach revealed enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism when the bacteria were located inside human cells . The targeted enrichment involved in this chemoproteomic approach is particularly beneficial for identifying low-abundance proteins and functional characterization of hypothetical proteins.

What considerations are important when designing primers for S. aureus atpA cloning and expression?

Table 2: Primer Design Considerations for S. aureus atpA

When designing primers for S. aureus atpA amplification and subsequent cloning, researchers should consider the following methodological approach:

• Analyze the full-length sequence of S. aureus atpA for optimal primer placement

• For partial atpA expression, carefully select the domain boundaries to maintain protein stability

• Include appropriate restriction enzyme sites that are absent in the target sequence

• Consider adding a 6x His-tag or other purification tags

• Verify primer specificity against the S. aureus genome to avoid non-specific amplification

• Optimize PCR conditions including annealing temperature and extension time

The choice between expressing the full-length or partial atpA protein depends on the research question. For structural studies, expressing stable domains may be preferable, while functional studies might require the complete protein or specifically the catalytic domain.

How can transcriptional analysis be applied to study atpA expression in S. aureus during infection?

To investigate atpA expression in S. aureus during infection scenarios, researchers can employ various transcriptional analysis methods:

• RT-PCR and qRT-PCR: Using specific primers targeting the atpA gene and appropriate reference genes for normalization

• RNA-Seq: For genome-wide transcriptional profiling, revealing co-expression patterns with other genes

• 5' RACE (Rapid Amplification of cDNA Ends): To map transcription start sites and understand promoter utilization

• Northern blotting: To identify specific transcript sizes and potential polycistronic messages

Based on similar studies in other organisms, transcriptional analysis of atpA typically reveals multiple, overlapping transcripts resulting from multiple promoters and mRNA-processing events . For instance, in C. reinhardtii, the atpA gene can be present as part of tetra-, tri-, di-, and monocistronic transcripts, suggesting complex transcriptional regulation . Similar complexity might be expected in S. aureus, especially during adaptation to different environments such as intracellular survival in macrophages or keratinocytes.

How does atpA contribute to S. aureus survival in macrophages and host adaptation?

S. aureus demonstrates remarkable adaptability within host cells, including macrophages, with ATP metabolism playing a crucial role in this adaptation. Experimental evolution studies have shown that S. aureus can develop adaptive phenotypes when repeatedly passaged through macrophages . While the specific contribution of atpA to these adaptations is not explicitly detailed in the search results, ATP-interacting proteins are implicated in the bacterial response to the intracellular environment.

Mechanistically, ATP synthase activity is likely modulated as part of the metabolic adaptation of S. aureus to the nutrient-limited and stressful environment inside macrophages. This adaptation may involve:

• Shifts in energy metabolism pathways

• Alterations in ATP synthase activity or expression

• Coordination with stress response systems

A study using a desthiobiotin-ATP probe revealed that when S. aureus resides inside human cells, there is enrichment in pathways required for nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism . This suggests that atpA and other ATP-related proteins are important for bacterial adaptation to the intracellular environment.

What are the challenges in differentiating between host and bacterial atpA activity during infection studies?

Table 3: Methodological Approaches to Differentiate Host and Bacterial atpA Activity

One of the significant challenges in studying S. aureus atpA during infection is distinguishing between bacterial and host ATP synthase activities. Activity-based protein profiling using a desthiobiotin-ATP probe offers a solution by allowing the selective labeling and isolation of active ATP-binding proteins from both host and pathogen in mixed samples . The subsequent proteomic analysis can differentiate between bacterial and host proteins based on their amino acid sequences.

To enhance the selectivity of this chemoproteomic analysis, researchers should implement stringent exclusion criteria, retaining only proteins that exhibit consistency across biological replicates. This approach yields a reliable set of proteins with consistent activity across experimental conditions, facilitating accurate comparative analysis .

How does S. aureus atpA activity change during the development of small colony variants (SCVs)?

Small colony variants (SCVs) of S. aureus represent an important adaptive phenotype associated with persistent infections and antimicrobial resistance. While the search results don't directly address atpA changes in SCVs, they do describe a novel SCV phenotype characterized by hyper-pigmentation resulting from a missense mutation in rsbW .

SCVs typically exhibit defects in electron transport and ATP synthesis, suggesting altered atpA expression or activity. Research approaches to investigate atpA changes in SCVs should include:

• Comparative proteomics of wild-type and SCV S. aureus strains

• Activity assays for ATP synthase function

• Transcriptional analysis of the atpA gene and its operon

• Metabolic profiling to assess energetic status

The experimental evolution approach described in the search results provides a valuable model for investigating how S. aureus adapts to selective pressures, including potential changes in atpA expression and activity . This model could be used to specifically examine how ATP synthase function evolves during adaptation to hostile environments such as within macrophages.

What are the optimal conditions for measuring recombinant S. aureus atpA enzymatic activity?

