Recombinant Staphylococcus aureus Putative peptidyl-prolyl cis-trans isomerase (SAR0916)

What is Staphylococcus aureus Putative peptidyl-prolyl cis-trans isomerase (SAR0916) and what is its role in bacterial physiology?

SAR0916 belongs to the peptidyl-prolyl cis/trans isomerase (PPIase) family in S. aureus, enzymes that catalyze the isomerization of peptide bonds preceding proline residues. These enzymes play crucial roles in protein folding by accelerating the typically slow cis-trans isomerization of prolyl peptide bonds. In S. aureus, PPIases like SAR0916 are particularly important for the proper folding of secreted virulence factors, which must be transported across the cell membrane in a denatured state and then correctly refolded in the extracellular environment to become functionally active .

Structurally, bacterial PPIases fall into different classes including cyclophilins, FK506-binding proteins (FKBPs), and parvulins. While the specific classification of SAR0916 is not explicitly stated in the available literature, its functional characterization suggests similarity to other staphylococcal PPIases that contribute to virulence factor activity.

How does SAR0916 compare with other characterized S. aureus PPIases?

S. aureus possesses several PPIases with distinct subcellular localizations and functional roles. Research has identified at least three key PPIases in S. aureus:

SAR0916 shares functional similarities with these characterized PPIases, particularly in its potential role in virulence factor folding. Genome analysis of community-associated methicillin-resistant S. aureus has shown that mutations in PPIase genes affect bacterial fitness under various conditions. For instance, ppiB mutants demonstrate decreased fitness in abscess models of infection, while prsA mutants show reduced fitness in human blood and during osteomyelitis infection .

What are the optimal conditions for recombinant expression of SAR0916?

For recombinant expression of S. aureus PPIases like SAR0916, the following methodological approach is recommended:

• Vector selection: The pET28a expression vector has been successfully used for the expression of S. aureus proteins, providing a His-tag for purification and strong inducible expression .

• Codon optimization: The amino acid sequence should be codon-optimized for expression in E. coli, which significantly improves protein yield. Commercial gene synthesis services can generate the synthetic gene with optimized codons .

• Expression system: E. coli BL21(DE3) is the preferred host strain for expression, typically cultured in LB medium supplemented with appropriate antibiotics.

• Induction conditions:

  • Culture temperature: 37°C for growth phase, reduced to 18-25°C post-induction

  • Induction at OD600 of 0.6-0.8

  • IPTG concentration: 0.5-1.0 mM

  • Post-induction time: 4-18 hours (overnight expression at lower temperatures often yields better results for soluble protein)

Careful optimization of these parameters is essential as they significantly impact the yield and solubility of the recombinant protein.

What purification strategy yields the highest purity and activity of recombinant SAR0916?

A multi-step purification process is recommended to obtain high-purity, active SAR0916:

• Cell lysis: Bacterial cells should be disrupted in a buffer containing:

  • 50 mM Tris-HCl, pH 8.0

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

• Initial purification: Immobilized Metal Affinity Chromatography (IMAC)

  • Ni-NTA or Co-NTA resin for His-tagged protein

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Wash buffer: Same as binding buffer with 20-40 mM imidazole

  • Elution buffer: Same as binding buffer with 250-300 mM imidazole

• Secondary purification: Size exclusion chromatography

  • Column: Superdex 75 or 200

  • Buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM DTT

• Quality control assessment:

  • SDS-PAGE for purity (>95% recommended)

  • Western blot for identity confirmation

  • Dynamic light scattering for aggregation analysis

  • Circular dichroism for secondary structure verification

• Activity verification: PPIase activity assay using the standard chymotrypsin-coupled assay with N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate

This purification strategy typically yields 10-20 mg of purified protein per liter of bacterial culture with >95% purity and preserved enzymatic activity.

How can the PPIase activity of SAR0916 be reliably measured in vitro?

The standard method for measuring PPIase activity is the chymotrypsin-coupled spectrophotometric assay:

Principle: The assay measures the rate of cis-to-trans isomerization of a proline-containing peptide substrate. Chymotrypsin specifically cleaves after the proline residue only when it's in the trans conformation, releasing p-nitroaniline which can be detected spectrophotometrically.

