Recombinant Staphylococcus aureus Probable CtpA-like serine protease (SACOL1455)

Basic Research Questions

• What is the CtpA-like serine protease (SACOL1455) in Staphylococcus aureus?

CtpA (SACOL1455) is the lone C-terminal processing protease in S. aureus, belonging to the S41 family of serine proteases. Highly conserved across all sequenced S. aureus strains, it plays a crucial role in bacterial physiology and pathogenesis. The full-length protein consists of 496 amino acids with distinct functional domains. Unlike most S. aureus proteases that are secreted, CtpA is localized to the bacterial cell wall where it contributes to cell wall integrity, stress tolerance, and virulence . This localization is consistent with its function in maintaining bacterial survival during infection and interaction with host components.

• What is the domain organization of the SACOL1455 protein?

The CtpA (SACOL1455) protein exhibits a modular domain structure consisting of:

• A protein binding PDZ domain located between amino acid residues 135 and 218

• An S41 CTP peptidase domain located between residues 231 and 395

• A peptidoglycan binding domain between residues 417 and 473

This domain organization is particularly significant as the peptidoglycan binding domain is unique to CTPs from Gram-positive bacteria and absent in Gram-negative bacterial homologs . The complete amino acid sequence includes: MDDKQHTSSSDDERAEIATSNQDQETNSSKRVHLKRWQFISILIGTILITAVITVVAYIF INQKISGLNKTDQSNLNKIENVYKILNSDYYKKQDSDKLSKAAIDGMVKELKDPYSEYLT KEQTKSFNEGVSGDFVGIGAEMQKKNDQIMVTSPMKGSPAERAGIRPKDVITKVNGKSIK GKALDEVVKDVRGKENTEVTLTVQRGSEEKDVKIKREKIHVKSVEYKKKGKVGVITINKF QNDTSGELKDAVLKAHKDGLKKIVLDLRNNPGGLLDEAVKMANIFIDKGKTVVKLEKGKD TEAIQTSNDALKEAKDMDISILVNEGSASASEVFTGALKDYNKAKVYGSKTFGKGVVQTT REFKDGSLLKYTEMKWLTPDGHYIHGKGIKPDVTIDTPKYQSLNVIPNTKTFKVGDDDKN IKTIKIGLSALGYKVDNESTQFDKALENQVKAFQQANKLEVTGEFNKETNNKFTELLVEK ANKHDDVLDKLINILK .

• How does the expression of ctpA vary under different conditions?

The expression of ctpA in S. aureus is dynamically regulated in response to environmental conditions:

• Expression reaches maximum levels during stationary phase of bacterial growth

• Significant upregulation occurs upon exposure to conditions encountered during infection

• Expression is induced when bacteria are exposed to peptidoglycan-targeting antibiotics

• Responsive to ex vivo interaction with components of the host immune system

This pattern of regulation suggests that CtpA expression is specifically triggered during infection and stress conditions, allowing S. aureus to adapt to challenging environments encountered within the host. The increased expression during exposure to cell wall-targeting antibiotics further supports its role in maintaining cell wall integrity under stress.

• What phenotypes are observed in S. aureus ctpA mutant strains?

S. aureus ctpA mutant strains exhibit several distinct phenotypes that highlight the importance of this protease:

• Decreased heat tolerance compared to wild-type strains

• Increased sensitivity to components of the host immune system

• Significantly attenuated virulence in animal models of infection

• Reduced bacterial burden in multiple organs during systemic infection, including reductions in the spleen (28-fold), brain (228-fold), heart (65-fold), and kidneys (6-fold)

• Altered cell wall stability and integrity

These phenotypic changes demonstrate that CtpA plays a multifaceted role in bacterial physiology and pathogenesis, particularly under stress conditions encountered during infection. The dramatic reduction in organ bacterial burden indicates that CtpA is critical for S. aureus virulence and survival within the host.

• What methods are used to produce recombinant SACOL1455 protein for research?

Recombinant SACOL1455 protein can be produced using the following methodology:

• Clone the full-length gene (coding for amino acids 1-496) from S. aureus genomic DNA

• Insert into an appropriate expression vector with an N-terminal His-tag

• Transform into E. coli expression strain

• Induce protein expression under optimized conditions

• Lyse cells and purify using nickel affinity chromatography

• Verify purity via SDS-PAGE analysis

• Store as lyophilized powder or in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• For long-term storage, add glycerol (5-50% final concentration) and store at -20°C/-80°C

This approach yields full-length recombinant protein suitable for enzymatic assays, structural studies, and functional characterization. Reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended before use in experimental procedures.