Table 4: Optimal Conditions for S. aureus atpA Activity Assays

Measuring the enzymatic activity of recombinant S. aureus atpA requires careful consideration of several methodological factors:

• Protein preparation: Ensure high purity and proper folding of the recombinant protein

• Assay selection:

  • ATP hydrolysis (reverse reaction) - typically measured by coupling ATP hydrolysis to NADH oxidation

  • ATP synthesis (forward reaction) - more challenging, requires establishing a proton gradient

• Controls: Include appropriate negative controls (heat-inactivated enzyme, known inhibitors)

• Data analysis: Calculate specific activity (μmol ATP hydrolyzed/min/mg protein)

How can researchers effectively study atpA transcription and translation during S. aureus infection?

To effectively study atpA transcription and translation during S. aureus infection, researchers can employ multiple complementary approaches:

• Transcriptional analysis:

  • RNA isolation from infected cells using methods that preserve bacterial RNA

  • RT-PCR or qRT-PCR with primers specific to S. aureus atpA

  • RNA-Seq to capture genome-wide transcriptional changes

  • Promoter mapping using 5' RACE or primer extension

• Translational analysis:

  • Western blotting with atpA-specific antibodies

  • Ribosome profiling to assess translational efficiency

  • Proteomics approaches, particularly activity-based protein profiling

  • Translational reporter fusions (e.g., atpA-GFP)

• In situ visualization:

  • Fluorescence in situ hybridization (FISH) for mRNA localization

  • Immunofluorescence for protein localization

When studying transcription specifically, researchers should be aware that bacterial genes like atpA can be transcribed as part of complex, polycistronic messages. For instance, in C. reinhardtii, the atpA gene is present in tetra-, tri-, di-, and monocistronic transcripts . Similar complexity might exist in S. aureus, necessitating careful primer design and analysis of multiple transcripts.

What are the best approaches for structural characterization of recombinant S. aureus atpA?

Table 5: Structural Characterization Methods for S. aureus atpA

Structural characterization of recombinant S. aureus atpA provides critical insights into its function and potential as a drug target. The methodological approach should follow these steps:

• Protein production optimization:

  • Express in a suitable system (E. coli, insect cells)

  • Optimize solubilization and purification conditions

  • Verify protein folding and homogeneity

• Initial biophysical characterization:

  • Circular dichroism to assess secondary structure

  • Size-exclusion chromatography to verify oligomeric state

  • Thermal shift assays to assess stability

• Advanced structural studies:

  • X-ray crystallography for atomic-resolution structures

  • Cryo-EM, particularly useful for the complete ATP synthase complex

  • NMR for dynamics studies of specific domains

• Functional validation:

  • Site-directed mutagenesis of key residues identified in structural studies

  • Activity assays to correlate structure with function

For the most comprehensive understanding, researchers should aim to characterize both the isolated atpA subunit and its context within the complete ATP synthase complex.

How should researchers interpret changes in atpA expression during S. aureus adaptation to host environments?

When interpreting changes in atpA expression during S. aureus adaptation to host environments, researchers should consider several factors:

• Context of adaptation: Different host cell types may elicit different bacterial responses. For example, S. aureus demonstrates tailored responses to the intracellular environment of different cell types like macrophages (THP-1) versus keratinocytes (HaCaT) .

• Temporal dynamics: Expression changes may be transient or sustained, reflecting different stages of adaptation.

• Correlation with phenotypic changes: Connect expression changes with observable phenotypes such as small colony variants, antibiotic resistance, or enhanced intracellular survival.

• Metabolic network effects: Consider atpA regulation in the context of broader metabolic reprogramming. ATP-binding proteins can be involved in nutrient acquisition, biosynthesis and metabolism of amino acids, and energy metabolism pathways that are enriched when S. aureus is located inside human cells .

• Host response interactions: Bacterial modulation of host processes may influence bacterial gene expression in return, creating feedback loops.

Changes in atpA expression should be interpreted as part of the bacteria's strategy to persist within specific host environments, potentially reflecting shifts in energy metabolism to adapt to nutrient availability or stress conditions.

What are common pitfalls in recombinant S. aureus atpA expression and purification, and how can they be addressed?

Table 6: Common Pitfalls and Solutions in Recombinant atpA Work

Common challenges in recombinant S. aureus atpA expression and purification include poor solubility, inclusion body formation, and loss of activity during purification. Methodological solutions include:

• For poor solubility:

  • Lower induction temperature (16-25°C)

  • Use solubility-enhancing fusion partners (e.g., MBP, SUMO)

  • Optimize expression conditions (media, induction time)

  • Consider cell-free expression systems

• For inclusion body recovery:

  • Develop efficient refolding protocols

  • Use mild solubilization conditions

  • Employ stepwise dialysis for refolding

• For activity preservation:

  • Include stabilizing agents in buffers (glycerol, reducing agents)

  • Minimize freeze-thaw cycles

  • Consider co-purification with interacting partners

  • Monitor activity throughout purification process

How can contradictory results in S. aureus atpA studies be reconciled?

