Recombinant Staphylococcus aureus Probable CtpA-like serine protease (SAB1275c)

What is Recombinant Staphylococcus aureus Probable CtpA-like serine protease (SAB1275c)?

Recombinant Staphylococcus aureus Probable CtpA-like serine protease (SAB1275c) is a full-length protein (496 amino acids) belonging to the S41 family of serine proteases. This protein is expressed recombinantly in E. coli with an N-terminal His-tag for purification purposes. The protein is derived from the pathogenic bacterium Staphylococcus aureus and functions as a C-terminal processing protease. It has been identified as the lone C-terminal processing protease in S. aureus and plays critical roles in bacterial virulence, stress response, and pathogenesis . The protein is encoded by the gene SAB1275c and has the UniProt ID Q2YXZ9 .

What is the cellular localization of CtpA in S. aureus and why is it significant?

CtpA in S. aureus is primarily localized to the bacterial cell wall, as demonstrated through subcellular fractionation studies and immunolocalization techniques. This localization is particularly significant because it suggests that CtpA may play a role in maintaining cell wall stability and integrity during infection . The cell wall localization also indicates that CtpA potentially processes surface-exposed proteins that interact with host factors during pathogenesis. Researchers studying the protein should consider this localization when designing experiments, particularly when investigating protein-protein interactions or potential substrates.

How should researchers handle and reconstitute recombinant SAB1275c protein for experimental use?

For optimal experimental outcomes, recombinant SAB1275c protein requires careful handling. The protein is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C upon receipt. Before opening, briefly centrifuge the vial to bring contents to the bottom. For reconstitution:

• Dissolve the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Add glycerol to a final concentration of 5-50% (50% is recommended) to prevent protein degradation.

• Aliquot the reconstituted protein for long-term storage at -20°C/-80°C.

• Avoid repeated freeze-thaw cycles as they can compromise protein integrity.

• Working aliquots may be stored at 4°C for up to one week .

The reconstituted protein is stored in a Tris/PBS-based buffer (pH 8.0) containing 6% Trehalose, which helps maintain protein stability .

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

CtpA plays a crucial role in S. aureus virulence through multiple mechanisms:

• Stress Tolerance: CtpA is essential for bacterial survival under stress conditions encountered during infection. Disruption of the ctpA gene leads to decreased heat tolerance, which may affect bacterial persistence in febrile hosts .

• Immune Evasion: CtpA mutant strains show increased sensitivity to components of the host immune system, suggesting that this protease is involved in mechanisms that help S. aureus evade host defenses .

• Virulence in Animal Models: Studies using murine models of infection have demonstrated that ctpA-deficient strains exhibit attenuated virulence compared to wild-type strains. This attenuation manifests as reduced bacterial burden in tissues, decreased inflammatory responses, and improved survival rates in infected animals .

• Gene Expression Regulation: CtpA expression is maximal when the bacterium is exposed to conditions typically encountered during infection, suggesting a regulated response to the host environment that optimizes bacterial survival and virulence .

These findings collectively establish CtpA as a critical virulence factor in S. aureus pathogenesis, making it a potential target for therapeutic intervention.

What methodological approaches can researchers use to study CtpA function in vitro?

To investigate CtpA function in vitro, researchers can employ several methodological approaches:

• Protease Activity Assays:

  • Use synthetic peptide substrates containing fluorogenic or chromogenic reporters

  • Monitor C-terminal cleavage events using mass spectrometry

  • Employ FRET-based assays to detect proteolytic activity in real-time

• Substrate Identification:

  • Perform proteomic analyses comparing wild-type and ctpA mutant strains

  • Use terminal amine isotopic labeling of substrates (TAILS) to identify processed proteins

  • Develop protein microarrays containing potential S. aureus substrates

• Structure-Function Analysis:

  • Express and purify truncated or point-mutated versions of CtpA

  • Perform X-ray crystallography or cryo-EM to determine three-dimensional structure

  • Use molecular docking to predict interactions with potential substrates or inhibitors

• Inhibitor Screening:

  • Develop high-throughput screening assays using recombinant CtpA

  • Test serine protease inhibitors for specific activity against CtpA

  • Perform structure-based drug design based on the active site architecture

These methodological approaches can provide valuable insights into CtpA's biochemical properties, substrate specificity, and potential as a therapeutic target.

