Recombinant Staphylococcus aureus ATP-dependent Clp protease proteolytic subunit (clpP)

What is Staphylococcus aureus ClpP and what is its function?

Staphylococcus aureus ClpP is the proteolytic subunit of the ATP-dependent Clp protease complex. It plays a crucial role in protein quality control by degrading accumulated and misfolded proteins, thereby facilitating bacterial adaptation to multiple stresses . ClpP is essential for the normal function of S. aureus, contributing to its pathogenicity and stress response capabilities.

The primary function of ClpP is proteolytic degradation of targeted proteins, but this activity depends on association with ATPase chaperones such as ClpX or ClpC, which recognize, unfold, and translocate substrates into the ClpP proteolytic chamber. This proteolytic activity is vital for cellular homeostasis, particularly under stress conditions when misfolded proteins accumulate. Additionally, ClpP participates in regulatory proteolysis, controlling the levels of specific regulators involved in virulence and stress responses .

What is the structural composition of S. aureus ClpP?

S. aureus ClpP forms a barrel-shaped tetradecameric complex consisting of two heptameric rings stacked face-to-face. This creates a central proteolytic chamber where protein degradation occurs. Each ClpP monomer contains a catalytic triad (Ser-His-Asp) typical of serine proteases, with the active sites facing the interior of the barrel.

Crystal structure studies reveal that cystargolide A can bind covalently to all 14 active sites of ClpP from S. aureus, providing insight into the molecular architecture of this enzyme . The entrance pores at both ends of the barrel are narrow, allowing only unfolded proteins to enter the proteolytic chamber, which is why ClpP requires ATP-dependent chaperones like ClpX or ClpC for substrate recognition and unfolding.

How does ClpP contribute to bacterial survival under various stress conditions?

ClpP is essential for S. aureus survival under various stress conditions, particularly temperature stress. Research shows that clpP deletion mutants (ΔclpP) exhibit significant growth defects at both reduced and elevated temperatures (20°C, 30°C, 37°C, 42°C, and 45°C) . The temperature sensitivity is especially pronounced at lower temperatures; at 20°C, the mutant initially grows at rates similar to the wild type for approximately 6 hours but subsequently ceases growth .

This temperature sensitivity suggests that ClpP plays a critical role in adapting to temperature fluctuations by degrading proteins that may become misfolded or dysfunctional at non-optimal temperatures. Beyond temperature stress, ClpP also contributes to survival under oxidative stress, nutrient limitation, and other environmental challenges that bacteria encounter during infection or in their natural habitats.

How does ClpP influence S. aureus virulence in infection models?

ClpP significantly impacts S. aureus virulence, as demonstrated by multiple infection models. Studies show that clpP deletion results in attenuated virulence in murine abscess models . This attenuation is linked to the regulatory impact of ClpP on the expression of genes encoding proteins involved in S. aureus pathogenicity.

The specific mechanisms of virulence attenuation include:

• Impaired production of secreted virulence factors

• Altered biofilm formation capabilities

• Reduced survival within host cells

• Compromised ability to respond to host immune defenses

Additionally, ClpP appears to be essential for long-term survival of S. aureus during infection, particularly under the stressful conditions encountered within host tissues . This makes ClpP an important factor in the establishment and persistence of S. aureus infections.

What is the relationship between ClpP and the agr virulence regulatory system?

ClpP plays a critical regulatory role in S. aureus pathogenicity through its influence on the agr (accessory gene regulator) system, which is a major virulence regulatory system in S. aureus. Research demonstrates that deletion of clpP results in repression of the global regulatory agr locus .

The agr system controls the expression of multiple virulence factors, particularly secreted toxins and enzymes. When clpP is deleted, expression of the agr system and agr-dependent extracellular virulence factors is diminished . This indicates that ClpP positively regulates agr expression, either directly or indirectly.

This relationship helps explain why clpP mutants exhibit reduced virulence, as the agr system is crucial for coordinating the expression of virulence factors during different stages of infection. The molecular mechanism underlying this regulation remains an area of active research, but likely involves the degradation of repressors or processing of activators of agr expression.

What phenotypic changes occur when clpP is deleted in S. aureus?

Deletion of clpP in S. aureus results in multiple phenotypic changes that affect both physiology and virulence:

These phenotypic changes highlight the multifaceted role of ClpP in S. aureus biology, affecting fundamental cellular processes as well as specialized virulence mechanisms. The pleiotropic nature of these changes suggests that ClpP influences multiple regulatory networks within the bacterium.

