Recombinant Staphylococcus aureus Probable CtpA-like serine protease (SAR1432)

What is the domain organization of S. aureus CtpA (SAR1432)?

• N-terminal dimerization region (NDR): Facilitates dimerization with another CtpA molecule

• PDZ domain: Involved in substrate recognition and binding

• Cap domain: A four-stranded β-sheet structure that controls access to the active site

• Protease core domain: Contains the catalytic Ser-Lys-Gln triad responsible for proteolytic activity

• C-terminal dimerization region (CDR): Contains the peptidoglycan binding domain

This domain architecture enables S. aureus CtpA to recognize specific substrates and localize to the bacterial cell wall where it performs its proteolytic function.

How is S. aureus CtpA localized within the bacterial cell?

S. aureus CtpA is primarily localized to the bacterial cell wall, where it can interact with its substrates. Studies have demonstrated that CtpA is membrane-anchored, with the majority of the protein, including the catalytic site, situated in the bacterial cell wall . This cellular localization distinctly differs from Gram-negative CTPs, which are typically found in the periplasmic space.

The peptidoglycan binding domain (residues 417-473) plays a crucial role in this localization by anchoring the enzyme to the peptidoglycan layer of the cell wall . The presence of this domain in Gram-positive CTPs but not in Gram-negative homologs highlights its importance for proper localization and function in the distinct cell envelope architecture of Gram-positive bacteria.

The cell wall localization of S. aureus CtpA strongly suggests that its proteolytic targets are also located in the bacterial cell wall, where it likely functions to maintain cell wall integrity through processing of cell wall-associated proteins . This localization is critical for its biological function and contributes to its role in virulence and stress resistance.

How does S. aureus CtpA compare to other bacterial C-terminal processing proteases?

Comparative analysis reveals both conserved features and unique aspects of S. aureus CtpA relative to other bacterial CTPs:

The higher sequence identity with B. subtilis CtpA (42.45%) compared to B. subtilis CtpB (31.76%) suggests that S. aureus CtpA may be functionally more similar to B. subtilis CtpA . The peptidoglycan binding domain represents a distinctive feature found exclusively in CTPs from Gram-positive bacteria, likely reflecting adaptation to their specific cell envelope architecture .

P. aeruginosa CtpA forms a hexamer (trimer of dimers) and requires interaction with the lipoprotein LbcA for activation , whereas B. subtilis CtpB forms a dimeric self-compartmentalizing ring structure that is activated by substrate binding . The activation mechanism of S. aureus CtpA remains to be fully elucidated but represents an important area for future research.

What is known about the catalytic mechanism of S. aureus CtpA?

S. aureus CtpA, like other S41 family proteases, employs a catalytic triad consisting of Ser-Lys-Gln for proteolytic activity. Based on homology with related CTPs, the catalytic serine (likely Ser-302) acts as a nucleophile to attack the carbonyl carbon of the scissile peptide bond in the substrate . The lysine residue (likely Lys-327) enhances the nucleophilicity of the serine by acting as a general base, while the glutamine residue (likely Gln-331) stabilizes the tetrahedral intermediate formed during catalysis .

The catalytic mechanism involves several key steps:

• Substrate recognition via the PDZ domain, which binds the C-terminal portion of the substrate

• Entry of the substrate through a narrow tunnel leading to the active site

• Nucleophilic attack by the catalytic serine on the scissile bond

• Formation and collapse of the tetrahedral intermediate

• Release of the cleaved products

In the inactive state, the catalytic triad residues are positioned beyond hydrogen-bonding distance. In P. aeruginosa CtpA, Ser-302 and Lys-327 are 4.2 Å apart, and Lys-327 and Gln-331 are 9.1 Å apart in the inactive conformation . Activation involves realignment of these residues to form a functional catalytic triad capable of peptide bond hydrolysis.

How is S. aureus CtpA activated and what is known about its regulatory mechanisms?