How can researchers create and validate CtpA mutant strains for functional studies?

Creating and validating CtpA mutant strains requires a systematic approach:

Creation of Mutant Strains:

• Allelic Exchange: Design primers to amplify upstream and downstream regions of the ctpA gene. Join these fragments to create a deletion construct lacking the ctpA coding sequence. Clone this construct into a temperature-sensitive shuttle vector and perform allelic exchange in S. aureus.

• CRISPR-Cas9 Methodology: Design guide RNAs targeting the ctpA gene and provide a repair template with the desired mutation or deletion. Transform S. aureus with the CRISPR-Cas9 system and select for cells with successful gene editing.

• Transposon Mutagenesis: Use a transposon library to identify insertion mutants in the ctpA gene, then confirm the insertion location by sequencing.

Validation Approaches:

• Genetic Verification: Perform PCR and sequencing to confirm the deletion or mutation of the ctpA gene.

• Transcriptional Analysis: Use RT-qPCR to verify the absence of ctpA transcript in mutant strains.

• Protein Expression: Perform Western blot analysis using anti-CtpA antibodies to confirm the absence of CtpA protein.

• Complementation Studies: Reintroduce the wild-type ctpA gene on a plasmid to restore the phenotype, confirming that observed defects are specifically due to ctpA deletion.

• Phenotypic Characterization:

  • Assess growth rates under standard and stress conditions

  • Evaluate heat tolerance (demonstrated to be decreased in ctpA mutants)

  • Test sensitivity to immune system components

  • Examine virulence in appropriate infection models

These approaches ensure that phenotypes observed in ctpA mutant strains are specifically attributable to the loss of CtpA function.

What are the challenges in studying CtpA proteolytic activity and how can they be overcome?

Studying CtpA proteolytic activity presents several challenges that researchers should address through careful experimental design:

Challenges:

• Substrate Specificity: Unlike many proteases, C-terminal processing proteases have highly specific recognition sequences, making substrate identification difficult.

• Activity Conditions: Determining the optimal pH, temperature, and ionic conditions for CtpA activity can be challenging, especially when replicating the bacterial cell wall environment.

• Protein Solubility: As a membrane-associated protein, recombinant CtpA may have solubility issues that affect in vitro assays.

• Distinguishing Direct vs. Indirect Effects: In vivo, distinguishing between direct CtpA proteolytic events and downstream effects can be difficult.

Solutions:

• Substrate Prediction:

  • Use bioinformatic approaches to predict potential substrates based on homology to known CTP substrates

  • Perform comparative proteomics between wild-type and ctpA mutant strains

  • Create synthetic peptide libraries based on C-terminal sequences of S. aureus proteins

• Activity Optimization:

  • Systematically test buffer conditions, detergents, and cofactors

  • Include cell wall components in assay buffers to mimic natural environment

  • Use protein engineering to create more soluble versions while maintaining activity

• Direct Substrate Identification:

  • Use activity-based protein profiling (ABPP) with biotinylated inhibitors

  • Develop "trap" mutants that bind but don't cleave substrates

  • Employ crosslinking approaches to capture transient enzyme-substrate complexes

By addressing these challenges methodically, researchers can gain deeper insights into CtpA's biological functions and substrate specificity.

How does CtpA expression change under different environmental conditions relevant to infection?

CtpA expression in S. aureus is dynamically regulated in response to environmental conditions encountered during infection, a critical aspect for researchers to consider when designing experiments:

Expression Patterns:

• Infection-Relevant Conditions: The ctpA gene shows maximal expression when S. aureus is exposed to conditions typically encountered during infection, such as temperature shifts, oxidative stress, and host immune factors .

• Temperature Response: Expression increases at 37°C (human body temperature) compared to lower temperatures, consistent with its role in pathogenesis.

• Growth Phase Dependency: CtpA expression varies throughout the bacterial growth cycle, with peak expression typically occurring in late exponential to early stationary phase.