Protocol:

• Prepare reaction buffer: 35 mM HEPES pH 7.8, 0.1 mg/mL bovine serum albumin

• Substrate preparation: 3-10 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide in trifluoroethanol containing 0.45 M LiCl

• Enzyme preparation: 10-100 nM purified SAR0916 in reaction buffer

• Add α-chymotrypsin (100-300 μM final concentration) to the reaction buffer

• Mix substrate and enzyme, immediately measure the increase in absorbance at 390 nm

• Calculate the catalytic efficiency using first-order rate constants

For more accurate measurements, the following controls should be included:

• Uncatalyzed reaction (substrate without enzyme)

• Heat-inactivated enzyme

• Reactions with known PPIase inhibitors (cyclosporin A for cyclophilins)

Data analysis: PPIase activity is calculated using the following equation:

$k_{cat}/K_m = (k_{obs} - k_0)/[E]$

Where:

• k_obs is the first-order rate constant of the catalyzed reaction

• k₀ is the first-order rate constant of the uncatalyzed reaction

• [E] is the enzyme concentration

What evidence supports the role of SAR0916 in virulence factor folding?

Studies of S. aureus PPIases have demonstrated their critical role in virulence factor folding and activity. While specific data for SAR0916 is limited, research on similar staphylococcal PPIases provides compelling evidence for their function in virulence:

• Direct demonstration of refolding acceleration: Purified S. aureus PpiB has been shown to directly interact with nuclease (Nuc) in vitro and accelerate its refolding rate .

• Deletion mutant phenotypes: Disruption of ppiB in S. aureus results in decreased nuclease activity in culture supernatants without altering the levels of Nuc protein, indicating that the enzyme is required for proper Nuc folding rather than expression or secretion .

• Correlation with virulence: Transposon-sequencing (TnSeq) studies have revealed that S. aureus PPIase mutants demonstrate decreased fitness in infection models. Specifically, ppiB mutants showed reduced fitness in abscess models, while prsA mutants exhibited decreased fitness in human blood and osteomyelitis infections .

To investigate SAR0916's specific role in virulence factor folding, researchers should:

• Generate a SAR0916 deletion mutant

• Assess the activity (not just presence) of multiple secreted virulence factors

• Perform complementation studies to confirm phenotypes

• Conduct in vitro refolding assays with purified SAR0916 and candidate virulence factors

How can SAR0916 be utilized in vaccine development against S. aureus infections?

SAR0916, as a PPIase involved in virulence factor folding, represents a potential vaccine candidate against S. aureus infections. A methodological approach for exploring its vaccine potential would include:

• Immunogenicity assessment:

  • Vaccinate mice with purified recombinant SAR0916

  • Evaluate antibody titers using ELISA

  • Assess T-cell responses through cytokine production analysis and lymphocyte proliferation assays

  • Characterize memory cell generation in draining lymph nodes

• Protection studies:

  • Challenge vaccinated animals with viable S. aureus

  • Measure bacterial loads in tissues

  • Assess disease severity and survival rates

  • Evaluate for sterilizing immunity vs. disease amelioration

Recent studies with S. aureus recombinant proteins have demonstrated that vaccination can generate memory cells in draining lymph nodes. For example, immunization with recombinant proteins F0F1 ATP synthase subunit α (SAS), succinyl-diaminopimelate (SDD), and cysteinyl-tRNA synthetase (CTS) in combination with GM-CSF DNA vaccine increased the percentage of IL-17A+ cells among CD44+ memory T cells, suggesting induction of protective immunity .

What mouse models are most appropriate for studying SAR0916 function in S. aureus virulence?

Several mouse models have been validated for studying S. aureus virulence factors and would be appropriate for investigating SAR0916 function:

• Systemic infection model:

  • Intravenous injection of S. aureus (wild-type vs. SAR0916 mutant)

  • Monitoring of bacterial burden in kidneys, liver, and spleen

  • Assessment of animal survival and weight loss

  • Histopathological examination of infected tissues

• Subcutaneous abscess model:

  • Subcutaneous injection of bacteria with cytodex beads

  • Measurement of abscess size and bacterial recovery

  • Particularly relevant as ppiB mutants show decreased fitness in this model

• Mastitis model:

  • Intra-mammary inoculation in lactating mice

  • Relevant for studying S. aureus as a major cause of bovine mastitis

  • Allows for assessment of local immune responses

• Osteomyelitis model:

  • Injection of bacteria into the tibial metaphysis

  • Micro-CT imaging to assess bone destruction

  • Relevant as prsA mutants show decreased fitness in this model

The choice of model should be guided by the specific aspect of SAR0916 function being investigated. For studying its role in secreted virulence factor folding, models that emphasize toxin-mediated pathology (like the skin abscess model) would be most informative.

How do environmental conditions affect SAR0916 expression and activity in S. aureus?

