Recombinant Staphylococcus aureus Probable transglycosylase isaA (isaA)

What is IsaA and what is its primary function in Staphylococcus aureus?

IsaA (Immunodominant staphylococcal antigen A) is a probable soluble lytic transglycosylase expressed by Staphylococcus aureus . As a housekeeping protein, it plays a crucial role in cell wall metabolism, specifically in peptidoglycan turnover and cell wall remodeling. The protein has been characterized as an immunodominant antigen, suggesting its significant interaction with the host immune system during infection . The enzyme participates in breaking glycosidic bonds in peptidoglycan, likely contributing to cell wall expansion during bacterial growth. Research indicates IsaA is ubiquitously expressed across various S. aureus strains, including methicillin-resistant variants, making it a conserved target for potential immunotherapeutic approaches .

What are the molecular characteristics of recombinant IsaA?

Recombinant IsaA has a molecular weight of approximately 28.8 kDa . The mature protein sequence spans amino acids 30-233 of the full protein sequence . When expressed recombinantly, IsaA is typically produced with affinity tags to facilitate purification and detection. Common configurations include N-terminal 10xHis-tagged and C-terminal Myc-tagged versions, which can be produced in E. coli expression systems . The full mature protein sequence is:

AEVNVDQAHLVDLAHNHQDQLNAAPIKDGAYDIHFVKDGFQYNFTSNGTTWSWSYEAANGQTAGFSNVAGADYTTSYNQGSNVQSVSYNAQSSNSNVEAVSAPTYHNYSTSTTSSSVRLSNGNTAGATGSSAAQIMAQRTGVSASTWAAIIARESNGQVNAYNPSGASGLFQTMPGWGPTNTVDQQINAAVKAYKAQGLGAWGF

Purified recombinant IsaA typically achieves greater than 85% purity as determined by SDS-PAGE analysis .

How conserved is IsaA across different S. aureus strains?

IsaA demonstrates remarkable conservation across diverse S. aureus strains, including both methicillin-sensitive and methicillin-resistant variants . Research has confirmed that monoclonal antibodies targeting IsaA can recognize the protein in multiple clinically relevant strains, including hospital-acquired MRSA (such as strains with SCCmec types II, III, and IV), community-acquired MRSA (CA-MRSA including strains MW2 and USA300), and even vancomycin-resistant S. aureus (VRSA) . This high degree of conservation makes IsaA an attractive target for broad-spectrum immunotherapeutic approaches against S. aureus infections. The consistent expression of IsaA across these diverse strains suggests its fundamental importance to S. aureus physiology and survival, reinforcing its potential as a universal target for anti-staphylococcal strategies.

What are the optimal approaches for designing IsaA-focused experiments?

When designing experiments centered on IsaA, researchers should implement a systematic design of experiments (DoE) approach to maximize efficiency and data quality. Begin by identifying key experimental variables that may influence outcomes, such as expression conditions, purification methods, or functional assays . For IsaA expression studies, crucial continuous variables include induction temperature, IPTG concentration, expression time, and media composition . Categorical variables might include different E. coli host strains or vector systems .

Set appropriate experimental bounds for each variable (e.g., temperature range of 16-37°C for expression) and normalize these to a coded scale (-1 to +1) to facilitate statistical analysis . Implement fractional factorial designs to efficiently screen multiple variables while minimizing experimental runs. For example, a 2^5-2 design would allow investigation of 5 factors with only 8 experiments instead of the full 32 required for comprehensive testing .

Ensure inclusion of appropriate controls, particularly an isogenic IsaA knockout strain (e.g., S. aureus MA12 ΔisaA::Em^r) to confirm antibody specificity and function . For optimization experiments, apply response surface methodology (RSM) to identify optimal conditions for IsaA expression, purification yield, or functional activity.

What methods are most effective for producing high-quality recombinant IsaA?

Production of high-quality recombinant IsaA requires careful optimization of expression and purification protocols. The preferred expression system is E. coli, typically using BL21(DE3) or similar strains . The protein should be cloned with appropriate affinity tags—commonly an N-terminal His-tag and C-terminal Myc-tag—to facilitate purification and detection .