The activation mechanism of S. aureus CtpA remains one of the most intriguing aspects of this enzyme and represents a significant knowledge gap. Unlike P. aeruginosa CtpA, which requires binding of the lipoprotein LbcA for activation , no equivalent activator has been identified for S. aureus CtpA.

In P. aeruginosa, the activation process involves several remarkable features:

• CtpA forms a hexamer (trimer of dimers) that is inactive in the absence of LbcA

• LbcA binding triggers relocation of the PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues

• Surprisingly, only one CtpA molecule in each dimer becomes activated upon LbcA binding

• A long loop from one CtpA dimer inserts into a neighboring dimer to facilitate activity

In contrast, B. subtilis CtpB is activated through a substrate-induced mechanism where binding of the substrate C-terminal peptide causes the PDZ domain to shift position, exposing the active site . This represents a self-compartmentalizing mechanism ensuring controlled proteolytic activity.

For S. aureus CtpA, several possibilities exist:

• It may be constitutively active

• It might require interaction with yet-unidentified protein partners

• It could be activated by substrate binding similar to B. subtilis CtpB

• Environmental factors (pH, ionic strength) might regulate its activity

Understanding the activation mechanism of S. aureus CtpA represents a crucial area for future research, as it could reveal novel regulatory mechanisms and potential targets for inhibition.

What experimental approaches can identify substrates of S. aureus CtpA?

Identifying the physiological substrates of S. aureus CtpA is essential for understanding its biological function. Several experimental strategies can be employed:

• Comparative proteomics analysis:

  • Compare the proteomes of wild-type and ctpA mutant S. aureus strains using techniques like 2D-gel electrophoresis or LC-MS/MS

  • Focus on proteins that show altered molecular weight or abundance between strains

  • Pay particular attention to cell wall-associated proteins, as they represent likely substrates based on CtpA localization

• Activity-based protein profiling:

  • Design activity-based probes that covalently bind to the active site of CtpA

  • Use these probes to capture CtpA-substrate complexes

  • Identify captured proteins through mass spectrometry analysis

• Targeted candidate approach:

  • Based on knowledge of CtpA function in other bacteria, test specific candidates

  • Cell wall hydrolases are promising candidates as they are known targets of P. aeruginosa CtpA

  • Penicillin-binding proteins (PBPs) might also be substrates, as PBP-3 is a known substrate of E. coli Prc

• Substrate trapping with inactive mutants:

  • Generate catalytically inactive CtpA (e.g., S302A mutant)

  • Use this mutant to trap substrates that bind but cannot be cleaved

  • Identify trapped proteins through co-immunoprecipitation followed by mass spectrometry

• C-terminal peptide library screening:

  • Since CTPs typically recognize the C-termini of their substrates, screen libraries of C-terminal peptides

  • Identify sequences that are efficiently cleaved by CtpA

  • Use this information to predict potential protein substrates in the S. aureus proteome

Understanding the substrates of S. aureus CtpA will provide crucial insights into its physiological roles and contribution to virulence, potentially revealing new therapeutic targets.

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

S. aureus CtpA plays a critical role in bacterial virulence through several mechanisms, as demonstrated by comprehensive studies of ctpA mutants:

• Attenuation in murine sepsis model:

  • Mice infected with ctpA mutant showed significantly higher survival rates (90%) compared to those infected with wild-type (40%)

  • Bacterial burden in organs of ctpA mutant-infected mice was dramatically reduced:

    • 228-fold decrease in brain

    • 65-fold decrease in heart

    • 28-fold decrease in spleen

    • 6-fold decrease in kidneys

• Decreased resistance to host immune factors:

  • The ctpA mutant exhibited 8.5-fold lower survival in human serum compared to wild-type

  • Complementation with functional ctpA restored serum resistance to wild-type levels (19% vs. 13.8%)

  • This indicates increased susceptibility to components of the humoral immune system

• Impaired stress tolerance:

  • The ctpA mutant showed 3-fold lower survival at 55°C compared to wild-type

  • Enhanced stress tolerance may contribute to survival during infection

• Expression regulation during infection:

  • ctpA expression is induced under conditions encountered during infection

  • Higher expression was observed in human serum and in intracellular environments

  • Expression was also induced in the presence of antibiotics targeting cell wall biosynthesis (phosphomycin and oxacillin)

The mechanisms underlying these phenotypes likely involve the maintenance of cell wall integrity and processing of virulence factors. The cell wall localization of CtpA suggests it may process or activate cell wall-associated virulence factors necessary for pathogenesis and resistance to host defenses . Additionally, CtpA may modulate the activity of cell wall hydrolases, similar to P. aeruginosa CtpA, which regulates cell wall cross-link hydrolases by degrading them .