Methodological Approaches to Study Expression:

• Transcriptional Analysis:

  • Use RT-qPCR to quantify ctpA mRNA levels under different conditions

  • Employ RNA-seq for genome-wide expression analysis

  • Create promoter-reporter fusions (e.g., ctpA promoter-GFP) to monitor expression in real-time

• Protein Level Analysis:

  • Perform Western blotting with anti-CtpA antibodies

  • Use quantitative proteomics to measure relative protein abundance

  • Employ immunofluorescence microscopy to visualize CtpA localization and abundance

• In vivo Expression:

  • Use in vivo expression technology (IVET) to identify promoter activity during infection

  • Recover bacteria from infected tissues and analyze ctpA expression

  • Employ animal models with reporter systems to track expression in real-time

Understanding these expression patterns helps researchers design more physiologically relevant experiments and potentially identify conditions for therapeutic targeting of CtpA function.

What animal models are suitable for studying the role of CtpA in S. aureus infections?

Several animal models have been validated for studying CtpA's role in S. aureus pathogenesis:

Murine Models:

• Systemic Infection Model: Intravenous injection of S. aureus wild-type or ctpA mutant strains has demonstrated that ctpA-deficient strains show attenuated virulence, with reduced bacterial burden in organs and improved survival rates .

• Skin and Soft Tissue Infection Model: Subcutaneous injection of bacteria to measure abscess formation, bacterial clearance, and local immune responses.

• Pneumonia Model: Intranasal or intratracheal instillation to assess pulmonary infection, particularly relevant for hospital-acquired pneumonia caused by S. aureus.

Alternative Models:

• Galleria mellonella (Wax Moth) Larvae: A cost-effective invertebrate model that correlates well with mammalian infection outcomes for initial virulence screening.

• Zebrafish Embryo Model: Transparent embryos allow real-time visualization of infection progression and immune cell interactions.

Experimental Design Considerations:

• Bacterial Preparation: Standardize growth conditions and inoculum preparation to ensure reproducibility.

• Genetic Background: Include appropriate controls (wild-type, complemented mutant) to confirm phenotypes are specifically due to ctpA mutation.

• Readouts:

  • Survival rates and time course

  • Bacterial burden in tissues

  • Inflammatory markers and cytokine profiles

  • Histopathological analysis of infected tissues

  • Immune cell recruitment and activation

• Timing: Monitor infections over appropriate time courses to capture both acute and prolonged effects of ctpA deficiency.

These animal models provide crucial insights into how CtpA contributes to different aspects of S. aureus pathogenesis in vivo.

What techniques can be used to identify physiological substrates of CtpA in S. aureus?

Identifying the physiological substrates of CtpA requires a multi-faceted approach combining proteomic, genetic, and biochemical techniques:

Comparative Proteomics:

• Differential Gel Electrophoresis (DIGE): Compare protein patterns between wild-type and ctpA mutant strains to identify proteins with altered migration patterns indicative of processing.

• Quantitative C-terminomics: Use specialized techniques like C-TAILS (C-terminal TAILS) to enrich and identify protein C-termini, comparing wild-type and ctpA mutant strains.

• Stable Isotope Labeling: Employ SILAC or similar approaches to quantitatively compare proteomes and identify proteins with altered abundance or processing.

Genetic Approaches:

• Suppressor Screens: Identify secondary mutations that restore phenotypes in ctpA mutant strains, potentially revealing functional pathways.

• Synthetic Lethality: Screen for genes that, when mutated alongside ctpA, cause more severe phenotypes, suggesting functional relationships.

Biochemical Validation:

• In vitro Processing Assays: Test candidate substrates with purified recombinant CtpA to confirm direct processing.

• Site-Directed Mutagenesis: Mutate predicted cleavage sites in candidate substrates to verify processing locations.

• Protein-Protein Interaction Studies: Use pull-down assays, bacterial two-hybrid systems, or crosslinking approaches to identify proteins that physically interact with CtpA.

Systems-Level Approaches:

• Integration of Multiple Datasets: Combine transcriptomic, proteomic, and phenotypic data to identify the most likely physiological substrates.

• Network Analysis: Map potential substrates into known virulence pathways to prioritize candidates for validation.

Through these complementary approaches, researchers can build a comprehensive understanding of CtpA's substrate network and its functional impact on S. aureus physiology and virulence.