For optimal expression, consider these methodological recommendations:

• Culture conditions: Use LB or Terrific Broth, with induction at OD600 0.6-0.8

• Induction: Lower temperatures (16-25°C) often improve solubility compared to standard 37°C

• IPTG concentration: 0.1-0.5 mM typically provides sufficient induction while minimizing inclusion body formation

• Harvest timing: 4-6 hours post-induction at 37°C, or overnight (16-18 hours) at lower temperatures

Purification should employ immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar resins, followed by size exclusion chromatography to achieve >85% purity . Quality control should include SDS-PAGE analysis, Western blotting with IsaA-specific antibodies, and functional assays to confirm transglycosylase activity. For applications requiring endotoxin-free preparations, additional purification steps such as Triton X-114 phase separation or endotoxin removal columns should be implemented.

How can researchers effectively validate IsaA knockout models?

Validation of IsaA knockout models is critical for studying IsaA function and confirming antibody specificity. When generating or using IsaA knockout strains (e.g., S. aureus MA12 ΔisaA::Em^r), implement a comprehensive validation strategy incorporating multiple complementary approaches :

• Genotypic confirmation: Perform PCR analysis using primers flanking the isaA gene and insertion site to verify successful disruption. Sequencing of the modified locus should confirm the precise genetic modification.

• Transcriptional validation: Conduct RT-PCR or RNA-Seq analysis to confirm absence of isaA transcripts in the knockout strain compared to wild-type.

• Protein expression verification: Perform Western blot analysis using IsaA-specific antibodies (such as UK-66P) to confirm absence of IsaA protein .

• Immunofluorescence microscopy: Use fluorescently-labeled anti-IsaA antibodies to demonstrate absence of surface staining in knockout strains compared to wild-type . This approach has successfully demonstrated that the IsaA mutant strain does not interact with IsaA-specific antibodies, as evidenced by lack of fluorescence on these cells .

• Functional complementation: Reintroduce the isaA gene via plasmid expression to restore the wild-type phenotype, confirming that observed effects are specifically due to IsaA absence.

This multi-faceted validation approach ensures the reliability of subsequent experiments using IsaA knockout models for mechanism studies or antibody specificity testing.

How does IsaA interact with the host immune system during S. aureus infection?

IsaA serves as an immunodominant antigen during S. aureus infection, actively engaging with the host immune system through multiple mechanisms . The protein's surface accessibility makes it readily recognizable by host immune surveillance. During infection, IsaA elicits strong antibody responses, with anti-IsaA antibodies detectable in human sera from patients with S. aureus infections .

Research demonstrates that monoclonal antibodies targeting IsaA (such as UK-66P of the IgG1 subclass) can effectively bind surface-exposed IsaA on viable S. aureus cells . These antibodies activate professional phagocytes and induce production of highly microbicidal reactive oxygen species (ROS) in a dose-dependent manner, facilitating bacterial killing . This opsonophagocytic activity represents a key mechanism by which anti-IsaA antibodies contribute to host defense.

In experimental mouse models, including catheter-related infection and sepsis survival models, passive immunization with anti-IsaA antibodies significantly reduced bacterial burden in tissues compared to untreated controls . This protective effect appears to involve both enhanced phagocyte recruitment and activation, as well as increased production of bactericidal ROS .

The conserved nature of IsaA across diverse S. aureus strains, including MRSA variants, makes it particularly valuable as an immune target with potential broad-spectrum activity against clinically relevant isolates .

What are the most suitable experimental models for studying IsaA-targeting therapeutic approaches?

For evaluating IsaA-targeting therapeutic approaches, several experimental models have proven particularly informative:

• Central venous catheter-related infection model: This mouse model closely mimics the clinicopathological features of human S. aureus catheter-associated infections . The model involves catheter placement followed by controlled bacterial challenge. Endpoints include bacterial burden in tissues, biofilm formation on catheters, and systemic dissemination. This model has successfully demonstrated protective effects of anti-IsaA antibody therapy .

• Sepsis survival model: This model assesses survival outcomes following systemic S. aureus challenge and has confirmed that anti-IsaA immunotherapy can significantly improve survival rates .

• Ex vivo phagocyte activation assays: These cellular assays measure the ability of anti-IsaA antibodies to enhance phagocyte function, including ROS production and bacterial killing capacity . Findings from these assays have shown that antibodies like UK-66P activate professional phagocytes and induce microbicidal ROS in a dose-dependent manner .

• Surface binding assays: Fluorescence microscopy using fluorescently-labeled anti-IsaA antibodies can visualize antibody binding to viable S. aureus cells, confirming surface accessibility of the target . This approach should include appropriate controls, such as IsaA knockout strains (S. aureus MA12 ΔisaA) and protein A knockout strains (Cowan I Δspa) to validate specificity .