The expression and activity of bacterial PPIases are often regulated in response to environmental stresses. For SAR0916, researchers should consider investigating the following factors:

• Temperature effects:

  • Expression level changes between 30°C, 37°C, and 42°C (fever temperature)

  • PPIase activity measurement at different temperatures

  • Thermal stability assessment using differential scanning fluorimetry

• pH adaptation:

  • Expression profiling across pH range 5.5-8.0

  • Activity measurement at physiologically relevant pH values

  • Correlation with infection site pH (e.g., acidic phagolysosomes)

• Oxygen availability:

  • Comparison of expression under aerobic, microaerobic, and anaerobic conditions

  • Correlation with redox state of the bacterial cytoplasm

• Nutrient limitation:

  • Expression during growth in minimal media vs. rich media

  • Response to iron limitation (common host defense mechanism)

• Host factor exposure:

  • Expression changes upon exposure to subinhibitory concentrations of antimicrobial peptides

  • Response to reactive oxygen species

Experimental approaches should include:

• qRT-PCR for transcript level measurement

• Western blotting for protein level assessment

• Reporter fusions (e.g., SAR0916 promoter-GFP) for real-time monitoring

• In vitro activity assays under varying conditions

What is the structural basis for substrate specificity of SAR0916 compared to other bacterial PPIases?

Understanding the structural determinants of SAR0916 substrate specificity requires a combination of structural biology and biochemical approaches:

• Structural determination:

  • X-ray crystallography of purified SAR0916

  • Alternative approach: Homology modeling based on related bacterial PPIases

  • NMR spectroscopy for dynamic regions

• Substrate binding characterization:

  • Isothermal titration calorimetry (ITC) with model peptide substrates

  • Surface plasmon resonance (SPR) for binding kinetics

  • Hydrogen-deuterium exchange mass spectrometry to identify binding interfaces

• Mutagenesis studies:

  • Identify conserved residues in the putative active site

  • Generate point mutations and assess activity changes

  • Correlate with structural information

• Substrate profiling:

  • Test activity against a library of proline-containing peptides

  • Establish a position-specific scoring matrix for preferred sequences

  • Compare with substrate preferences of other S. aureus PPIases

Based on studies of related bacterial PPIases, SAR0916 likely recognizes specific amino acid sequences surrounding the target proline residue, with preferences for particular residues at the P1 (preceding) and P1' (following) positions. Determining these preferences would enable prediction of potential physiological substrates and help elucidate its specific role in S. aureus virulence factor folding.

How can CRISPR-Cas9 genome editing be optimized for studying SAR0916 function in S. aureus?

CRISPR-Cas9 technology offers precise genome editing capabilities for studying SAR0916 function:

• Vector system selection:

  • Temperature-sensitive plasmids like pMAD or pIMAY for S. aureus

  • Dual-plasmid systems with separate Cas9 and sgRNA components

• sgRNA design considerations:

  • Target unique sequences to avoid off-target effects

  • Aim for GC content between 40-60%

  • Verify PAM availability (NGG for SpCas9)

  • Screen multiple sgRNAs for each target

• Editing strategies:

  • Gene knockout: Complete deletion of SAR0916

  • Point mutations: Alter specific catalytic residues

  • Domain swapping: Replace domains with those from other PPIases

  • Promoter replacement: For controlled expression

  • Epitope tagging: For localization and interaction studies

• Verification methods:

  • PCR screening of transformants

  • Sanger sequencing of edited regions

  • Whole genome sequencing to check for off-target effects

  • Western blotting for protein expression verification

• Complementation controls:

  • Ectopic expression from another locus

  • Plasmid-based complementation

  • Expression of catalytically inactive variants

This approach allows precise genetic manipulation to study SAR0916 function without the polar effects often associated with traditional insertional mutagenesis methods.

What proteomics approaches can identify the complete set of SAR0916 substrates in S. aureus?

Identifying the complete set of SAR0916 substrates requires multifaceted proteomics approaches:

• Comparative secretome analysis:

  • Wild-type vs. SAR0916 knockout strain

  • Focus on proteins with altered abundance or activity

  • 2D gel electrophoresis followed by mass spectrometry

  • Label-free quantitative LC-MS/MS

• Interactome mapping:

  • Affinity purification-mass spectrometry: Using tagged SAR0916

  • Proximity labeling: BioID or APEX2 fusions to label proteins in proximity

  • Crosslinking-MS: To capture transient enzyme-substrate interactions

  • Co-immunoprecipitation: With antibodies against SAR0916

• Activity-based protein profiling:

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

  • Click chemistry approaches for substrate enrichment

  • Identification of labeled proteins by mass spectrometry

• Fold change analysis:

  • Compare protein folding states using limited proteolysis

  • Hydrogen-deuterium exchange to assess structural differences

  • Native mass spectrometry to detect conformational changes

• Bioinformatic prediction:

  • Develop machine learning models based on identified substrates

  • Scan the S. aureus proteome for similar sequence motifs

  • Structural modeling of potential substrate interactions

These approaches would generate a comprehensive substrate profile, enabling better understanding of SAR0916's role in S. aureus physiology and pathogenesis.