• What assays can be used to detect and measure CtpA activity?

Several methodological approaches can be used to detect and quantify CtpA activity:

• Cleavage assays: Incubate purified CtpA with potential substrate proteins, stop the reaction with SDS-containing buffer, and analyze cleavage products via SDS-PAGE and Western blotting using specific antisera .

• Inhibition studies: Test serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) or aprotinin at different concentrations to confirm the serine protease mechanism .

• Activity-based profiling: Use biotinylated or "clickable" peptides containing NHS carbamates as activity-based probes to detect active CtpA .

• Mass spectrometry: Analyze proteolytic fragments to determine precise cleavage sites and substrate preferences.

• Zymography: Detect proteolytic activity using substrate-containing gels to visualize zones of clearing.

• Proteomics approaches: Apply CLIPPER 2.0 or similar tools for comprehensive degradomics data analysis and substrate identification .

Selection of the appropriate assay depends on the specific research question, available resources, and experimental context.

• What are the major similarities and differences between S. aureus CtpA and homologous proteins in other bacteria?

CtpA proteins show both conservation and divergence across bacterial species:

Key differences include:

• S. aureus possesses a single CTP, while many bacteria have multiple C-terminal processing proteases

• The peptidoglycan binding domain is unique to Gram-positive bacterial CTPs

• S. aureus CtpA is localized to the cell wall, while in Gram-negative bacteria, homologous proteins are periplasmic

These differences likely reflect adaptations to the distinct cell envelope architectures of Gram-positive versus Gram-negative bacteria.

• How does CtpA contribute to S. aureus virulence?

CtpA contributes to S. aureus virulence through several mechanisms:

• Maintenance of cell wall integrity: Helps preserve bacterial structure during exposure to host defense mechanisms

• Stress tolerance: Enhances bacterial survival under various stress conditions encountered during infection

• Immune evasion: Contributes to resistance against components of the host immune system

• Systemic survival: Enables bacterial persistence and replication in multiple organ systems

In murine infection models, disruption of the ctpA gene leads to significantly decreased virulence, with markedly reduced bacterial burden in multiple organs including spleen, brain, heart, and kidneys . The dramatic attenuation in virulence (6-228 fold reduction in organ burden) demonstrates that CtpA is a critical virulence determinant in S. aureus.

Advanced Research Questions

• How does CtpA interact with proteolytic cascades in S. aureus?

CtpA likely participates in proteolytic cascades within S. aureus, though its specific role is still being fully characterized. Based on research in other bacterial systems:

• In Gram-negative bacteria such as Pseudomonas, CtpA functions upstream of the Prc protease in proteolytic cascades

• CtpA appears to function as a regulator that prevents Prc-mediated proteolysis of certain substrates

• The proteolytic network involving CtpA may influence multiple cellular processes including:

  • Cell-surface signaling

  • Stress responses

  • Virulence factor processing

  • Cell wall maintenance

The complete proteolytic network involving CtpA in S. aureus remains to be fully elucidated, but the significant phenotypic effects of ctpA mutation suggest it plays a central role in bacterial physiology and virulence. Research in Pseudomonas has shown that "CtpA functions upstream of Prc in the proteolytic cascade and seems to prevent the Prc-mediated proteolysis of the CSS anti-σ factor" , suggesting potential regulatory interactions rather than just direct substrate processing.

• How can activity-based probes be used to study CtpA function?

Activity-based probes (ABPs) represent powerful tools for studying serine proteases like CtpA:

• ABP design principles for serine proteases:

  • Peptides containing N-alkyl glycine NHS carbamates function as potent irreversible inhibitors that can be adapted as ABPs

  • These probes can incorporate biotin tags for detection or "clickable" moieties for downstream modification

  • The reactive NHS-carbamate group forms a covalent bond with the active site serine

• Specific ABP applications for CtpA research:

  • Detection of active CtpA in complex biological samples

  • Profiling CtpA activity under different physiological conditions

  • Identification of conditions that modulate CtpA activity

  • Screening for potential inhibitors

• Experimental workflow:

  • Synthesize peptide ABPs containing appropriate specificity elements and reporting groups

  • Incubate with bacterial lysates or purified CtpA

  • Analyze labeled proteins via SDS-PAGE and detection methods appropriate for the incorporated tag

  • Validate specificity using competitive inhibitors or genetic controls

The advantage of ABPs is that they report on functional enzyme activity rather than just protein presence, enabling more nuanced studies of CtpA regulation and function .

