srtA Antibody

What is Sortase A (SrtA) and why is it significant in bacterial research?

Sortase A (SrtA) is a transpeptidase enzyme produced by many Gram-positive bacteria, including Staphylococcus aureus and Group A Streptococci (GAS). SrtA recognizes secreted proteins containing an LPXTG motif (where X is any amino acid) and covalently attaches these proteins to cell wall peptidoglycan . The significance of SrtA lies in its essential role in bacterial virulence and pathogenicity, as it anchors numerous surface proteins involved in bacterial adhesion, colonization, and immune evasion . This makes SrtA a potential target for vaccine development and antimicrobial strategies. Analysis of 24 isolates across 12 GAS serotypes has demonstrated that SrtA is highly conserved, suggesting its fundamental role in bacterial physiology and pathogenesis .

How does SrtA function at the molecular level?

At the molecular level, SrtA catalyzes a transpeptidation reaction through a distinctive two-step mechanism:

• First, SrtA cleaves the peptide bond between threonine (T) and glycine (G) within the LPXTG sorting motif of target proteins .

• Second, it forms a new amide bond between the carboxyl group of threonine and one of two possible nucleophiles:

  • The ε-amino group of the pentaglycine (Gly5) cross-bridge in peptidoglycan precursors (Lipid II)

  • The N-terminal amino group of oligoglycine-containing peptides in laboratory applications

The reaction proceeds through an acyl-enzyme intermediate, where the substrate protein becomes covalently linked to SrtA's active site cysteine before being transferred to the nucleophilic amine group . This mechanism enables SrtA to function as a highly specific "molecular stapler" in both natural bacterial processes and bioengineering applications.

What are the key differences between wild-type SrtA and engineered SrtA variants for research applications?

Wild-type SrtA (WT SrtA) and engineered variants differ significantly in their kinetic properties and practical utility for research applications:

The pentamutant SrtA (5M SrtA with P94R/D160N/D165A/K190E/K196T mutations) represents a significant improvement over wild-type, while newer variants like 1M SrtA (N127K) have shown even greater efficiency in specific applications such as antibody labeling . These engineered variants have made SrtA-mediated ligations more practical for various biotechnology applications, particularly for antibody conjugation and protein PEGylation .

How effective is SrtA as a vaccine target against Group A Streptococcal (GAS) infections?

Intranasal vaccination with SrtA has demonstrated significant efficacy against GAS infections in mouse models. Research has shown that:

• SrtA vaccination reduced colonization of nasal-associated lymphoid tissue (NALT) by heterologous serotypes of GAS

• Immunized mice showed 1-2 log reduction in bacterial load 24 hours after challenge with various GAS serotypes (M1, M28, M49, and M12)

• Protection was observed across multiple GAS serotypes, indicating cross-serotype immunity

The effectiveness stems from SrtA's high conservation across GAS strains and its ability to induce robust Th17-mediated immune responses. This approach addresses a critical challenge in GAS vaccine development: overcoming the limitations of M protein-based vaccines that are restricted by serotype specificity. With more than 150 M-serotypes of GAS identified worldwide, SrtA represents a promising alternative strategy for developing broadly protective vaccines .

What immune mechanisms are involved in SrtA-mediated protection against bacterial infections?

SrtA vaccination induces a distinctive Th17-biased and antibody-independent protective immune response, which is characterized by:

• Th17 cell activation: Vaccination significantly increases CD4+ IL-17A+ cells in nasal-associated lymphoid tissue (NALT). These cells expanded from approximately 3-5% to 15-20% of CD4+ T cells in B6 mice and 6-8% in BALB/c mice following SrtA/CTB immunization .

• IL-17A dependency: Neutralization of IL-17A with antibodies abrogates the protective effect of vaccination, confirming the critical role of this cytokine. In experimental models, anti-IL-17A antibody treatment resulted in a 2-log increase in bacterial load compared to isotype controls .

• Neutrophil recruitment: SrtA-induced protection correlates with rapid neutrophil influx into NALT of immunized mice following bacterial challenge, enhancing bacterial clearance .

• Antibody independence: Despite generating SrtA-specific IgG and IgA antibodies, the protection is primarily T cell-mediated. Evidence shows that:

  • B cell-deficient (μMT) mice cleared bacteria as efficiently as wild-type mice after vaccination

  • Adoptive transfer of B cells from immunized mice failed to confer protection to naive recipients

This unique immune profile suggests that SrtA vaccination activates mechanisms that can bypass the bacterial antiphagocytic defenses, offering potential advantages over conventional antibody-dependent vaccine strategies .

