Recombinant Staphylococcus aureus Alkyl hydroperoxide reductase subunit C (ahpC)

What is the function of AhpC in Staphylococcus aureus?

AhpC functions as a key enzymatic component in S. aureus's defense against oxidative stress. As part of the alkyl hydroperoxide reductase system, AhpC detoxifies hydrogen peroxide (H₂O₂) and organic peroxides, preventing cellular damage. Research has demonstrated that AhpC provides residual catalase activity, particularly significant in strains lacking the primary catalase KatA . This contributes to the bacterium's ability to neutralize reactive oxygen species generated during host immune responses.

The protein is regulated as part of the PerR regulon, with expression increasing under oxidative stress conditions . Importantly, AhpC works in conjunction with AhpF in a NADH/NADPH-dependent manner to reduce hydroperoxides to their corresponding alcohols, thereby protecting cellular components from oxidative damage. This antioxidant function is critical for S. aureus survival both during infection and environmental persistence .

How can recombinant S. aureus AhpC be expressed and purified?

Recombinant expression of S. aureus AhpC typically employs Escherichia coli as the host organism. The methodology involves:

• Gene cloning: The ahpC gene can be PCR-amplified from S. aureus genomic DNA using specific primers that incorporate appropriate restriction sites. As demonstrated in previous studies, primers similar to AhpC+1 and AhpC+2 can be designed to amplify the ahpC gene along with its native promoter region .

• Vector construction: The amplified gene is digested with appropriate restriction enzymes (commonly BamHI as used in research protocols) and ligated into a compatible expression vector. Shuttle vectors like pSK5630 have been successfully used for this purpose .

• Host transformation: The recombinant vector is transformed into an E. coli expression strain. For functional studies in S. aureus, the construct can be first transformed into S. aureus RN4220 (a restriction-deficient intermediate host) and then transferred to the target S. aureus strain via phage transduction using φ11 .

• Protein expression: For biochemical studies, expression is typically induced in E. coli using IPTG or similar inducers when using T7-based expression systems.

• Purification: The recombinant protein can be purified using affinity chromatography (commonly His-tag or GST-tag approaches), followed by size-exclusion chromatography to obtain highly pure protein.

The purification strategy should be optimized to maintain the enzyme's native conformation and activity, typically including reducing agents to protect the protein's thiol groups.

What experimental approaches are used to measure AhpC activity?

AhpC activity can be measured through several complementary approaches:

• Peroxidase activity assay: This spectrophotometric method measures the consumption of NADPH (340 nm) in the presence of AhpF, AhpC, and hydroperoxide substrates. The reaction rate indicates the peroxidase activity of the AhpC-AhpF system.

• H₂O₂ scavenging assay: The disappearance of hydrogen peroxide can be monitored using ferrous oxidation xylenol orange (FOX) assay or with horseradish peroxidase-based assays that produce colorimetric or fluorometric readouts.

• Growth and sensitivity testing: Functional complementation studies in ahpC mutants have been used to assess activity. For example, the sensitivity of S. aureus to paraquat (which generates superoxide radicals) can be measured using disk diffusion assays to evaluate the functionality of recombinant AhpC .

• Catalase activity detection: As AhpC provides residual catalase activity, spectrophotometric assays measuring the degradation of H₂O₂ (240 nm) can be employed to quantify this activity in purified recombinant protein or in bacterial lysates.

These methodological approaches provide complementary data on both the biochemical activity of purified recombinant AhpC and its functional role in bacterial oxidative stress resistance.

How does AhpC contribute to S. aureus virulence and pathogenesis?

AhpC plays a multifaceted role in S. aureus virulence through several mechanisms:

Interestingly, research has shown that double katA ahpC mutants, despite having severe growth defects in aerobic laboratory conditions, are not significantly attenuated in certain infection models. This suggests that during specific infection scenarios, the oxygen availability may be reduced, making these oxidative stress defense mechanisms less critical .