What structural changes occur during substrate binding and catalysis?

Based on structural studies of homologous CTPs, several significant conformational changes likely occur during substrate binding and catalysis by S. aureus CtpA:

• PDZ domain repositioning:

  • In the inactive state, the PDZ domain blocks access to the active site

  • Upon substrate binding, the PDZ domain shifts position to allow substrate entry

  • This conformational change exposes the narrow tunnel leading to the catalytic site

• Realignment of catalytic residues:

  • In the inactive state, the catalytic residues (Ser-Lys-Gln) are positioned beyond hydrogen-bonding distance

  • In P. aeruginosa CtpA, Ser-302 and Lys-327 are 4.2 Å apart in the inactive state

  • Activation involves repositioning of these residues to form a functional catalytic triad

• Substrate binding pocket remodeling:

  • The substrate binding pocket undergoes structural rearrangement to accommodate the substrate

  • In P. aeruginosa CtpA, LbcA binding triggers remodeling of the substrate binding pocket

  • Similar remodeling likely occurs in S. aureus CtpA, either spontaneously or upon interaction with regulatory factors

• Substrate tunnel opening:

  • The narrow tunnel that guides the substrate peptide to the active site widens

  • This tunnel is formed between the cap and body regions of the protease core domain

  • Opening of this tunnel is essential for substrate access to the catalytic site

These structural changes ensure that proteolytic activity is tightly regulated and only occurs upon proper substrate recognition. Detailed structural studies of S. aureus CtpA in complex with substrates or substrate analogs would provide valuable insights into these conformational changes and the molecular basis of substrate specificity.

What approaches can be used to design inhibitors of S. aureus CtpA?

Given the importance of CtpA in S. aureus virulence, developing specific inhibitors represents a promising strategy for anti-virulence therapies. Several approaches can be employed:

• Structure-based design:

  • Develop homology models of S. aureus CtpA based on related CTPs

  • Identify key features of the active site and substrate binding pocket

  • Design small molecules that bind to the active site or allosteric sites

  • Use molecular docking and molecular dynamics simulations to optimize candidate inhibitors

• Mechanism-based inhibitors:

  • Design compounds that mimic the transition state of the proteolytic reaction

  • Develop covalent inhibitors that react with the catalytic serine

  • Examples include chloromethyl ketones, boronic acids, or β-lactones

• Peptide-based inhibitors:

  • Design peptides or peptidomimetics that mimic the C-terminal sequence of natural substrates

  • Incorporate non-cleavable isosteres of the scissile bond

  • Include moieties that enhance binding to the PDZ domain

• Target protein-protein interactions:

  • In P. aeruginosa, disrupting the CtpA-LbcA interaction inhibits protease activity

  • If S. aureus CtpA requires protein partners for activation, these interactions could be targeted

  • Alternatively, if S. aureus CtpA functions as an oligomer, disrupting oligomerization could inhibit activity

• Evaluation strategies:

  • Develop in vitro assays using purified recombinant CtpA and synthetic substrates

  • Test promising compounds in cellular assays to assess their ability to phenocopy ctpA mutants

  • Evaluate lead compounds in animal infection models to determine in vivo efficacy

Since ctpA mutants show attenuated virulence but can still grow under laboratory conditions , CtpA inhibitors would likely function as anti-virulence agents rather than traditional antibiotics. This approach might reduce selective pressure for resistance development while still effectively combating infection.

How does site-directed mutagenesis inform our understanding of CtpA function?