What are the technical considerations for preparing SrtA for vaccination studies?

When preparing SrtA for vaccination studies, researchers should consider several technical factors that influence immunogenicity and experimental outcomes:

• Protein purification: Recombinant SrtA should be purified to >90% homogeneity, typically using His-tag affinity chromatography followed by size exclusion chromatography. The Bradford assay can be used to determine protein concentration, with standard curves having R² values between 0.98-0.99 for accuracy .

• Endotoxin removal: LPS contamination must be reduced to <0.1 EU/μg SrtA protein to avoid confounding immune responses. Comparative studies have shown that LPS-cleaned SrtA and LPS-containing SrtA preparations induce equivalent protection, confirming that observed effects are due to SrtA rather than contaminants .

• Adjuvant selection: Cholera toxin B subunit (CTB) has been demonstrated as an effective mucosal adjuvant for SrtA vaccination. Experimental data indicate that SrtA alone or CTB alone provided minimal protection, while SrtA/CTB combination significantly enhanced GAS clearance and Th17 responses .

• Immunization schedule: Optimal protocols involve three intranasal immunizations at one-week intervals, with bacterial challenge performed 7-10 days after the final immunization .

• Dose optimization: Typical effective doses are 10-20 μg SrtA with 1-2 μg CTB per immunization in mouse models, though dose-response studies should be conducted for each experimental system .

These technical considerations are essential for ensuring reproducible results and valid interpretations of SrtA vaccination efficacy in research studies.

What are the advantages of SrtA-mediated conjugation over conventional antibody labeling methods?

SrtA-mediated conjugation offers several distinct advantages over conventional antibody labeling methods:

Research has demonstrated that SrtA-conjugated antibodies maintain their antigen and Fc-receptor interactions while achieving potent functional activity . For antibody-drug conjugates (ADCs) and other applications requiring precise control over modification site and stoichiometry, SrtA-mediated conjugation provides significant advantages that translate to improved product quality and therapeutic potential .

How can SrtA be optimized for efficient antibody labeling?

Optimizing SrtA for efficient antibody labeling involves several key strategies:

• Enzyme engineering: Evolved SrtA variants demonstrate dramatically improved conjugation efficiency:

  • The 1M SrtA-pG variant (N127K mutation) showed superior labeling efficiency with only 0.4-1.0 molar equivalents of enzyme relative to antibody

  • The 5M SrtA pentamutant (P94R/D160N/D165A/K190E/K196T) provides 120-fold improvement in catalytic efficiency over wild-type SrtA

• Proximity-based enhancement: Fusing SrtA to antibody-binding domains significantly improves conjugation:

  • SrtA fused to protein G (SrtA-pG) shows enhanced labeling of human IgG compared to unfused SrtA

  • This proximity effect restricts labeling to specific lysine residues (five on the heavy chain and one on the light chain of human IgG1)

• Reaction optimization: Key parameters include:

  • Optimal molar ratios: 1:1 SrtA:antibody and 25-400 μM LPETG-substrate

  • Buffer composition: 50 mM Tris, pH 7.5, 150 mM NaCl, 10 mM CaCl₂

  • Temperature: Typically 37°C for 1-4 hours depending on the SrtA variant

  • LPXTG substrate concentration: Higher concentrations accelerate reaction rates but may increase hydrolysis side reactions

• Domain organization: Testing different fusion protein architectures (e.g., SrtA-pG versus pG-SrtA) to identify optimal configurations for specific antibody targets

These optimization strategies have transformed SrtA from a laboratory curiosity to a practical tool for antibody engineering, enabling efficient production of homogeneous antibody conjugates for research and potential therapeutic applications .

What experimental controls are essential when using SrtA for antibody labeling?

When using SrtA for antibody labeling, several experimental controls are essential for ensuring valid and interpretable results:

• Substrate-only controls: Reactions containing the LPXTG substrate and antibody without SrtA to detect non-enzymatic labeling or non-specific interactions .

• SrtA-only controls: Reactions containing SrtA and antibody without the LPXTG substrate to identify potential direct SrtA-antibody interactions .

