Recombinant Staphylococcus aureus Uncharacterized peptidase SAR1786 (SAR1786)

What is SAR1786 and why is it significant for S. aureus research?

SAR1786 is an uncharacterized peptidase identified in Staphylococcus aureus strain MRSA252, a clinically significant methicillin-resistant strain . Peptidases are important for bacterial survival and virulence, making them potential therapeutic targets. While SAR1786 remains largely uncharacterized, studying this enzyme may provide insights into S. aureus pathogenicity mechanisms, particularly since S. aureus is a leading cause of both community-acquired and hospital-acquired bloodstream infections with mortality rates ranging from 15-40% .

Research on peptidases like SAR1786 is particularly valuable because S. aureus has developed multiple strategies to evade host immune systems, including biofilm formation, intracellular persistence in host cells, and the formation of small colony variants (SCVs) with increased antibiotic tolerance . Understanding the biochemical functions of all peptidases in this pathogen may reveal novel intervention points.

What is the molecular classification of SAR1786?

Based on sequence analysis, SAR1786 has been classified as a putative peptidase in S. aureus MRSA252 . While the exact peptidase family classification is not explicitly stated in available data, its identification through BLAST searches suggests homology with known peptidase domains. For comparison, many bacterial peptidases belong to specific families such as M20B (as seen in other S. aureus peptidases) or serine peptidases (as studied in other pathogens) .

To definitively determine the classification, researchers should:

• Perform comprehensive sequence alignment with known peptidase families

• Identify conserved catalytic domains and motifs

• Conduct phylogenetic analysis to establish evolutionary relationships

• Verify functional characteristics experimentally

What are the optimal conditions for recombinant expression of SAR1786?

Expression System Selection:

Optimization Protocol:

• Clone the SAR1786 gene into an expression vector containing a His-tag or other affinity tag

• Transform into the selected expression host

• Test expression at different temperatures (16°C, 25°C, 37°C)

• Vary IPTG concentrations (0.1-1.0 mM) for induction

• Optimize induction time (4-24 hours)

• Analyze protein expression via SDS-PAGE and Western blotting

• Assess solubility in various buffer conditions

Similar approaches have been successful for other bacterial peptidases, where careful optimization of expression conditions was essential for obtaining functionally active enzyme .

What purification strategy yields the highest purity and activity for SAR1786?

A multi-step purification protocol is recommended:

• Initial Capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, with imidazole gradient (20-250 mM)

• Intermediate Purification:

  • Ion exchange chromatography (IEX) based on theoretical pI

  • Size exclusion chromatography (SEC) to remove aggregates

• Activity Preservation:

  • Include protease inhibitors (excluding those that would inhibit the target peptidase)

  • Maintain reducing conditions with 1-5 mM DTT or 2-ME if cysteine residues are present

  • Determine thermal stability and optimize storage conditions accordingly

The purification strategy should be validated by assessing enzyme activity at each step, as activity loss during purification is a common challenge with peptidases .

How can the catalytic activity of SAR1786 be measured reliably?

Due to the uncharacterized nature of SAR1786, a systematic approach to identify substrate specificity is necessary:

Exploratory Substrate Screening:

• Test a panel of chromogenic or fluorogenic peptide substrates with different amino acid compositions

• Analyze hydrolysis of various peptide bonds using HPLC-based peptide mapping

• Employ a combinatorial peptide library to identify preferred cleavage sites

Quantitative Activity Assay Protocol:

• Incubate purified SAR1786 (0.1-10 μg) with identified substrate

• Maintain reaction conditions: pH 7.4, 37°C, appropriate buffer system

• Monitor product formation via:

  • Absorbance changes for chromogenic substrates

  • Fluorescence intensity for fluorogenic substrates

  • HPLC or LC-MS analysis for unlabeled peptides

• Calculate kinetic parameters (Km, kcat, kcat/Km)

Similar methodologies have been successfully applied to characterize other bacterial peptidases, such as the serine peptidases in T. brucei, where activity assays were crucial for validating the functional significance of the enzymes .

What is the substrate specificity profile of SAR1786 and how does it compare to other S. aureus peptidases?