What is the compensatory regulatory relationship between AhpC and KatA in S. aureus?

S. aureus has evolved a sophisticated compensatory regulatory mechanism between AhpC and KatA that enhances the bacterium's oxidative stress response:

• Mutually compensatory expression: When either ahpC or katA is mutated, the expression of the remaining gene increases to compensate for the lost function. This ensures continued protection against oxidative damage .

• PerR-mediated regulation: This compensatory regulation occurs through the PerR repressor. In a katA mutant, increased intracellular H₂O₂ levels cause derepression of the PerR regulon, leading to upregulation of ahpC expression .

• Functional redundancy with distinct roles: While both enzymes detoxify H₂O₂, they show different kinetic properties and substrate preferences. AhpC can provide residual catalase activity in a katA mutant, demonstrating functional overlap despite structural differences .

• Synergistic effects: The double katA ahpC mutant exhibits a severe growth defect under aerobic conditions due to the complete inability to scavenge hydrogen peroxide. This leads to H₂O₂ accumulation in the medium, resulting in DNA damage and impaired growth .

• Differential roles in infection contexts: Research has demonstrated that while both enzymes are required for environmental persistence and nasal colonization, their importance varies in different infection models, suggesting context-specific roles during pathogenesis .

This regulatory interplay represents a sophisticated adaptation that enhances S. aureus resilience against oxidative stress through redundancy and compensatory mechanisms.

How does the pleiotropic regulator CymR influence AhpC expression and function?

CymR functions as a major pleiotropic repressor that influences AhpC expression and oxidative stress response through several interconnected mechanisms:

• Indirect regulation of the PerR regulon: Transcriptome analysis has revealed that deletion of cymR results in upregulation of genes in the PerR regulon, including ahpFC. This demonstrates a regulatory connection between sulfur metabolism (controlled by CymR) and oxidative stress response genes .

• Metabolic impact on redox homeostasis: CymR regulates cysteine metabolism, which affects the intracellular redox state. A ΔcymR mutant exhibits increased intracellular cysteine pools and hydrogen sulfide formation, which influences the cellular response to oxidative stressors .

• Enhanced sensitivity to oxidative stress: Despite upregulation of ahpFC in the ΔcymR mutant, these bacteria show increased sensitivity to hydrogen peroxide, disulfide, tellurite and copper stresses. This counterintuitive finding suggests that proper regulation of sulfur metabolism by CymR is essential for mounting an effective oxidative stress response .

• Impact on virulence: The ΔcymR mutant shows enhanced survival inside macrophages but severely impaired virulence in mouse models. This indicates that the precise regulation of oxidative stress response genes, including ahpC, is critical for successful pathogenesis .

• Integration of metabolic and stress responses: CymR represents a key link between sulfur metabolism and oxidative stress defense, integrating these critical cellular functions for optimal bacterial fitness in various environments.

This regulatory relationship highlights how S. aureus coordinates metabolic processes with stress responses through interconnected regulatory networks, with AhpC functioning within this complex system.

What structural features of S. aureus AhpC contribute to its catalytic mechanism?

S. aureus AhpC belongs to the 2-Cys peroxiredoxin family, with several structural features crucial for its function:

• Redox-active cysteine residues: The enzyme contains conserved peroxidatic and resolving cysteine residues that form a catalytic cycle. The peroxidatic cysteine reacts with hydroperoxides to form a sulfenic acid intermediate, which then forms a disulfide bond with the resolving cysteine.

• Oligomeric structure: AhpC typically functions as a homodimer in its reduced state but can form higher-order oligomers (decamers) depending on its redox status. This structural flexibility is important for its catalytic cycle and regulation.

• Active site architecture: The peroxidatic cysteine resides in a specialized pocket with surrounding residues that modulate its reactivity and substrate specificity. This architecture contributes to AhpC's ability to react rapidly with hydroperoxides.

• Conformational changes: During catalysis, AhpC undergoes significant conformational changes between "fully folded" (FF) and "locally unfolded" (LU) states, allowing the formation and resolution of the disulfide bond.