Site-directed mutagenesis represents a powerful approach to probe the structure-function relationships of S. aureus CtpA:

• Catalytic triad mutations:

  • Mutation of the catalytic serine (S302A based on homology) eliminates proteolytic activity

  • In P. aeruginosa, the S302A mutant forms a hexamer but remains in an inactive configuration

  • Mutation of the catalytic lysine (K327A) or glutamine (Q331A) would disrupt the catalytic triad and reduce or abolish activity

  • These mutations produce valuable tools for structural studies by stabilizing enzyme-substrate complexes

• PDZ domain mutations:

  • Mutations in the substrate-binding groove of the PDZ domain affect substrate recognition

  • These can alter substrate specificity or completely abolish substrate binding

  • Such mutants can distinguish between PDZ-dependent and PDZ-independent functions of CtpA

• Peptidoglycan binding domain mutations:

  • Mutations in the peptidoglycan binding domain (residues 417-473) likely affect cellular localization

  • These mutants might show normal in vitro activity but reduced in vivo function

  • Such studies could reveal the importance of proper localization for CtpA function

• Dimerization interface mutations:

  • Mutations in the N-terminal or C-terminal dimerization regions could disrupt oligomerization

  • In P. aeruginosa CtpA, the C-terminal dimerization interface involves β-strand S10 and helix H6

  • Similar residues could be targeted in S. aureus CtpA to assess the importance of oligomerization

• Loop region mutations:

  • In P. aeruginosa CtpA, a long loop from one dimer inserts into a neighboring dimer

  • If similar loops exist in S. aureus CtpA, mutating these regions could provide insights into activation mechanisms

Systematic mutagenesis combined with biochemical characterization, localization studies, and in vivo virulence assays would provide a comprehensive understanding of structure-function relationships in S. aureus CtpA and identify critical residues for inhibitor design.

What expression systems are optimal for producing recombinant S. aureus CtpA?

Selecting the appropriate expression system is crucial for obtaining functional recombinant S. aureus CtpA. Several systems can be considered:

• E. coli expression system:

  • Most commonly used for recombinant protein production

  • Successfully used to produce His-tagged S. aureus CtpA (SAR1432)

  • Recommended protocol:

    • Clone the full-length sar1432 gene (coding for residues 1-496) into a pET vector with an N-terminal His-tag

    • Transform into E. coli BL21(DE3) or Rosetta(DE3) for rare codon optimization

    • Grow cultures to mid-log phase (OD600 ~0.6-0.8) at 37°C

    • Induce with 0.5 mM IPTG

    • Continue expression at reduced temperature (18-25°C) for 16-18 hours

  • Advantages: High yield, well-established protocols, cost-effective

  • Disadvantages: May form inclusion bodies, potential folding issues

• Gram-positive expression systems:

  • B. subtilis or L. lactis provide a more native environment for proper folding

  • May be valuable if post-translational modifications are required

  • Advantages: Better folding, potentially higher activity

  • Disadvantages: Lower yields, more complex protocols

• Cell-free expression systems:

  • Allow rapid production without cell culture

  • Useful for screening expression constructs

  • Advantages: Rapid, avoids toxicity issues

  • Disadvantages: Expensive, typically lower yields

For optimal expression of functional S. aureus CtpA, modifications to the standard protocol may be required:

• Solubility enhancement:

  • Use fusion tags like MBP, SUMO, or TrxA to improve solubility

  • Include low concentrations of non-ionic detergents in lysis buffer

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Stability considerations:

  • Include protease inhibitors to prevent degradation

  • Add stabilizing agents (5-10% glycerol, 1-5 mM DTT)

  • Work at 4°C throughout purification

The successful expression of recombinant S. aureus CtpA provides the foundation for biochemical, structural, and functional studies of this important virulence factor.

How can the protease activity of S. aureus CtpA be quantitatively measured?