What are the standard methods to measure ClpP proteolytic activity in vitro?

Several standardized methods are employed to measure ClpP proteolytic activity in vitro:

• Fluorogenic peptide substrates: The most common method utilizes fluorogenic peptide substrates that release fluorescent molecules upon cleavage. Activity is measured as relative fluorescence units per microgram of ClpP per hour (RFU/μg of ClpP/h) . This approach allows for quantitative assessment of ClpP activation or inhibition by various compounds.

• Protein substrate degradation assays: These involve incubating recombinant ClpP with protein substrates such as α-casein (5 μM), followed by SDS-PAGE and silver staining to visualize protein degradation . A typical reaction volume is 50 μL with a final concentration of 10 ng/μL ClpP and 1% DMSO as a vehicle control.

• Dose-response studies: These determine the EC50 of compounds that activate or inhibit ClpP by measuring activity across a concentration gradient. Background activity (samples treated with vehicle) is subtracted from experimental data .

For activation studies with compounds like ONC201, the protocol typically involves preincubating recombinant ClpP for 1 hour at 37°C with the compound or vehicle control, followed by addition of substrate and further incubation .

How can recombinant S. aureus ClpP be expressed and purified for functional studies?

Expression and purification of recombinant S. aureus ClpP typically follows these steps:

• Cloning: The clpP gene is amplified from S. aureus genomic DNA and cloned into an expression vector (commonly pET-based) with an appropriate affinity tag (His-tag, GST-tag).

• Expression: The construct is transformed into E. coli expression strains (BL21(DE3) or derivatives). Expression is typically induced with IPTG (0.1-1 mM) when cultures reach mid-log phase (OD600 ~0.6-0.8), followed by incubation at 18-25°C for 16-20 hours to minimize inclusion body formation.

• Cell lysis: Bacterial cells are harvested by centrifugation and lysed using methods such as sonication, French press, or chemical lysis in buffer containing protease inhibitors.

• Purification: A multi-step purification process typically includes:

  • Affinity chromatography (Ni-NTA for His-tagged proteins)

  • Ion exchange chromatography

  • Size exclusion chromatography to obtain the tetradecameric complex

• Quality control: Assess purity by SDS-PAGE, verify oligomeric state by native PAGE or gel filtration, and confirm activity using standard peptidase assays.

Purified ClpP should be stored in buffer containing glycerol (10-20%) at -80°C in small aliquots to maintain activity through multiple freeze-thaw cycles. Proper storage and handling are crucial as the oligomeric structure of ClpP is essential for its activity.

What techniques are used to study ClpP structure and inhibitor binding?

Several techniques are employed to investigate ClpP structure and its interaction with inhibitors:

• X-ray crystallography: This has been pivotal in determining the structure of ClpP and its complexes with inhibitors. Crystal structures have revealed that cystargolide A binds covalently to all 14 active sites of ClpP from S. aureus and other bacterial species . These structures provide valuable insights into the molecular mechanism of ClpP inhibition by β-lactones, which are the predominant class of ClpP inhibitors.

• Cryo-electron microscopy (cryo-EM): This technique is increasingly used to study large protein complexes like ClpP with its partner ATPases (ClpX, ClpC).

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach identifies regions of conformational change upon inhibitor binding.

• Thermal shift assays: These assess the impact of inhibitors on protein stability.

• Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC): These methods quantify binding kinetics and thermodynamics of ClpP-inhibitor interactions.

• Molecular docking and dynamics simulations: These computational approaches predict binding modes and conformational changes induced by inhibitors.

The combination of these techniques provides comprehensive understanding of how inhibitors like cystargolides and β-lactones interact with and inhibit ClpP, facilitating rational drug design targeting this protease.

What compounds are known to activate or inhibit S. aureus ClpP?

Several classes of compounds have been identified that can modulate S. aureus ClpP activity:

The anticancer compound ONC201 and related TR compounds strongly activate ClpP's peptidase activity, with TR compounds displaying approximately 10-100 fold increased potency compared to ONC201 . The mechanism involves direct binding to ClpP, though structural details of this interaction require further investigation.

Cystargolides represent an important class of natural product inhibitors. Crystal structures reveal that cystargolide A covalently binds to all 14 active sites of ClpP from S. aureus, providing insights into the molecular mechanism of inhibition by β-lactones .

How does temperature influence ClpP expression and activity in S. aureus?

Temperature significantly impacts both ClpP expression and activity in S. aureus:

• ClpP expression: Studies indicate that transcriptional activation of clpP occurs in response to temperature stress. The gene is upregulated both at elevated temperatures (heat shock response) and at reduced temperatures, suggesting its role in adaptation to temperature fluctuations.