• Interaction surfaces: AhpC has specific structural features that enable interaction with AhpF, its physiological electron donor, facilitating efficient catalytic cycling.

Understanding these structural features has implications for developing inhibitors that could potentially disrupt this important antioxidant defense mechanism in S. aureus.

What methods are used to study the role of AhpC in S. aureus infection models?

Researchers employ diverse methodological approaches to investigate AhpC's role in S. aureus pathogenesis:

These methodological approaches provide complementary data on AhpC's multifaceted roles in bacterial physiology and pathogenesis.

How can site-directed mutagenesis be used to study critical residues in S. aureus AhpC?

Site-directed mutagenesis represents a powerful approach for investigating structure-function relationships in S. aureus AhpC. The methodology involves:

• Identification of target residues: Based on sequence alignments with homologous proteins and structural analysis, researchers can identify catalytically important residues. For AhpC, these typically include:

  • The conserved peroxidatic cysteine

  • The resolving cysteine

  • Residues involved in substrate binding

  • Residues mediating oligomerization or interaction with AhpF

• Primer design for mutagenesis: Overlapping primers containing the desired mutation are designed. For instance, to convert a cysteine to serine (a common substitution that maintains structure while eliminating thiol reactivity), primers incorporating the required nucleotide changes are created.

• PCR-based mutagenesis: Several techniques can be employed:

  • QuikChange™ site-directed mutagenesis

  • Overlap extension PCR

  • Gibson Assembly with mutagenic primers

• Verification of mutations: The mutated constructs are sequenced to confirm the presence of the desired mutation and absence of unwanted changes.

• Expression and purification: Mutant proteins are expressed and purified following protocols established for the wild-type protein.

• Functional characterization: Comparative analysis between wild-type and mutant proteins includes:

  • Peroxidase activity assays

  • Structural analysis (circular dichroism, thermal stability)

  • Oligomerization assessment (size-exclusion chromatography)

  • Redox state analysis

• In vivo complementation: Mutant ahpC genes can be introduced into ahpC-deficient S. aureus to assess functional complementation under oxidative stress conditions.

This methodological approach has been instrumental in elucidating the catalytic mechanism of AhpC and identifying residues essential for its antioxidant function.

What analytical techniques are used to study AhpC-KatA compensatory mechanisms?

Investigating the complex compensatory relationship between AhpC and KatA requires multiple analytical approaches:

• Gene expression analysis:

  • Quantitative RT-PCR to measure ahpC expression in katA mutants and vice versa

  • RNA-Seq for genome-wide transcriptional changes

  • Reporter gene fusions (e.g., lacZ, gfp) to monitor gene expression in different genetic backgrounds

• Protein level assessment:

  • Western blotting with specific antibodies to quantify AhpC protein levels

  • Proteomics approaches to assess global protein changes

  • Pulse-chase experiments to determine protein stability and turnover

• Enzymatic activity measurements:

  • Catalase activity assays using spectrophotometric methods

  • Peroxidase activity assays with various substrates

  • H₂O₂ scavenging assays in whole cells and cell extracts

• Genetic approaches:

  • Construction of single and double mutants (ahpC, katA, ahpC/katA)

  • Complementation studies with controlled expression systems

  • Regulatory mutants (e.g., perR) to dissect the regulatory network

• Physiological stress responses:

  • Growth curves under varying oxidative stress conditions

  • Survival assays following exposure to H₂O₂ or organic peroxides

  • Measurement of intracellular ROS levels using fluorescent probes

• Molecular mechanisms of regulation:

  • Electrophoretic mobility shift assays to study PerR binding to promoter regions

  • DNase I footprinting to identify precise binding sites

  • Chromatin immunoprecipitation to examine in vivo protein-DNA interactions

These complementary approaches provide a comprehensive understanding of how S. aureus coordinates the expression and activity of these two critical antioxidant enzymes to maintain redox homeostasis.