Developing reliable assays for S. aureus CtpA activity is essential for characterizing its biochemical properties and screening for inhibitors. Several complementary approaches can be used:

• Fluorogenic peptide substrates:

  • Design peptides containing a fluorophore and quencher separated by a potential cleavage sequence

  • Upon cleavage, fluorescence increases as the fluorophore is separated from the quencher

  • Example setup: A peptide containing the C-terminal sequence of a predicted natural substrate with EDANS (fluorophore) and DABCYL (quencher)

  • Advantages: High sensitivity, real-time monitoring, high-throughput compatible

• Protein substrate cleavage assays:

  • Incubate CtpA with potential protein substrates and analyze by SDS-PAGE

  • Monitor disappearance of substrate and appearance of cleavage products

  • Example protocol:

    • Mix purified CtpA (0.1-1 μM) with protein substrate (5-10 μM)

    • Incubate at 37°C in buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2)

    • Remove aliquots at various time points (0-60 min)

    • Analyze by SDS-PAGE and quantify band intensities

  • Advantages: Uses native substrates, provides information on cleavage sites

• Mass spectrometry-based assays:

  • Identify exact cleavage sites using LC-MS/MS analysis of reaction products

  • Enables precise characterization of substrate specificity

  • Advantages: High specificity, identifies cleavage sites precisely

  • Disadvantages: Lower throughput, requires specialized equipment

Standard reaction conditions for activity assays might include:

• Buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM divalent cations (Ca2+ or Mg2+)

• Temperature: 37°C (physiological for S. aureus)

• Enzyme concentration: 10-100 nM

• Substrate concentration: 1-100 μM (for kinetic analysis)

These assays enable determination of important kinetic parameters such as kcat, KM, and inhibition constants, providing a quantitative basis for understanding CtpA function and inhibitor effectiveness.

What purification strategies yield high-purity, active S. aureus CtpA?

Purification of high-purity, active S. aureus CtpA requires a multi-step strategy. The following protocol is recommended:

• Cell lysis and initial clarification:

  • Resuspend bacterial cell pellet in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT, protease inhibitor cocktail)

  • Lyse cells by sonication or high-pressure homogenization

  • Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

  • Filter supernatant through a 0.45 μm filter

• Immobilized metal affinity chromatography (IMAC):

  • For His-tagged CtpA, use Ni-NTA or Co-NTA resin

  • Bind protein in batch or using a pre-packed column

  • Wash extensively with wash buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole)

  • Elute with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250-300 mM imidazole)

• Size exclusion chromatography (SEC):

  • Further purify CtpA using a Superdex 200 column

  • Use buffer compatible with downstream applications (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol)

  • Analyze fractions by SDS-PAGE and pool those containing pure CtpA

• Quality control assessments:

  • Verify purity by SDS-PAGE (>95% purity)

  • Confirm identity by mass spectrometry

  • Assess activity using enzymatic assays

  • Check for proper folding using circular dichroism spectroscopy

Specific challenges and solutions for S. aureus CtpA purification:

• Solubility issues: If CtpA forms inclusion bodies:

  • Lower induction temperature (16-18°C)

  • Use solubility-enhancing tags (MBP, SUMO)

  • Add detergents (0.1% Triton X-100) to lysis buffer

• Proteolytic degradation: To minimize self-proteolysis:

  • Include protease inhibitor cocktail in all buffers

  • Work quickly and maintain samples at 4°C

  • Consider purifying catalytically inactive mutants (S302A) for structural studies

• Storage considerations:

  • Concentrate protein to 1-5 mg/ml

  • Add glycerol to 10-20% final concentration

  • Flash-freeze in liquid nitrogen and store at -80°C

  • Avoid multiple freeze-thaw cycles

This purification strategy should yield highly pure, active S. aureus CtpA suitable for biochemical, structural, and functional studies.

What approaches can be used for structural studies of S. aureus CtpA?

Structural characterization of S. aureus CtpA can be performed using multiple complementary techniques:

A comprehensive structural characterization would ideally combine multiple approaches:

Such multi-technique structural characterization would provide invaluable insights into the molecular basis of S. aureus CtpA function, regulation, and substrate specificity, potentially informing the development of specific inhibitors.