• Growth phenotypes: ClpP is required for growth at both reduced and elevated temperatures. Deletion mutants (ΔclpP) exhibit growth defects across a range of temperatures (20°C-45°C), with particular sensitivity to reduced temperatures . At 20°C, the mutant grows normally for approximately 6 hours before growth cessation .

• Physiological role: The temperature sensitivity of ΔclpP mutants suggests that ClpP plays a crucial role in degrading temperature-sensitive proteins that may misfold or become non-functional at non-optimal temperatures.

• Activity regulation: The enzymatic activity of ClpP itself may be temperature-dependent, with optimal activity corresponding to the physiological temperature of S. aureus.

These temperature effects highlight the importance of ClpP in bacterial adaptation to environmental temperature changes, which is particularly relevant for pathogens like S. aureus that must transition between different temperature environments during infection.

What is the molecular mechanism of ClpP inhibition by β-lactones?

The molecular mechanism of ClpP inhibition by β-lactones, such as cystargolides, has been elucidated through structural studies:

This mechanism explains why β-lactones are the predominant class of ClpP inhibitors and provides a framework for the rational design of more potent and selective inhibitors targeting ClpP in pathogenic bacteria.

How can ClpP be targeted for antimicrobial development against S. aureus?

ClpP presents a promising target for antimicrobial development against S. aureus through several strategic approaches:

• Inhibition strategy: Direct inhibition of ClpP using compounds like cystargolides that covalently bind to the active sites can disrupt protein homeostasis, particularly under stress conditions . Since ClpP is required for growth and virulence, its inhibition may attenuate infection.

• Activation strategy: Paradoxically, hyperactivation of ClpP using compounds like ADEP antibiotics can result in uncontrolled proteolysis and bacterial death. These activators cause ClpP to function independently of its ATP-dependent chaperones, leading to degradation of essential proteins.

• Anti-virulence approach: Since ClpP regulates the expression of virulence factors through the agr system , targeting ClpP may reduce virulence without imposing strong selective pressure for resistance development.

• Combination therapy: ClpP modulators might be particularly effective when combined with conventional antibiotics, potentially overcoming resistance mechanisms and reducing effective dosages.

• Structure-guided design: Crystal structures showing inhibitor binding to all 14 active sites of S. aureus ClpP provide valuable templates for structure-based drug design of improved inhibitors with enhanced specificity and pharmacokinetic properties.

The therapeutic potential of targeting ClpP is enhanced by its conservation among pathogenic bacteria while being structurally distinct from human proteases, potentially allowing for selective targeting.

What role does ClpP play in S. aureus stress response and antibiotic resistance?

ClpP is central to S. aureus stress response and may influence antibiotic resistance through multiple mechanisms:

• General stress response: ClpP helps manage protein damage arising from various stresses (oxidative, heat, pH) by degrading misfolded proteins. This is evidenced by the growth defects of ΔclpP strains under various stress conditions, particularly temperature stress .

• Regulatory functions: Beyond protein quality control, ClpP regulates stress response pathways by controlling the levels of regulatory proteins. The global transcriptional profile of ΔclpP strains reveals altered expression of genes involved in adaptation to multiple stresses .

• Antibiotic tolerance: There is evidence that ClpP contributes to tolerance against certain antibiotics by:

  • Degrading proteins damaged by antibiotic action

  • Regulating expression of stress response genes that confer protection

  • Modulating the persistence phenotype, where a subpopulation of bacteria enters a dormant, antibiotic-tolerant state

• Biofilm formation: ClpP influences biofilm formation, a growth mode that inherently increases antibiotic tolerance. Alterations in ClpP function may affect biofilm development and consequently antibiotic susceptibility.

Understanding the role of ClpP in stress response and antibiotic tolerance may reveal new strategies to enhance the efficacy of existing antibiotics against S. aureus, particularly persistent or recalcitrant infections.

How does research on S. aureus ClpP inform our understanding of ClpP in other pathogenic bacteria?

Research on S. aureus ClpP provides important insights that extend to understanding ClpP function in other pathogenic bacteria:

• Conserved mechanisms: The basic structure and function of ClpP are highly conserved across bacterial species. Studies revealing that cystargolide A binds to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii highlight structural similarities that can inform broad-spectrum therapeutic approaches.

• Divergent regulation: Despite structural conservation, regulatory mechanisms controlling ClpP expression and activity may differ between species. Comparing these differences enhances our understanding of how different pathogens adapt to their specific ecological niches.