What approaches can be used to investigate the kinetic properties of recombinant S. aureus AhpC?

Investigating the kinetic properties of recombinant S. aureus AhpC requires a multi-faceted approach:

• Steady-state kinetic analysis:

  • Determination of Michaelis-Menten parameters (Km, kcat, kcat/Km) for various hydroperoxide substrates

  • Analysis of the dependence of reaction rates on AhpF concentration

  • Assessment of the effects of pH, temperature, and ionic strength on enzyme activity

• Pre-steady-state kinetics:

  • Stopped-flow spectroscopy to measure rapid reaction phases

  • Rapid quench techniques to trap reaction intermediates

  • Analysis of the rate-limiting steps in the catalytic cycle

• Substrate specificity profiling:

  • Comparative kinetic analysis with different hydroperoxides (H₂O₂, organic hydroperoxides, lipid hydroperoxides)

  • Competition assays to determine relative substrate preferences

  • Structure-activity relationship studies with substrate analogs

• Redox potential measurements:

  • Determination of the redox potential of the catalytic cysteines

  • Analysis of how redox potential affects reactivity with different substrates

  • Investigation of factors that modulate the redox properties of the enzyme

• Inhibition studies:

  • Characterization of competitive and non-competitive inhibitors

  • Determination of inhibition constants (Ki)

  • Analysis of the mechanisms of inhibition

The following table summarizes typical kinetic parameters for recombinant S. aureus AhpC with various substrates:

Note: These values represent typical ranges based on studies of AhpC from various bacterial species, as specific values for S. aureus AhpC may vary depending on the exact experimental conditions.

How can researchers interpret contradictory findings regarding AhpC's role in virulence?

When faced with seemingly contradictory results regarding AhpC's role in virulence, researchers should employ several analytical strategies:

• Contextual analysis of infection models:

  • Different infection models may assess distinct aspects of pathogenesis. For example, research has shown that while ahpC/katA double mutants have severe growth defects in aerobic laboratory conditions, they are not attenuated in certain infection models .

  • The apparent contradiction may reflect differences in oxygen availability between in vitro conditions and in vivo microenvironments .

  • Researchers should consider whether models evaluate acute versus chronic infection, different anatomical sites, or distinct host immune responses.

• Genetic background considerations:

  • S. aureus strain differences can significantly impact experimental outcomes.

  • The presence of compensatory mutations or regulatory adaptations may mask the effects of ahpC deletion.

  • Complete genetic characterization of mutant strains is essential for proper interpretation.

• Methodological variations:

  • Differences in mutant construction techniques (clean deletion vs. insertion) can affect phenotypic outcomes.

  • Complementation approaches (chromosomal vs. plasmid-based) may provide different levels of gene expression.

  • The timing of measurements during infection can reveal temporal aspects of AhpC's importance.

• Regulatory network analysis:

  • The compensatory relationship between AhpC and KatA means that single mutant phenotypes may be masked .

  • Global regulators like PerR and CymR further complicate the interpretation by affecting multiple stress response systems simultaneously .

  • Systems biology approaches can help unravel these complex regulatory interactions.

• Host factor considerations:

  • Host genetic background and immune status significantly impact infection outcomes.

  • The specific reactive oxygen species profile generated by different host cells may influence the relative importance of AhpC.

  • The metabolic state of the host tissue creates unique microenvironments with varying oxidative challenges.

By systematically addressing these factors, researchers can reconcile apparently contradictory findings and develop a more nuanced understanding of AhpC's role in S. aureus virulence across different contexts.

What statistical approaches are most appropriate for analyzing AhpC enzyme kinetics data?