• What experimental models are most suitable for studying CtpA's role in S. aureus pathogenesis?

Multiple experimental models can be employed to investigate CtpA's role in S. aureus pathogenesis:

• In vitro models:

  • Human cell line infections (e.g., A549 lung epithelial cells)

  • Primary human neutrophil killing assays

  • Human serum resistance assays measuring complement deposition and bacterial survival

  • Phagocytosis assays with GFP-expressing S. aureus strains

• Ex vivo models:

  • Human blood survival assays

  • Neutrophil extracellular trap (NET) formation and bacterial killing assays

• In vivo models:

  • Murine systemic infection model (demonstrated 6-228 fold reductions in organ bacterial burden)

  • Skin and soft tissue infection models

  • Zebrafish embryo infection model (used successfully for studying related proteases)

Each model system offers distinct advantages, and selection should be based on the specific research question. The murine model has been successfully used to demonstrate CtpA's role in virulence, showing significant attenuation in ctpA mutant strains, with dramatically reduced bacterial burden in multiple organs .

• How do researchers identify and validate CtpA substrates?

Identifying authentic CtpA substrates requires multiple complementary approaches:

• Substrate specificity profiling:

  • Synthetic peptide libraries to identify preferred cleavage motifs

  • Positional scanning to determine amino acid preferences at each position

  • Activity-based probes with varying specificity elements

• Candidate substrate approaches:

  • In vitro cleavage assays with purified potential substrates

  • Western blot analysis to detect processing differences between wild-type and ctpA mutant strains

  • Mass spectrometry to identify precise cleavage sites

• Unbiased substrate identification:

  • Proteomic comparison of wild-type and ctpA mutant strains

  • Terminal proteomics to identify proteins with altered C-termini

  • CLIPPER 2.0 software for peptide-level annotation and data analysis of mass spectrometry-based proteomics data

  • Analysis of proteoform certainty based on protein accessions

• Substrate validation:

  • Site-directed mutagenesis of putative cleavage sites

  • Complementation studies in ctpA mutant backgrounds

  • Functional assays to determine the consequence of substrate processing

The CLIPPER 2.0 computational tool is particularly valuable for this work as it "facilitates peptide-level annotation and data analysis" and "enables fast and automated database information retrieval, statistical and network analysis, as well as visualization of terminomic datasets" .

• How does CtpA influence S. aureus survival under various stress conditions?

CtpA contributes significantly to S. aureus survival under several stress conditions:

• Heat stress:

  • ctpA mutants exhibit decreased heat tolerance

  • CtpA may process heat-damaged proteins or stabilize cell wall structures at elevated temperatures

  • This function is consistent with the role of related proteases in other bacteria

• Immune stress:

  • Enhanced sensitivity to components of the host immune system in ctpA mutants

  • CtpA may protect against antimicrobial peptides, complement components, or other host defense molecules

  • Similar to the SplB protease that targets host complement proteins , CtpA may contribute to immune evasion

• Antibiotic stress:

  • CtpA expression is induced by peptidoglycan-targeting antibiotics

  • May contribute to cell wall remodeling in response to antibiotic challenge

  • Could process specific proteins involved in antibiotic resistance mechanisms

The multifaceted role of CtpA in stress tolerance explains its importance for S. aureus adaptation during infection and highlights its potential as a therapeutic target for novel anti-virulence strategies.

• How can researchers design effective inhibitors for CtpA?

Designing effective CtpA inhibitors requires a methodical approach:

• Understanding the active site:

  • Crystal structure determination of CtpA

  • Homology modeling based on related S41 proteases

  • Substrate specificity profiling to identify key recognition elements

• Inhibitor design strategies:

  • Peptide-based inhibitors targeting the substrate binding pocket

  • Small molecule screening for active site binders

  • Covalent inhibitors targeting the catalytic serine (similar to PMSF or aprotinin)

  • Structure-based design of transition state analogues

• Testing and validation:

  • In vitro enzymatic assays with purified CtpA

  • Cellular assays measuring effects on bacterial survival and virulence factor production

  • Assessment of specificity against other serine proteases

  • Evaluation of stability, bioavailability, and toxicity

• Biological validation:

  • Comparison of inhibitor effects with genetic deletion phenotypes

  • Combination studies with antibiotics

  • Efficacy testing in infection models

This approach could lead to novel anti-virulence compounds that sensitize S. aureus to host defenses without directly killing bacteria, potentially reducing selective pressure for resistance development.