For quantitative assessment of antibody-IsaA binding kinetics, surface plasmon resonance (Biacore) analysis provides precise affinity measurements that correlate with therapeutic efficacy .

What approaches can be used to determine IsaA antibody binding kinetics and affinity?

Determining IsaA antibody binding kinetics and affinity is crucial for therapeutic antibody development. Surface plasmon resonance (SPR) using platforms such as the Biacore system represents the gold standard for these measurements . The methodology involves:

• Antibody immobilization: Reversibly immobilize anti-IsaA antibodies (e.g., UK-66P) using an anti-mouse Fc antibody covalently coupled to the sensor surface. This approach typically achieves high density immobilization (approximately 18,700 resonance units) on CM5 sensor surfaces .

• Analyte preparation: Prepare purified recombinant IsaA (rIsaA) at various concentrations in running buffer.

• Kinetic analysis: Measure association and dissociation rates by injecting increasing concentrations of rIsaA over the immobilized antibody surface. Association rates (ka) are measured during binding, while dissociation rates (kd) are measured during the subsequent buffer flow.

• Data analysis: Calculate binding affinity (KD = kd/ka) using appropriate fitting models, typically a 1:1 Langmuir binding model.

Alternative methodologies include isothermal titration calorimetry (ITC) for thermodynamic parameters and enzyme-linked immunosorbent assays (ELISA) for relative binding assessments. Bio-layer interferometry (BLI) offers an alternative optical-based approach with real-time, label-free detection.

For validating antibody specificity, include controls such as IsaA knockout strains (S. aureus MA12 ΔisaA::Em^r) and protein A knockout strains (Cowan I Δspa::Tc^r) to ensure observed binding is specific to the IsaA antigen rather than non-specific interactions .

What evidence supports IsaA as a viable target for anti-staphylococcal immunotherapy?

Multiple lines of evidence strongly support IsaA as a promising target for anti-staphylococcal immunotherapy:

• Conservation across strains: IsaA is highly conserved across diverse S. aureus strains, including methicillin-sensitive and methicillin-resistant variants, community-acquired MRSA, and vancomycin-resistant S. aureus . This conservation suggests a fundamental role in bacterial physiology and provides a basis for broad-spectrum therapeutic activity.

• Surface accessibility: Despite being classified as a housekeeping protein, IsaA demonstrates surface accessibility, making it available for antibody binding on intact bacterial cells . Fluorescence microscopy studies using monoclonal antibodies have confirmed binding to surface-exposed IsaA on viable S. aureus .

• Therapeutic efficacy in animal models: Anti-IsaA monoclonal antibodies (particularly IgG1 subclass antibodies like UK-66P) have demonstrated significant protection in multiple mouse infection models . In central venous catheter-related infection models and sepsis survival models, passive immunization with anti-IsaA antibodies significantly reduced bacterial burden in host tissues compared to untreated controls .

• Mechanism of action: Anti-IsaA antibodies have been shown to activate professional phagocytes and induce production of highly microbicidal reactive oxygen species in a dose-dependent manner, resulting in effective bacterial killing . This well-defined mechanism provides a rational basis for therapeutic development.

• Proof-of-concept studies: Research has established proof of concept that "monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models" .

These findings collectively indicate that IsaA represents a viable and promising target for antibody-based prophylaxis or adjunctive treatment of human S. aureus infections.

What methodological approaches can optimize antibody-based targeting of IsaA?

Optimizing antibody-based targeting of IsaA requires systematic methodological approaches across multiple research domains:

• Antibody engineering and selection:

  • Select antibody isotypes with optimal effector functions (IgG1 has demonstrated efficacy in mouse models)

  • Screen for antibodies with high affinity to IsaA using surface plasmon resonance (Biacore analysis)

  • Evaluate cross-reactivity against diverse S. aureus clinical isolates including MRSA variants

  • Consider humanization of promising murine antibodies for clinical translation

• Epitope mapping and optimization:

  • Identify the specific epitopes recognized by effective antibodies

  • Prioritize epitopes that are conserved across S. aureus strains

  • Characterize epitope accessibility on the bacterial surface

  • Evaluate epitope stability under various physiological conditions

• Functional assessment:

  • Test antibody-dependent activation of phagocytes and production of reactive oxygen species