Analysis of AhpC enzyme kinetics requires robust statistical approaches to ensure accurate interpretation:

• Regression analysis for kinetic parameters:

  • Non-linear regression using the Michaelis-Menten equation to determine Km and Vmax values

  • Lineweaver-Burk, Eadie-Hofstee, or Hanes-Woolf transformations as complementary approaches

  • Global fitting of multiple datasets when analyzing inhibition patterns

• Statistical comparison of kinetic parameters:

  • ANOVA with post-hoc tests for comparing multiple experimental conditions

  • t-tests (paired or unpaired) for direct comparisons between two conditions

  • Multiple regression analysis to identify factors significantly affecting enzyme activity

• Error analysis and propagation:

  • Calculation of standard errors for derived parameters (kcat, kcat/Km)

  • Monte Carlo simulations to estimate parameter uncertainty

  • Bootstrapping approaches for non-parametric estimation of confidence intervals

• Outlier detection and management:

  • Grubbs' test or Dixon's Q test for identification of statistical outliers

  • Robust regression methods that minimize the influence of outliers

  • Careful documentation and justification for any data exclusion

• Model discrimination:

  • Akaike Information Criterion (AIC) or Bayesian Information Criterion (BIC) for comparing alternative kinetic models

  • F-test for nested models to determine if additional parameters significantly improve the fit

  • Residual analysis to assess the appropriateness of the selected model

• Experimental design considerations:

  • Power analysis to determine appropriate sample sizes

  • Factorial designs to efficiently explore multiple variables

  • Response surface methodology for optimization studies

When reporting kinetic data for recombinant S. aureus AhpC, researchers should include:

• Complete description of statistical methods

• Sample sizes and number of independent experiments

• Measures of central tendency and dispersion

• Confidence intervals for key parameters

• Statistical significance levels for comparative analyses

This rigorous statistical approach ensures reproducible and reliable characterization of AhpC enzymatic properties.

How should researchers interpret the relationship between AhpC function and S. aureus metabolic state?

The relationship between AhpC function and S. aureus metabolic state represents a complex interplay that requires nuanced interpretation:

• Integration with central metabolism:

  • AhpC activity depends on NADPH availability, linking it directly to the pentose phosphate pathway and other NADPH-generating systems

  • Changes in carbon source utilization alter the redox balance and consequently the demand for AhpC activity

  • Researchers should assess metabolic flux distribution when interpreting AhpC function across different growth conditions

• Oxygen availability effects:

  • S. aureus exhibits remarkable metabolic flexibility across aerobic and anaerobic conditions

  • The importance of AhpC varies with oxygen tension, explaining why ahpC/katA mutants show severe growth defects aerobically but remain viable under low-oxygen conditions

  • Interpretation should consider the precise oxygen availability in experimental systems

• Connection to sulfur metabolism:

  • The CymR regulator links sulfur metabolism to oxidative stress responses

  • Intracellular cysteine pools and hydrogen sulfide production affect the cellular redox state and AhpC function

  • Analysis should incorporate sulfur metabolite measurements when evaluating AhpC activity

• Growth phase considerations:

  • AhpC expression and importance varies across growth phases

  • Exponential versus stationary phase cells show different oxidative stress vulnerabilities

  • Temporal analysis across growth phases provides more comprehensive understanding

• Metabolomic analysis approaches:

  • Untargeted metabolomics can reveal unexpected connections between AhpC and metabolic pathways

  • Stable isotope labeling helps track metabolic flux changes in ahpC mutants

  • Integration of transcriptomic and metabolomic data provides systems-level insights

• Biofilm versus planktonic considerations:

  • S. aureus in biofilms exhibits distinct metabolic states with altered redox parameters

  • AhpC importance may differ significantly between planktonic and biofilm growth

  • Interpretation should specify the physiological state being examined

What emerging technologies could advance our understanding of AhpC function in S. aureus?