• Antibody target knockout cell lines: For functional testing of labeled antibodies, target knockout cell lines are critical to confirm target-dependent effects and rule out off-target activity .

• Stability controls: Labeled antibodies should be tested for aggregation and stability under various storage conditions (4°C, -20°C, -80°C) and in physiological buffers (PBS, serum) .

• Functional validation: Multiple assays should confirm that:

  • Antigen binding is preserved (measured by ELISA or surface plasmon resonance)

  • Fc-receptor interactions remain intact (using FcγR binding assays)

  • Biological activity is maintained (cell-based functional assays)

• Product characterization controls:

  • Size exclusion chromatography to confirm absence of aggregation or fragmentation

  • Mass spectrometry to verify exact conjugation sites and stoichiometry

  • Western blot with multiple detection methods (e.g., anti-His, protein-L HRP, streptavidin-HRP) to confirm specificity of labeling

These controls are particularly important when using proximity-based SrtA approaches, where understanding the specificity and consequences of labeling is essential for interpreting experimental outcomes and ensuring reproducibility .

How does SrtA contribute to bacterial virulence mechanisms?

SrtA plays a multifaceted role in bacterial virulence through several mechanisms:

• Cell wall anchoring of virulence factors: SrtA anchors up to 12 GAS cell wall proteins containing LPXTG-like motifs, including:

  • M proteins (major virulence factors)

  • Protein F (adhesin)

  • C5a peptidase (ScpA) (immune evasion)

  • Protein G-related α2-macroglobin-binding protein (GRAB)

  • Pili (adhesion structures)

• Mediating host-pathogen interactions: SrtA-anchored surface proteins facilitate:

  • Adhesion to host tissues and extracellular matrix components

  • Colonization of mucosal surfaces

  • Evasion of host immune responses

• Virulence attenuation in knockout models: Studies across multiple bacterial species demonstrate that:

  • S. aureus ΔsrtA mutants show decreased virulence in murine infection models

  • S. lugdunensis SrtA-deficient mutants exhibit significantly reduced virulence in rat infective endocarditis models

  • S. gordonii insertional inactivation of srtA results in marked reduction of specific anchored surface proteins and decreased adherence

The critical role of SrtA in virulence is further supported by its conservation across pathogenic Gram-positive bacteria and the significant attenuation of infection observed when the srtA gene is inactivated . This makes SrtA an attractive target for anti-virulence therapeutics that could disrupt bacterial colonization without selecting for resistance in the same manner as conventional antibiotics .

How do human antibody responses to SrtA develop during bacterial infections?

Human antibody responses to SrtA during bacterial infections show complex patterns that reflect both exposure history and individual immunological factors:

• Development of anti-SrtA antibodies: Human populations exhibit detectable antibodies against numerous staphylococcal antigens, including SrtA, with significant heterogeneity in responses .

• Dynamics of antibody responses: Anti-SrtA antibody responses:

  • Develop early in childhood, indicating early exposure to SrtA-producing bacteria

  • Show no consistent correlation between IgG and IgA responses, suggesting independent regulation of these isotypes

  • Exhibit high inter-individual variability spanning several orders of magnitude

• Factors influencing anti-SrtA antibody levels:

  • Colonization status significantly impacts antibody levels, with carriers showing distinct antibody profiles

  • Host factors like sex, smoking status, age, BMI, and serum glucose influence antibody levels, though these explain only a small portion of the observed variability

• Isotype distribution: Both IgG and IgA responses against SrtA are detected in serum and mucosal secretions, with some antigens eliciting preferentially more IgG than IgA and vice versa .

Interestingly, despite generating antibody responses, humans experience recurrent infections with different bacterial serotypes, suggesting that natural antibody responses to bacterial antigens like SrtA may not confer robust protective immunity. This underscores the complexity of host-pathogen interactions and highlights challenges in developing antibody-based protective strategies against these infections .

What methodologies are most effective for studying SrtA's contribution to bacterial pathogenesis?

