Recombinant Staphylococcus aureus Putative peptidyl-prolyl cis-trans isomerase (SAS0824)

What is the primary function of peptidyl-prolyl cis-trans isomerases in bacteria like S. aureus?

Peptidyl-prolyl isomerases (PPIases) catalyze the cis-trans isomerization of peptide bonds N-terminal to proline residues in polypeptide chains. This isomerization represents a rate-limiting step in protein folding and is crucial for proper protein conformation. In S. aureus, as in other organisms, PPIases like SAS0824 facilitate conformational changes in target proteins, potentially affecting multiple cellular processes including protein folding, signal transduction, and transcriptional regulation . Methodologically, their activity can be assessed using spectroscopic techniques to monitor changes in conformation of proline-containing peptide substrates, with enzyme kinetics typically following Michaelis-Menten behavior.

How does the structure of SAS0824 compare to other bacterial peptidyl-prolyl isomerases?

SAS0824 belongs to the parvulin-like family of PPIases, which differ structurally from other PPIase families such as cyclophilins and FK506-binding proteins. The parvulin-like PPIases typically feature a conserved PPIase domain with a characteristic β-sheet core surrounded by α-helices. When comparing SAS0824 to other bacterial PPIases, researchers should consider:

• Domain organization and size (often smaller than eukaryotic counterparts)

• Conservation of catalytic residues

• Presence of substrate-binding pockets

• Structural elements that determine substrate specificity

Comparative analysis using structural alignment tools and homology modeling based on crystallized PPIases provides insights into the unique features of SAS0824. Unlike Pin1 (a eukaryotic PPIase), SAS0824 may lack the stringent phosphorylation-dependent substrate recognition .

What are the optimal conditions for expressing and purifying recombinant SAS0824?

For optimal expression and purification of recombinant SAS0824:

Expression System Selection:

• E. coli BL21(DE3) typically yields high expression levels

• Consider using pET expression vectors with N-terminal His-tag for purification ease

Expression Conditions:

• IPTG concentration: 0.5-1.0 mM

• Temperature: 18-25°C (lower temperatures often reduce inclusion body formation)

• Duration: 4-16 hours (overnight expression at lower temperatures often improves solubility)

Purification Protocol:

• Lyse cells in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• Perform initial purification using Ni-NTA affinity chromatography

• Apply size exclusion chromatography for higher purity

• Consider ion-exchange chromatography as a polishing step

Stability Considerations:

• Add 5-10% glycerol to storage buffers

• Store at -80°C in small aliquots to avoid freeze-thaw cycles

• Assess enzyme activity immediately after purification and after storage

Protein purity should be evaluated by SDS-PAGE (>95% purity) and activity assays to ensure functionality.

What enzymatic assays are most reliable for measuring SAS0824 activity in vitro?

Multiple assays can quantify SAS0824 PPIase activity, each with specific advantages:

Protease-Coupled Assay:

• Principle: Measures the rate of chymotrypsin cleavage of a proline-containing peptide substrate

• Substrate: Typically succinyl-Ala-Xaa-Pro-Phe-p-nitroanilide (where Xaa can vary)

• Detection: Spectrophotometric monitoring of p-nitroanilide release at 390 nm

• Advantages: Simple, quantitative

• Limitations: Indirect measurement, potential protease interference

NMR-Based Assays:

• Principle: Direct observation of cis-trans isomerization

• Method: Time-resolved NMR to monitor populations of cis and trans conformers

• Advantages: Direct measurement, no coupling enzymes required

• Limitations: Requires specialized equipment, lower throughput

Fluorescence-Based Assays:

• Substrates: Fluorescently labeled peptides with proline residues

• Detection: Changes in fluorescence upon isomerization

• Advantages: Higher sensitivity, potential for high-throughput screening

• Setup: Monitor fluorescence intensity changes at excitation/emission wavelengths appropriate for the chosen fluorophore

For all assays, include proper controls including heat-inactivated enzyme, competitive inhibitors, and validation with known PPIase enzymes.

How does SAS0824 potentially contribute to S. aureus virulence and pathogenesis?