Several cutting-edge technologies hold promise for deeper insights into AhpC function:

• CRISPR-Cas9 genome editing:

  • Precise engineering of single nucleotide mutations in the native ahpC gene

  • Creation of conditional knockdowns using CRISPRi

  • Multiplexed mutation analysis to identify synthetic lethal interactions

  • Genome-wide screens to identify genes affecting AhpC function

• Advanced structural biology approaches:

  • Cryo-electron microscopy to visualize AhpC-AhpF complexes

  • Time-resolved X-ray crystallography to capture catalytic intermediates

  • Hydrogen-deuterium exchange mass spectrometry to analyze conformational dynamics

  • Integrative structural biology combining multiple techniques for complete structural models

• Single-cell technologies:

  • Single-cell RNA-seq to examine heterogeneity in ahpC expression within bacterial populations

  • Fluorescent biosensors to monitor real-time peroxide levels and AhpC activity in living cells

  • Microfluidic devices to study oxidative stress responses with precise environmental control

  • Single-cell proteomics to analyze protein-level adaptations

• Systems biology approaches:

  • Multi-omics integration (transcriptomics, proteomics, metabolomics) to build comprehensive models

  • Machine learning for prediction of AhpC-dependent phenotypes across conditions

  • Flux balance analysis to quantify metabolic impacts of AhpC activity

  • Network analysis to position AhpC within the broader oxidative stress response system

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize AhpC localization and dynamics

  • Correlative light and electron microscopy to connect function with ultrastructure

  • Intravital imaging to monitor S. aureus oxidative stress responses during infection

  • Label-free imaging to detect metabolic changes associated with AhpC activity

• Synthetic biology strategies:

  • Designer AhpC variants with altered catalytic properties or regulation

  • Synthetic gene circuits to probe AhpC regulation dynamics

  • Cell-free expression systems for high-throughput AhpC variant characterization

  • Biosensors based on AhpC for detecting peroxide levels in various environments

These emerging technologies will enable researchers to address longstanding questions about AhpC function and potentially reveal new therapeutic targets within the oxidative stress defense network of S. aureus.

What are the potential therapeutic applications targeting S. aureus AhpC?

Targeting S. aureus AhpC offers several promising therapeutic strategies:

• Direct inhibitor development:

  • Structure-based design of small molecules targeting the AhpC active site

  • Allosteric inhibitors disrupting the AhpC-AhpF interaction

  • Covalent inhibitors targeting the catalytic cysteines

  • Peptide inhibitors disrupting AhpC oligomerization

• Anti-virulence approaches:

  • Compounds that dysregulate the compensatory relationship between AhpC and KatA

  • Agents that prevent AhpC upregulation during infection

  • Molecules targeting the PerR regulatory system to disrupt oxidative stress responses

  • Development of adjuvants that enhance oxidative killing by immune cells

• Combination therapy strategies:

  • Pairing AhpC inhibitors with conventional antibiotics to enhance efficacy

  • Dual targeting of AhpC and KatA to overcome compensatory mechanisms

  • Combining AhpC inhibitors with agents that generate reactive oxygen species

  • Sequential therapy approaches exploiting temporal vulnerability windows

• Vaccine development:

  • AhpC-based subunit vaccines

  • Attenuated S. aureus strains with modified AhpC for live-attenuated vaccines

  • Epitope identification for targeted immune responses

  • Development of antibodies disrupting AhpC function

• Diagnostic applications:

  • AhpC-based biomarkers for S. aureus detection

  • Monitoring AhpC expression as an indicator of antibiotic effectiveness

  • Point-of-care tests detecting AhpC activity in clinical samples

  • Techniques to identify S. aureus strains with altered AhpC function

• Novel screening platforms:

  • High-throughput screens for AhpC inhibitors

  • Phenotypic screening approaches targeting oxidative stress vulnerability

  • Fragment-based drug discovery focused on the AhpC active site

  • Natural product libraries screening for AhpC modulators

Therapeutic development must consider:

• The compensatory mechanisms between AhpC and KatA

• Potential off-target effects on human peroxiredoxins

• The context-dependent importance of AhpC in different infection scenarios

• Resistance development through alternative oxidative stress defense pathways

By addressing these considerations, AhpC-targeted approaches could contribute to addressing the critical need for new anti-staphylococcal therapies in the face of increasing antibiotic resistance .