How can researchers develop and screen for inhibitors of CtpA activity?

Developing inhibitors of CtpA represents a potential therapeutic strategy against S. aureus infections:

Inhibitor Design Strategies:

• Structure-Based Design: If the three-dimensional structure of CtpA is available, use computational approaches to design molecules that fit the active site.

• Peptidomimetic Approach: Design modified peptides based on known substrate sequences that compete for the active site but resist cleavage.

• Natural Product Screening: Test libraries of natural compounds, particularly those with known antimicrobial properties, for CtpA inhibitory activity.

Screening Methodologies:

• Biochemical Assays:

  • Develop fluorogenic or chromogenic substrates for high-throughput screening

  • Use FRET-based assays to detect inhibition of proteolytic activity

  • Employ thermal shift assays to identify compounds that bind to and stabilize CtpA

• Cell-Based Assays:

  • Screen for compounds that phenocopy ctpA mutation in S. aureus

  • Use reporter strains where CtpA activity is linked to a detectable signal

  • Test for compounds that specifically sensitize S. aureus to stress conditions where CtpA is known to be important

• In silico Screening:

  • Perform virtual screening of compound libraries against the CtpA active site

  • Use molecular dynamics simulations to refine understanding of inhibitor binding

Validation and Optimization:

• Structure-Activity Relationship Studies: Systematically modify promising inhibitors to improve potency and selectivity.

• Specificity Testing: Confirm that inhibitors specifically target CtpA rather than other serine proteases.

• Cellular Penetration: Evaluate the ability of inhibitors to reach their target within bacterial cells.

• In vivo Efficacy: Test promising inhibitors in animal models of S. aureus infection.

Development of specific CtpA inhibitors could provide new therapeutic options against multidrug-resistant S. aureus strains.

How does S. aureus CtpA compare to homologous proteases in other bacterial species?

Comparing S. aureus CtpA with homologous proteases provides valuable evolutionary and functional insights:

Comparative Table of Bacterial CTPs:

Key Evolutionary Insights:

• Gram-positive vs. Gram-negative Differences: In Gram-negative bacteria, CTPs are typically located in the periplasm, while in Gram-positives like S. aureus, they associate with the cell wall or membrane .

• Functional Divergence: While most bacterial CTPs are involved in stress responses, their specific functions have diverged. S. aureus has a single CTP (CtpA) compared to B. subtilis with both CtpA and CtpB, suggesting potential functional consolidation .

• Conservation of Catalytic Domains: The catalytic domains tend to be more conserved across species, while substrate recognition domains show greater variability, reflecting adaptation to different physiological roles.

Functional Implications:

• Unique Role in S. aureus: As the lone CTP in S. aureus, CtpA likely performs functions that may be distributed among multiple proteases in other species .

• Virulence Connection: The critical role of CtpA in S. aureus virulence parallels findings in some Gram-negative pathogens, suggesting convergent evolution of virulence mechanisms involving CTPs.

• Substrate Diversity: Differences in substrate recognition domains suggest that S. aureus CtpA may have unique substrate preferences compared to its homologs.

This comparative analysis highlights both the evolutionary conservation of CTP functions and the species-specific adaptations that make S. aureus CtpA a unique virulence factor and potential therapeutic target.

What are critical quality control parameters for recombinant SAB1275c in research applications?

Ensuring the quality of recombinant SAB1275c is essential for reliable experimental results:

Critical Quality Control Parameters:

• Purity Assessment:

  • SDS-PAGE analysis should confirm >90% purity as specified

  • Consider additional methods like size-exclusion chromatography for higher resolution

  • Check for presence of contaminating proteases that could confound activity assays

• Identity Confirmation:

  • Western blotting with anti-His tag antibodies and/or specific anti-CtpA antibodies

  • Mass spectrometry to confirm molecular weight and sequence coverage

  • N-terminal sequencing to verify the intact start site

• Activity Verification:

  • Develop and use a standardized activity assay with a defined substrate

  • Determine specific activity (units/mg) for batch-to-batch comparison

  • Test activity under various buffer conditions to ensure optimal performance

• Stability Assessment:

  • Monitor stability at different temperatures and time points

  • Test freeze-thaw stability to validate storage recommendations

  • Evaluate long-term stability of the reconstituted protein

Experimental Controls:

• Negative Controls:

  • Heat-inactivated protein (typically 95°C for 10 minutes)

  • Protein treated with serine protease inhibitors

  • Buffer-only controls for all assays

• Positive Controls:

  • Commercial serine proteases with known activity

  • Previously validated batches of SAB1275c

• Standard Curve Generation:

  • Create standard curves for quantitative assays

  • Include internal standards for day-to-day normalization

Implementing these quality control measures ensures consistent, reliable results across experiments and between different research groups studying this important virulence factor.

How can researchers optimize expression and purification of recombinant SAB1275c for structural studies?

Optimizing expression and purification of recombinant SAB1275c for structural studies requires specialized strategies:

Expression Optimization:

• Expression System Selection:

  • E. coli BL21(DE3) or derivatives for high-level expression

  • Consider Shuffle T7 strains for enhanced disulfide bond formation

  • For challenging expression, test insect or mammalian cell systems

• Construct Design:

  • Test multiple truncation constructs to identify stable domains

  • Consider fusion partners (MBP, SUMO) to enhance solubility

  • Include TEV or PreScission protease sites for tag removal

  • Test both N- and C-terminal His-tags to determine optimal configuration

• Expression Conditions:

  • Screen multiple temperatures (15°C, 20°C, 25°C, 30°C, 37°C)

  • Test various induction methods (IPTG concentration, auto-induction)

  • Optimize media formulations (LB, TB, M9, specialized media)

  • Consider co-expression with chaperones for improved folding

Purification Strategies:

• Multi-step Purification:

  • Initial IMAC (immobilized metal affinity chromatography) using the His-tag

  • Ion exchange chromatography as a secondary step

  • Size exclusion chromatography for final polishing and buffer exchange

  • Consider on-column refolding for inclusion body purification

• Buffer Optimization:

  • Screen various pH conditions (pH 6.0-9.0)

  • Test different salt concentrations (0-500 mM NaCl)

  • Add stabilizing agents (glycerol, trehalose, specific ions)

  • Include reducing agents if necessary (DTT, TCEP)

• Quality Assessment for Structural Studies:

  • Dynamic light scattering to verify monodispersity

  • Thermal shift assays to identify stabilizing conditions

  • Circular dichroism to confirm secondary structure

  • Limited proteolysis to identify stable domains

• Crystallization Preparation:

  • Concentrate to 5-20 mg/mL (test multiple concentrations)

  • Remove His-tag if it interferes with crystallization

  • Verify protein homogeneity by native PAGE

  • Perform pre-crystallization tests to determine optimal concentration

By methodically optimizing these parameters, researchers can obtain high-quality recombinant SAB1275c suitable for crystallization, NMR studies, or cryo-EM analysis, ultimately advancing our structural understanding of this important virulence factor.

What are the most promising future research directions for CtpA in S. aureus?

Several promising research directions can advance our understanding of CtpA and its potential as a therapeutic target:

• Comprehensive Substrate Identification: Employing advanced proteomics to fully characterize the substrate repertoire of CtpA will reveal its mechanistic role in virulence. This includes identifying both direct substrates and downstream effects of CtpA processing .

• Structural Characterization: Determining the three-dimensional structure of S. aureus CtpA through X-ray crystallography or cryo-EM would facilitate structure-based drug design efforts.

• Host-Pathogen Interactions: Investigating how CtpA influences interactions with host immune components could uncover novel immune evasion mechanisms and potential intervention points .

• Development of Specific Inhibitors: Designing and testing specific inhibitors of CtpA activity could lead to novel anti-virulence therapeutics that don't promote resistance like traditional antibiotics.

• Combination Therapies: Exploring how CtpA inhibition might sensitize S. aureus to conventional antibiotics or host immune defenses could lead to more effective combination therapies.

• Regulatory Networks: Elucidating how CtpA expression and activity are regulated during infection would provide deeper insights into S. aureus adaptation to the host environment .

• Comparative Studies: Extending investigations to other Staphylococcal species would reveal conserved and species-specific roles of CtpA in pathogenesis.