SAS0824, as a peptidyl-prolyl isomerase, may contribute to S. aureus virulence through several mechanisms:

Protein Folding and Stability:

• May facilitate proper folding of virulence factors and toxins

• Could stabilize secreted proteins involved in host invasion

Stress Response:

• May participate in bacterial adaptation to environmental stresses during infection

• Could enhance survival under host-imposed stresses (oxidative, pH, temperature)

Regulatory Functions:

• Potential role in modulating activity of transcription factors through conformational changes

• May influence expression of virulence genes through transcriptional regulation

Host-Pathogen Interactions:

• Possible involvement in modifying host proteins to facilitate infection

• May interfere with host cell signaling pathways

Similar to findings with other pathogens, PPIases may facilitate virulence by catalyzing conformational changes in key proteins. Experimental approaches to investigate these roles include:

• Generation of SAS0824 knockout strains and assessment of virulence in animal models

• Transcriptomic and proteomic analysis comparing wild-type and knockout strains

• Identification of SAS0824 protein substrates using chemical crosslinking or co-immunoprecipitation

• Evaluation of SAS0824 inhibitors on S. aureus growth and virulence

What is known about SAS0824 expression patterns during different stages of S. aureus infection?

Current research suggests that PPIase expression in pathogens often changes during infection phases. For SAS0824 specifically, researchers should investigate:

Expression Profiling:

• Analyze gene expression using RT-qPCR at different infection stages

• Apply RNA-seq to compare transcriptomes during colonization versus invasive infection

• Use proteomics to quantify SAS0824 protein levels under various conditions

Regulatory Mechanisms:

• Identify potential transcription factors regulating SAS0824 expression

• Determine if SAS0824 is regulated by stress response systems (similar to other virulence factors)

Infection Models:

• Compare SAS0824 expression in biofilm versus planktonic growth (relevant for device-associated infections)

• Analyze expression during intracellular persistence within host cells

• Monitor temporal expression patterns throughout infection progression

While specific data on SAS0824 expression patterns may be limited, approaches used for other S. aureus virulence factors can be applied. The expression of many S. aureus virulence factors is regulated by global regulators such as agr, sarA, and sae, and SAS0824 might follow similar patterns .

Can SAS0824 be targeted for vaccine development against S. aureus infections?

The potential of SAS0824 as a vaccine target requires consideration of several factors:

Antigen Properties:

• Surface accessibility: Determine if SAS0824 is accessible to antibodies or located intracellularly

• Conservation: Analyze sequence conservation across S. aureus strains (highly conserved targets may provide broader protection)

• Immunogenicity: Assess ability to elicit robust immune responses

Vaccine Development Approach:

• Recombinant protein vaccines: Full-length SAS0824 or immunogenic epitopes

• Conjugate vaccines: Consider bioconjugation to other S. aureus antigens (capsular polysaccharides)

• Multi-antigen approach: Include SAS0824 as part of a multivalent formulation targeting multiple virulence factors

Evaluation Strategy:

• Determine antibody titers and functional activity in animal models

• Assess protection in multiple infection models (bacteremia, skin infection, pneumonia)

• Evaluate T-cell responses (both humoral and cellular immunity may be important)

The development of effective S. aureus vaccines has proven challenging, with several high-profile clinical trial failures . Therefore, any SAS0824-based vaccine approach should consider lessons from previous attempts, particularly the importance of generating both humoral and cellular immunity. Combination with other antigens might increase efficacy, similar to the rFSAV approach that includes five recombinant S. aureus antigens .

What approaches can be used to identify specific inhibitors of SAS0824 activity?

Identifying SAS0824 inhibitors requires systematic screening and validation approaches:

Initial Screening Methods:

• High-throughput enzymatic assays using fluorescent substrates

• Fragment-based screening to identify molecular scaffolds with inhibitory potential

• In silico screening using modeled or crystallized SAS0824 structure

• Repurposing screens of known PPIase inhibitors from other systems

Structure-Activity Relationship Studies:

• Use medicinal chemistry to optimize lead compounds

• Focus on specificity for bacterial versus human PPIases

• Consider properties relevant for antimicrobial activity (membrane permeability, stability)

Validation of Inhibitor Activity:

• Biochemical validation using purified enzyme

• Cellular assays to confirm target engagement in living bacteria

• Assessment of effects on S. aureus growth and virulence

• Evaluation of toxicity against mammalian cells

Advanced Characterization:

• Determine binding mode through crystallography or NMR

• Assess effects on bacterial proteome using thermal proteome profiling

• Test in animal infection models for in vivo efficacy

Since PPIases play roles in human cells as well, selectivity is critical. Focus on structural differences between bacterial and human PPIases to develop selective inhibitors with minimal off-target effects .