• How does CtpA compare with other S. aureus proteases in terms of function and contribution to virulence?

S. aureus produces multiple proteases that contribute to virulence through distinct mechanisms:

Key differences:

These differences highlight the complementary roles of different proteases in S. aureus pathogenesis, with CtpA playing a foundational role in bacterial fitness during infection .

• What methodological approaches can be used to study the role of CtpA in biofilm formation?

Investigating CtpA's potential role in biofilm formation requires multiple complementary approaches:

• Static biofilm assays:

  • Compare wild-type and ctpA mutant strains for biofilm formation in microtiter plates

  • Quantify biomass using crystal violet staining

  • Assess biofilm architecture through confocal microscopy

  • Evaluate extracellular matrix composition with specific stains

• Flow cell biofilm systems:

  • Analyze biofilm development under dynamic conditions

  • Assess structural integrity under shear stress

  • Monitor biofilm maturation and dispersal over time

• Molecular analyses:

  • Transcriptomics to identify biofilm-related genes affected by ctpA mutation

  • Proteomics to characterize extracellular matrix protein differences

  • Targeted analysis of known biofilm components (e.g., polysaccharide intercellular adhesin, extracellular DNA)

• Genetic approaches:

  • Complementation studies with wild-type and catalytically inactive CtpA

  • Epistasis analysis with known biofilm regulators

  • Construction of reporter strains to monitor biofilm-related gene expression

Because cell wall proteins are important for biofilm formation and CtpA is a cell wall-associated protease that influences cell wall integrity, it may play a significant role in biofilm development or maturation.

• How can mass spectrometry-based proteomics be optimized for studying CtpA function?

Optimizing mass spectrometry-based proteomics for CtpA research requires specialized approaches:

• Sample preparation considerations:

  • Combine multiple proteolytic enzymes (not just trypsin) to increase coverage

  • Use enrichment strategies for cell wall proteins

  • Apply targeted approaches for C-terminal peptide analysis

  • Implement stable isotope labeling for quantitative comparisons

• MS analysis parameters:

  • Utilize data-dependent and data-independent acquisition methods

  • Optimize MS/MS fragmentation for improved peptide identification

  • Implement targeted approaches for known CtpA substrates

• Data analysis techniques:

  • Apply CLIPPER 2.0 for comprehensive peptide-level annotation

  • Use MSqRob or similar tools for statistical analysis of quantitative data

  • Implement algorithms to identify differential protein processing

  • Analyze protein terminal peptides specifically to identify processing events

• Validation approaches:

  • Confirm processing events with targeted MS assays

  • Perform parallel protein immunoblotting for key proteins

  • Correlate MS findings with functional assays

These methodologies enable comprehensive characterization of CtpA's impact on the S. aureus proteome and identification of direct and indirect effects on protein processing.

• What is the relationship between CtpA activity and host serine protease responses during infection?

The interplay between bacterial CtpA and host serine proteases represents an important aspect of host-pathogen interactions:

• Host serine proteases in infection:

  • Neutrophil elastase, cathepsin G, and proteinase 3 are key antimicrobial proteases

  • Plasmin and other serine proteases can influence tissue remodeling during infection

  • Serine proteases contribute to both pro- and anti-inflammatory responses

• Potential interactions with CtpA:

  • CtpA may process bacterial surface proteins to reduce recognition by host proteases

  • CtpA could modify bacterial components that activate host proteases

  • Host proteases might target bacterial proteins processed by CtpA

• Research methodologies:

  • Compare wild-type and ctpA mutant responses to purified host proteases

  • Analyze differential susceptibility to neutrophil killing

  • Examine alterations in bacterial surface proteins in response to host proteases

  • Investigate potential synergistic or antagonistic relationships between bacterial and host proteases

• Clinical relevance:

  • Analysis of proteolytic activity in clinical samples from infected patients

  • Evaluation of serine protease patterns in plasma from patients with S. aureus infections

  • Correlation between protease activity and infection outcomes

Recent research has shown complex relationships between bacterial proteases and host response, with evidence that "the concentration, potential activity of plasminogen, and the total amount of serine proteases in plasma from BC patients were greater than the values of the corresponding indicators in healthy donors" , suggesting dysregulated proteolysis during infection.