While specific data on SAR1786 substrate specificity is limited, a comparative analysis approach can be used:

Methodology for Determining Specificity:

• Use positional scanning synthetic combinatorial libraries (PS-SCL)

• Perform cleavage site mapping using mass spectrometry

• Employ bioinformatic prediction tools based on sequence homology

Comparative Analysis Framework:

Research on other bacterial peptidases suggests that substrate specificity is often linked to specific functional roles in bacterial physiology and pathogenicity . For instance, signal peptide peptidases cleave after specific motifs (A-X-A) in signal sequences, which is critical for protein secretion and processing .

Is SAR1786 essential for S. aureus survival and virulence?

To determine whether SAR1786 is essential for S. aureus survival and virulence, researchers should employ a systematic approach similar to that used for other bacterial peptidases:

In Vitro Essentiality Assessment:

• Generate SAR1786 knockout mutants using CRISPR-Cas9 or allelic replacement

• Compare growth curves of wild-type and mutant strains under various conditions

• Assess biofilm formation capability

• Evaluate stress response (oxidative, temperature, pH, antimicrobial)

In Vivo Virulence Evaluation:

• Use murine or other appropriate infection models

• Compare bacterial load in tissues

• Measure survival rates and disease progression

• Assess the formation of small colony variants (SCVs) which are associated with persistent infections

Studies of other peptidases in pathogens have revealed that some are indeed essential for survival. For example, signal peptide peptidase (SPP1) in Trypanosoma brucei was found to be essential for parasite survival both in vitro and in vivo, with its catalytic activity being crucial . Similarly, M20B family peptidases in S. aureus have been identified as important for full virulence .

How does SAR1786 contribute to antimicrobial resistance mechanisms in MRSA?

The potential role of SAR1786 in antimicrobial resistance should be investigated through:

Experimental Approaches:

• Compare expression levels of SAR1786 in susceptible vs. resistant strains

• Analyze SAR1786 expression changes upon antibiotic exposure

• Determine if SAR1786 knockout affects minimum inhibitory concentrations (MICs)

• Investigate potential interactions with known resistance determinants

S. aureus strains, particularly MRSA, display complex resistance mechanisms including biofilm formation and the development of small colony variants with increased antibiotic tolerance . These mechanisms enable persistent infections and treatment failures. If SAR1786 plays a role in protein processing related to these adaptive responses, it could be indirectly involved in resistance.

For context, vancomycin resistance in S. aureus is defined by a minimum inhibitory concentration (MIC) of ≥16 μg/ml, with intermediate susceptibility (VISA) at 4-8 μg/ml . Any changes in these values upon SAR1786 manipulation would suggest involvement in resistance mechanisms.

What structural features determine the specificity and activity of SAR1786?

To elucidate the structural features of SAR1786:

Structure Determination Methods:

• X-ray crystallography of purified protein (2.0 Å resolution or better)

• Cryo-electron microscopy for larger complexes

• NMR spectroscopy for dynamic regions

• Homology modeling if experimental structures are unavailable

Critical Structural Elements to Identify:

• Catalytic domain architecture

• Active site residues

• Substrate binding pocket characteristics

• Potential regulatory domains or sites for post-translational modifications

For comparison, studies of serine peptidases have shown that the active site serine is essential for catalytic activity. In T. brucei SPP1, mutation of the active site serine to glycine resulted in complete loss of function, demonstrating the critical nature of this residue . Similar structure-function relationships likely exist for SAR1786.

What are the most effective approaches for developing selective inhibitors against SAR1786?

Structure-Based Inhibitor Design Strategy:

• Target Identification Phase:

  • Characterize the active site architecture using structural studies

  • Identify unique features distinct from human peptidases

  • Determine key catalytic residues through site-directed mutagenesis

• Inhibitor Design and Screening:

  • Perform virtual screening against the active site model

  • Design transition-state analogs based on preferred substrates

  • Develop a focused library of potential inhibitors based on structural insights

• Validation and Optimization:

  • Determine IC50 and Ki values for lead compounds

  • Assess selectivity against human peptidases

  • Optimize pharmacokinetic properties

• Efficacy Testing:

  • Evaluate antibacterial activity in vitro

  • Test impact on biofilm formation

  • Assess effectiveness in animal infection models

The development of peptidase inhibitors has proven successful as a therapeutic strategy in various contexts. The inhibition approach should consider the specific catalytic mechanism of SAR1786, which would need to be experimentally determined .

How can CRISPR-Cas9 technologies be applied to study SAR1786 function in S. aureus?