Contradictory results in S. aureus atpA studies may arise from multiple factors, and researchers should adopt a systematic approach to reconcile such discrepancies:

• Strain differences: Different S. aureus strains may exhibit varying regulation and function of atpA. The search results mention that S. aureus strains representing major human epidemic clones were used in experimental evolution studies . Researchers should carefully document strain information and consider strain-specific effects.

• Experimental conditions: Growth conditions, media composition, and host cell types can significantly influence bacterial gene expression and protein function. For instance, S. aureus demonstrates different adaptations when interacting with macrophages versus keratinocytes .

• Methodological variations: Different techniques may measure different aspects of atpA biology:

  • Transcriptomic approaches measure mRNA levels

  • Proteomic approaches measure protein abundance

  • Activity-based methods measure functional protein states

  • These different layers may not always correlate perfectly

• Conditional phenotypes: Some S. aureus adaptations may be conditional and unstable in nutrient-replete conditions in vitro. The search results describe a novel SCV phenotype that rapidly converted from hyper-pigmented SCV to a non-pigmented large colony variant via spontaneous sigB deletion events .

• Technical reconciliation approaches:

  • Perform side-by-side comparisons using standardized protocols

  • Use multiple complementary techniques to address the same question

  • Conduct collaborative studies across laboratories

  • Develop consensus reporting standards for S. aureus atpA research

By carefully considering these factors and adopting a multi-technique approach, researchers can develop a more comprehensive and consistent understanding of S. aureus atpA biology.

What are promising approaches for targeting S. aureus atpA in antimicrobial development?

The critical role of ATP synthase in bacterial metabolism makes atpA a potential target for novel antimicrobial development. Promising research approaches include:

• Structure-based drug design:

  • Use structural data on S. aureus atpA to identify unique binding sites

  • Design selective inhibitors that target bacterial but not human ATP synthase

  • Explore allosteric inhibitors that may have a different resistance profile

• Natural product exploration:

  • Screen for natural compounds that specifically inhibit bacterial ATP synthase

  • Investigate existing ATP synthase inhibitors (e.g., oligomycin, bedaquiline) for activity against S. aureus

• Combination approaches:

  • Develop ATP synthase inhibitors that synergize with existing antibiotics

  • Target atpA in combination with other metabolic pathways

• Anti-virulence strategies:

  • Target atpA modulation during host adaptation without directly killing bacteria

  • Explore potential to prevent formation of persistent phenotypes like SCVs

Given that ATP synthase is essential for energy metabolism, especially under aerobic conditions, compounds targeting atpA could potentially address antibiotic-resistant S. aureus infections, including those caused by small colony variants.

How might CRISPR-Cas9 technology be applied to study atpA function in S. aureus?

CRISPR-Cas9 technology offers powerful approaches for investigating atpA function in S. aureus through precise genetic manipulation:

• Gene knockout and knockdown studies:

  • Create complete atpA knockout strains (if viable) or conditional knockdowns

  • Generate domain-specific mutations to dissect functional regions

  • Develop inducible systems to control atpA expression temporally

• Base editing applications:

  • Introduce specific point mutations without double-strand breaks

  • Create libraries of atpA variants to study structure-function relationships

  • Correct or introduce mutations identified in adapted strains

• CRISPRi for gene regulation studies:

  • Repress atpA expression without genetic modification

  • Study dosage effects on bacterial physiology and virulence

  • Create multiplexed systems to study atpA in context with other ATP synthase genes

• Reporter integration:

  • Insert fluorescent or luminescent reporters to monitor atpA expression

  • Tag the native protein to study localization and interaction partners

These CRISPR-based approaches can help determine the essentiality of atpA under different conditions, identify genetic interactions, and validate it as a potential antimicrobial target.

What new insights might systems biology approaches provide about atpA's role in S. aureus pathogenesis?

Systems biology approaches offer holistic perspectives on atpA's role in S. aureus pathogenesis by integrating multiple data types:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Build network models of atpA interactions with other cellular systems

  • Identify emergent properties not evident from single-omics approaches

• Flux balance analysis:

  • Model metabolic fluxes during infection with varying atpA expression

  • Predict metabolic vulnerabilities when ATP synthesis is compromised

  • Simulate the effects of potential inhibitors

• Host-pathogen interaction networks:

  • Map how atpA and ATP synthase activity influence host cell responses

  • Identify signaling pathways affected by bacterial energy metabolism

  • Develop predictive models of infection outcomes based on ATP synthase activity

• Evolutionary systems biology:

  • Study how atpA evolves during experimental evolution in macrophages

  • Identify compensatory mutations when ATP synthase is inhibited

  • Predict resistance mechanisms to ATP synthase inhibitors

Systems biology approaches could reveal how atpA contributes to S. aureus adaptation during infection, particularly in the context of metabolic reprogramming observed when bacteria transition to intracellular environments . These insights could inform both fundamental understanding of pathogenesis and development of intervention strategies.