What computational methods are most effective for predicting SAS0824 substrate specificity?

Predicting SAS0824 substrate specificity requires sophisticated computational approaches:

Sequence-Based Methods:

• Position-specific scoring matrices to identify potential proline-containing motifs

• Machine learning algorithms trained on known PPIase substrates

• Analysis of surface charge distribution and hydrophobicity patterns around proline residues

Structure-Based Approaches:

• Molecular docking of potential substrate peptides to SAS0824 model

• Molecular dynamics simulations to assess binding stability and conformational changes

• Free energy calculations to estimate binding affinities

Integrated Prediction Pipeline:

• Generate homology model of SAS0824 based on related PPIases

• Identify conserved catalytic residues and substrate-binding pocket

• Perform virtual screening of S. aureus proteome to identify proline-containing segments

• Apply molecular dynamics to assess substrate binding stability

• Validate predictions experimentally with synthetic peptides

Experimental Validation:

• Synthesize predicted substrate peptides and measure isomerization rates

• Use proteomic approaches (e.g., stable isotope labeling) to identify proteins affected by SAS0824 deletion

• Employ peptide arrays to determine sequence preferences

Unlike Pin1, which specifically recognizes phosphorylated Ser/Thr-Pro motifs , bacterial PPIases often have broader substrate specificity, requiring more complex prediction algorithms that consider structural context beyond simple sequence motifs .

How can researchers investigate the conformational changes induced by SAS0824 in target proteins?

Investigating conformational changes in SAS0824 substrates requires specialized biophysical techniques:

High-Resolution Structural Methods:

• X-ray crystallography of substrate proteins with and without SAS0824

• NMR spectroscopy to monitor changes in chemical shifts upon isomerization

• Cryo-electron microscopy for larger protein complexes

Spectroscopic Approaches:

• Circular dichroism (CD) to detect secondary structure changes

• Fluorescence resonance energy transfer (FRET) using labeled substrates

• Intrinsic tryptophan fluorescence to monitor conformational changes

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

• Measures solvent accessibility changes in proteins

• Can identify regions undergoing conformational changes

• Time-resolved approach allows kinetic analysis of isomerization

Computational Methods:

• Molecular dynamics simulations to model cis-trans transitions

• Normal mode analysis to identify conformational flexibility

• Markov state modeling to characterize conformational ensembles

Experimental Setup for Monitoring Isomerization:

• Prepare purified target protein with confirmed proline in cis conformation

• Add catalytic amounts of SAS0824

• Monitor conformational changes using time-resolved biophysical techniques

• Compare with uncatalyzed isomerization and with isomerization in the presence of inhibitors

These approaches can reveal how SAS0824-catalyzed isomerization affects target protein structure and function, providing insights into its role in S. aureus biology .

How does SAS0824 function compare to eukaryotic peptidyl-prolyl isomerases like Pin1?

SAS0824 and eukaryotic Pin1 share the fundamental peptidyl-prolyl isomerase activity but differ in several important aspects:

Substrate Specificity:

• Pin1 specifically recognizes and isomerizes phosphorylated Ser/Thr-Pro motifs

• SAS0824 likely has broader substrate specificity without phosphorylation requirements

• This difference reflects their distinct biological roles

Domain Structure:

• Pin1 contains both a WW domain (for phosphoprotein binding) and a PPIase domain

• SAS0824 likely lacks the WW domain, containing primarily the catalytic PPIase domain

• These structural differences influence target recognition mechanisms

Biological Functions:

• Pin1 regulates cell cycle progression, transcriptional regulation, and has been implicated in various diseases including cancer and viral infections

• SAS0824 likely functions in protein folding, stress responses, and potentially virulence in S. aureus

• Evolution has adapted these enzymes for species-specific regulatory roles

Inhibitor Sensitivity:

• Pin1 is inhibited by juglone, PiB, and various synthetic compounds

• SAS0824 inhibitor profiles remain to be fully characterized

• Differential inhibitor sensitivity can be exploited for selective targeting

This comparative analysis provides valuable insights for researchers developing selective inhibitors or studying evolutionary conservation of PPIase functions across kingdoms .

What experimental approaches can determine if SAS0824 is essential for S. aureus survival under different environmental conditions?