CRISPR-Cas9 Experimental Design for SAR1786 Research:

• Gene Knockout Studies:

  • Design sgRNAs targeting the SAR1786 gene

  • Construct a CRISPR-Cas9 delivery system compatible with S. aureus

  • Generate knockout mutants and confirm deletion via PCR and sequencing

  • Perform comprehensive phenotypic characterization

• CRISPRi for Conditional Knockdown:

  • Employ catalytically inactive Cas9 (dCas9) fused to a repressor domain

  • Target the SAR1786 promoter region

  • Create an inducible system to control expression levels

  • Monitor effects of partial knockdown on growth and virulence

• Domain Function Analysis:

  • Use precise CRISPR editing to introduce point mutations

  • Target predicted catalytic residues and substrate binding sites

  • Create domain deletion variants

  • Assess functional consequences of each modification

• Promoter Studies:

  • Integrate reporter genes downstream of the native promoter

  • Monitor expression under various conditions

  • Identify regulatory elements controlling expression

Similar approaches were successfully used in studying essential genes in other pathogens, such as the signal peptide peptidase in T. brucei, where RNAi was employed to demonstrate essentiality . CRISPR-based methods offer greater precision and versatility for studying gene function in S. aureus.

What is the regulatory network controlling SAR1786 expression during infection?

Research Methodology for Regulatory Network Analysis:

• Transcriptional Profiling:

  • Perform RNA-seq under various infection-relevant conditions

  • Compare expression in bloodstream infections vs. biofilm growth

  • Analyze expression changes in response to host factors

• Promoter Characterization:

  • Identify transcription start sites using 5' RACE

  • Map binding sites for regulatory proteins using ChIP-seq

  • Construct promoter-reporter fusions to verify regulatory elements

• Regulatory Protein Identification:

  • Conduct DNA pull-down assays followed by mass spectrometry

  • Perform yeast one-hybrid screens

  • Validate interactions using EMSAs and footprinting

• Integration with Known Regulatory Networks:

  • Assess correlation with virulence regulators (Agr, SarA, SaeRS)

  • Examine coordination with stress response pathways

  • Investigate potential quorum sensing control mechanisms

S. aureus employs complex regulatory networks to control virulence factor expression during infection, adapting to different host environments and stresses . Understanding how SAR1786 fits into these networks would provide insights into its role during pathogenesis and potentially reveal new intervention strategies.

What are the critical challenges in translating SAR1786 research to clinical applications?

Key Research-to-Clinical Translation Challenges:

• Target Validation Hurdles:

  • Demonstrating essentiality in clinical isolates, not just laboratory strains

  • Confirming relevance in polymicrobial infection contexts

  • Validating function in persistent infections and small colony variants

• Inhibitor Development Barriers:

  • Achieving selectivity over human peptidases

  • Ensuring penetration through biofilms and into intracellular niches

  • Preventing resistance development

  • Demonstrating efficacy against diverse clinical isolates

• Translational Research Priorities:

  • Developing high-throughput screening assays for inhibitor discovery

  • Establishing appropriate animal models that recapitulate human infections

  • Integrating SAR1786 research with broader antimicrobial strategies

The established importance of other S. aureus peptidases in virulence suggests that SAR1786 could be a valuable therapeutic target, particularly in the context of bloodstream infections which have mortality rates of 15-40% .

How can systems biology approaches advance our understanding of SAR1786 in the context of S. aureus pathogenicity?

Integrated Systems Biology Framework:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Map changes in metabolic pathways upon SAR1786 manipulation

  • Identify compensatory mechanisms when SAR1786 is inhibited

  • Create regulatory network models incorporating SAR1786

• Infection Dynamics Modeling:

  • Develop mathematical models of SAR1786 contribution to growth and persistence

  • Simulate impacts of inhibition under various conditions

  • Predict optimal combination therapy approaches

• Host-Pathogen Interaction Analysis:

  • Characterize host immune responses to SAR1786-deficient strains

  • Identify potential impacts on intracellular persistence capabilities

  • Assess effects on biofilm formation and small colony variant development

• Evolutionary Analysis:

  • Examine conservation and variation of SAR1786 across S. aureus lineages

  • Assess potential for horizontal gene transfer

  • Predict evolutionary responses to targeted inhibition

Systems biology approaches can provide a comprehensive understanding of how SAR1786 functions within the broader context of S. aureus pathogenicity, potentially revealing unexpected relationships and novel intervention strategies.