• Variable virulence contributions: While ClpP contributes to virulence in multiple pathogens including S. aureus, Salmonella enterica, Streptococcus pneumoniae, and Listeria monocytogenes , the specific virulence factors regulated by ClpP vary between species. These differences reflect the unique virulence strategies employed by each pathogen.

• Therapeutic implications: Understanding both the conservation and divergence in ClpP structure and function across species aids in developing targeted antimicrobial strategies. Compounds that exploit conserved features may offer broad-spectrum activity, while those targeting species-specific aspects may provide selective activity.

• Evolutionary insights: Comparative studies of ClpP across pathogenic bacteria reveal evolutionary adaptations in protein quality control systems that contribute to pathogenesis and bacterial fitness in diverse environments.

This cross-species perspective on ClpP enhances our fundamental understanding of bacterial physiology and provides a broader foundation for therapeutic targeting of this crucial protease system.

What are the key unanswered questions about S. aureus ClpP that require further investigation?

Despite significant advances, several critical questions about S. aureus ClpP remain unanswered:

• Substrate specificity: What determines the specificity of ClpP for different protein substrates during normal growth versus stress conditions? A comprehensive identification of ClpP substrates under various conditions would provide valuable insights into its regulatory roles.

• Regulatory networks: How does ClpP influence the agr virulence regulatory system at the molecular level? The mechanisms connecting ClpP activity to virulence gene expression remain incompletely understood.

• Structure-function relationships: How do specific structural features of S. aureus ClpP contribute to its function, and how do they differ from ClpP in other species? Further structural studies comparing ClpP from different bacteria may reveal important species-specific adaptations.

• In vivo dynamics: How is ClpP activity modulated in vivo during infection? Real-time monitoring of ClpP activity in the host environment represents a technical challenge that could provide valuable insights.

• Resistance mechanisms: Can S. aureus develop resistance to ClpP-targeting compounds, and if so, through what mechanisms? Understanding potential resistance pathways is crucial for therapeutic development.

These questions represent important areas for future research that will enhance our understanding of this critical protease and its potential as a therapeutic target.

What innovative methodologies might advance ClpP research?

Emerging methodologies could significantly advance our understanding of S. aureus ClpP:

• CRISPR interference (CRISPRi): This approach allows for tunable repression of clpP expression rather than complete deletion, potentially revealing phenotypes that might be masked in knockout studies due to compensatory mechanisms.

• Proximity-dependent biotin identification (BioID): This technique could identify proteins that interact transiently with ClpP or its chaperones in vivo, providing insights into the dynamic ClpP interactome.

• Single-cell proteomic approaches: These methods could reveal heterogeneity in ClpP activity within bacterial populations, particularly in biofilms or during infection.

• Advanced imaging techniques: Techniques such as super-resolution microscopy could visualize the subcellular localization and dynamics of ClpP under different conditions.

• Chemical biology approaches: Development of activity-based probes specific for ClpP would allow monitoring of its activity in complex biological samples.

• Integrative multi-omics: Combining transcriptomics, proteomics, and metabolomics approaches could provide a comprehensive view of the cellular consequences of ClpP modulation.

Implementation of these innovative methodologies would address technical limitations of current approaches and potentially resolve longstanding questions about ClpP function in S. aureus.

How might computational approaches enhance understanding of ClpP function and drug development?

Computational approaches offer powerful tools for advancing ClpP research and therapeutic development:

• Molecular dynamics simulations: These can provide insights into conformational changes associated with ClpP activation or inhibition, and the mechanisms by which compounds like ONC201 enhance its activity .

• Machine learning for substrate prediction: AI-based approaches could predict potential ClpP substrates based on primary sequence or structural features, guiding experimental validation.

• Network analysis: Computational modeling of regulatory networks involving ClpP could reveal indirect effects of ClpP modulation on bacterial physiology and virulence.

• Virtual screening: High-throughput computational screening could identify novel ClpP modulators from chemical libraries, accelerating drug discovery efforts.

• Quantum mechanics/molecular mechanics (QM/MM): These methods could elucidate the detailed reaction mechanisms of ClpP inhibition by compounds like cystargolides , informing the design of improved inhibitors.

• Systems biology approaches: Integration of multi-omics data through computational modeling could predict the systems-level impact of ClpP modulation under various conditions.

These computational approaches, particularly when integrated with experimental validation, have the potential to significantly accelerate both fundamental research on ClpP and the development of ClpP-targeting therapeutics for S. aureus infections.