To determine the essentiality of SAS0824 under various conditions, researchers should employ:

Genetic Manipulation Approaches:

• CRISPR-Cas9 gene editing to create clean deletions

• Conditional knockdown systems (e.g., inducible antisense RNA)

• Complementation studies to confirm phenotype specificity

• Transposon mutagenesis libraries for high-throughput screening

Environmental Challenge Assays:

• Growth curve analysis under standard and stress conditions:

  • Oxidative stress (H₂O₂, paraquat)

  • Temperature stress (heat shock, cold shock)

  • Nutrient limitation

  • pH extremes

  • Antimicrobial challenges

• Biofilm formation assays

• Host cell interaction models

Competitive Fitness Experiments:

• Co-culture wild-type and mutant strains

• Use strain-specific markers to track population dynamics

• Calculate competitive index under various conditions

Comprehensive Phenotypic Profiling:

• Transcriptomic analysis (RNA-seq) comparing wild-type and SAS0824 mutants

• Proteomic profiling to identify affected pathways

• Metabolomic analysis to detect metabolic perturbations

In vivo Models:

• Invertebrate infection models (e.g., Galleria mellonella)

• Specialized mouse infection models (skin infection, bacteremia, etc.)

• Tissue-specific colonization and persistence assessment

These approaches will reveal conditions under which SAS0824 becomes critical for bacterial survival, potentially identifying environmental niches where targeting this enzyme would be most effective .

What are the most common pitfalls in studying SAS0824 and how can researchers overcome them?

Researchers studying SAS0824 face several technical challenges:

Expression and Purification Issues:

• Pitfall: Insoluble protein expression or inclusion body formation

• Solution: Optimize expression conditions (lower temperature, reduced inducer concentration), use solubility-enhancing tags, or consider refolding protocols from inclusion bodies

Activity Assessment Challenges:

• Pitfall: Low signal-to-noise ratio in enzymatic assays

• Solution: Optimize buffer conditions, increase enzyme concentration, use more sensitive detection methods, or consider alternative substrates with better kinetic properties

Specificity Determination:

• Pitfall: Difficulty identifying physiological substrates

• Solution: Implement crosslinking approaches, develop substrate trapping mutants, or use proximity labeling techniques like BioID

Structural Analysis Barriers:

• Pitfall: Challenges in obtaining crystal structures

• Solution: Screen multiple constructs with varied N/C-terminal boundaries, use surface entropy reduction, or consider NMR for solution structure

Genetic Manipulation Challenges:

• Pitfall: Difficulty generating clean knockouts if gene is essential

• Solution: Use conditional expression systems, CRISPR interference, or antisense RNA approaches

Reproducibility Issues:

• Pitfall: Variability in activity under different conditions

• Solution: Standardize protocols, include appropriate controls, consider the influence of metal ions or redox state on activity

By anticipating these common pitfalls, researchers can design more robust experimental approaches to characterize SAS0824 structure, function, and biological significance.

How can researchers differentiate between direct and indirect effects when studying SAS0824 mutants?

Differentiating direct from indirect effects requires systematic experimental design:

Complementation Studies:

• Generate clean deletion mutant (ΔSAS0824)

• Complement with wild-type SAS0824

• Include catalytically inactive SAS0824 mutant (point mutation in active site)

• Compare phenotypes across all strains

Time-Resolved Analysis:

• Monitor phenotypic changes immediately after SAS0824 inhibition/depletion

• Early changes are more likely to represent direct effects

• Late changes often reflect secondary adaptations

Substrate Identification and Validation:

• Use biochemical approaches to identify direct SAS0824 substrates

• Confirm interaction using pulldown or co-immunoprecipitation techniques

• Validate using in vitro isomerization assays with purified components

Conditional Expression Systems:

• Employ inducible or repressible promoters to control SAS0824 expression

• Perform dose-response experiments to identify threshold effects

• Track temporal changes in phenotype after modulating expression

In vitro Reconstitution:

• Reconstitute proposed biochemical pathways with purified components

• Test whether SAS0824 directly catalyzes the reaction of interest

• Compare kinetics with and without SAS0824

Control Experiments:

• Include deletion mutants of other PPIases as specificity controls

• Test effects of chemical PPIase inhibitors for comparison

• Use structurally similar but catalytically inactive proteins as controls

This systematic approach helps distinguish direct enzymatic effects from secondary consequences, enabling more accurate characterization of SAS0824's biological functions.