  • Measure opsonophagocytic killing capacity

  • Assess antibody-dependent cellular cytotoxicity

  • Evaluate complement activation potential

• In vivo evaluation:

  • Use clinically relevant infection models such as catheter-related infection and sepsis models

  • Determine optimal dosing regimens, including prophylactic versus therapeutic administration

  • Assess potential synergy with conventional antibiotics

  • Evaluate efficacy against multiple S. aureus strains with varying virulence profiles

• Formulation optimization:

  • Develop stable liquid or lyophilized formulations

  • Evaluate long-term stability under various storage conditions

  • Optimize buffer composition to maintain antibody functionality

  • Consider advanced delivery approaches for local infection sites

These methodological approaches collectively provide a comprehensive framework for optimizing antibody-based targeting of IsaA for potential clinical applications in S. aureus infection management.

What analytical techniques are most informative for characterizing IsaA structure and function?

Comprehensive characterization of IsaA structure and function requires integration of multiple analytical techniques:

• Structural analysis:

  • X-ray crystallography: Provides high-resolution three-dimensional structure, revealing catalytic sites and potential epitopes

  • Nuclear Magnetic Resonance (NMR): Offers insights into protein dynamics and ligand interactions in solution

  • Cryo-electron microscopy: Can visualize IsaA in different conformational states or in complex with antibodies

  • Circular dichroism (CD) spectroscopy: Assesses secondary structure content and thermal stability

  • Small-angle X-ray scattering (SAXS): Provides low-resolution structural information in solution

• Functional characterization:

  • Enzymatic assays: Measure transglycosylase activity using fluorescently labeled peptidoglycan substrates

  • Site-directed mutagenesis: Identify catalytic residues by systematic mutation and activity testing

  • Isothermal titration calorimetry (ITC): Determine binding thermodynamics with substrates or inhibitors

  • Surface plasmon resonance (SPR): Assess real-time binding kinetics with interaction partners

• Cellular localization:

  • Immunofluorescence microscopy: Visualize IsaA distribution on bacterial surface using specific antibodies

  • Cell fractionation: Determine subcellular distribution between membrane, cell wall, and secreted fractions

  • Electron microscopy with immunogold labeling: Provide high-resolution localization data

• Interaction studies:

  • Immunoprecipitation coupled with mass spectrometry: Identify IsaA-interacting proteins

  • Bacterial two-hybrid systems: Screen for protein-protein interactions

  • Cross-linking studies: Capture transient interactions in the cellular context

• In silico approaches:

  • Molecular dynamics simulations: Model IsaA dynamics and substrate interactions

  • Homology modeling: Predict structure based on related transglycosylases

  • Bioinformatic analysis: Identify conserved domains and regulatory elements

Integration of these complementary techniques provides comprehensive insights into IsaA structure, function, and potential as a therapeutic target.

What are the current knowledge gaps and future research priorities for IsaA?

Despite significant advances in IsaA research, several important knowledge gaps remain that represent priorities for future investigation:

• Structural characterization: While IsaA has been identified as a probable transglycosylase, high-resolution structural data remains limited. Determining the three-dimensional structure would facilitate rational drug design and antibody engineering approaches. Crystallography or cryo-EM studies of IsaA alone and in complex with substrates or antibodies should be prioritized.

• Precise biological function: The exact physiological role of IsaA in S. aureus biology requires further elucidation. Comprehensive phenotypic characterization of IsaA knockouts under various stress conditions and growth phases would provide valuable insights. Transcriptomic and proteomic profiling of these mutants could reveal compensatory mechanisms and functional networks.

• Epitope mapping: Detailed mapping of protective epitopes recognized by effective antibodies such as UK-66P would enable more targeted therapeutic development . This should include analysis of epitope conservation across diverse clinical isolates and investigation of potential escape mutations.

• Translational challenges: While mouse models have demonstrated efficacy of anti-IsaA antibodies, translation to human therapeutics requires addressing species-specific differences in immune responses . Humanized antibody development and testing in more clinically relevant models should be pursued.

• Combination approaches: Investigating potential synergies between anti-IsaA antibodies and conventional antibiotics or other immunotherapeutic agents could enhance therapeutic efficacy. Systematic combination studies with various antibiotic classes should be conducted.

• Resistance mechanisms: Understanding potential bacterial adaptations to evade IsaA-targeted therapies is crucial for long-term efficacy. Serial passage experiments and analysis of clinical isolates from treatment failures would provide valuable insights.