Several complementary methodologies have proven effective for investigating SrtA's contribution to bacterial pathogenesis:

• Genetic manipulation approaches:

  • Insertional inactivation of srtA using homologous recombination

  • Construction of markerless deletion mutants (ΔsrtA)

  • Complementation studies with plasmid-encoded wild-type srtA

• Protein detection methods:

  • Western blotting using anti-SrtA antibodies raised against purified SrtA ΔN (N-terminal truncated)

  • Cell fractionation to separate membrane, wall, and supernatant fractions for localizing SrtA and its substrates

  • Immunofluorescence microscopy to visualize SrtA-anchored proteins on the bacterial surface

• Functional assays:

  • Cell adhesion assays to quantify bacterial attachment to host cell lines

  • Biofilm formation assays to assess intercellular aggregation

  • Transmission electron microscopy to examine bacterial surface structures

• In vivo infection models:

  • Murine colonization models for nasal-associated lymphoid tissue (NALT)

  • Rat endocarditis models for assessing virulence in deep tissue infection

  • Adoptive transfer studies to evaluate immune protection mechanisms

• Substrate specificity analysis:

  • In vitro transpeptidation assays using purified SrtA and synthetic peptide substrates

  • Mass spectrometry to identify SrtA-anchored proteins in bacterial cell walls

These methodologies have collectively established SrtA's crucial role in bacterial pathogenesis across multiple species and identified it as a potential target for novel anti-virulence strategies. By combining genetic, biochemical, and in vivo approaches, researchers have built a comprehensive understanding of SrtA's functions and the consequences of its inhibition .

How can conflicting data regarding the role of antibodies in SrtA-mediated protection be reconciled?

Reconciling conflicting data on antibodies in SrtA-mediated protection requires careful experimental design and consideration of multiple factors:

• Distinguishing direct vs. indirect antibody effects:

  • Experimental approach: Compare protection in wild-type vs. B cell-deficient (μMT) mice after SrtA immunization

  • Key findings: μMT mice cleared bacteria as efficiently as wild-type mice despite lacking antibodies, suggesting direct antibody-mediated protection is not essential

  • Complementary evidence: Adoptive transfer of B cells from immunized mice failed to confer protection to naive recipients

• Exposure history and immunization protocols:

  • Single vs. multiple exposures: A single pharyngeal infection in baboons resulted in antibody-dependent protection against homologous but not heterologous GAS serotypes

  • Multiple exposures: Repeated inoculations (either with live GAS or SrtA/CTB) were required to achieve high levels of GAS-specific memory Th17 cells and cross-serotype protection

  • Hypothesis: The conflicting data may reflect differences in the immune mechanisms activated by single versus multiple exposures

• Accessibility of SrtA to antibodies:

  • Structural considerations: SrtA is located inside the cell wall, potentially limiting antibody access

  • Analogous systems: Similar antibody-independent, T cell-biased protection has been observed against other pathogens with internal conserved antigens (e.g., influenza, S. aureus, S. pneumoniae)

• Experimental models and readouts:

  • Site of infection: Mucosal versus systemic infection models may reveal different roles for antibodies

  • Readout timing: Early (24h) versus late measurements may reflect different clearance mechanisms

This reconciliation suggests a model where SrtA-specific antibodies, despite being generated, play a limited role in mucosal protection, while Th17-mediated neutrophil recruitment provides the dominant protective mechanism, particularly against heterologous strains. This model may explain why children typically experience multiple streptococcal infections before developing effective immunity .

What are the most promising strategies for enhancing SrtA enzymatic activity for biotechnology applications?

Advanced strategies for enhancing SrtA enzymatic activity for biotechnology applications include:

• Directed evolution approaches:

  • Error-prone PCR has yielded SrtA variants with dramatically improved catalytic efficiency

  • Key mutations identified:

    • 5M variant (P94R/D160N/D165A/K190E/K196T): 120-fold improvement in catalytic efficiency

    • 1M variant (N127K): Superior antibody labeling efficiency

    • 3M variant (T156S/D176E/D170E): Enhanced conjugation to specific antibody targets

  • These mutations enhance substrate binding and/or catalytic turnover

• Structural biology-guided engineering:

  • Crystal structure analysis of SrtA-substrate complexes has identified key residues for substrate recognition and catalysis

  • Rational design of the active site to reduce calcium dependence or enhance substrate binding

  • Engineering the β6/β7 loop region that undergoes conformational changes during catalysis

• Proximity-based enhancement:

  • Fusion of SrtA to substrate-binding domains:

    • SrtA-protein G fusions (SrtA-pG) show dramatically improved antibody labeling specificity and efficiency

    • Domain organization optimization (N-terminal vs. C-terminal fusions) for specific targets

  • Advantages: Enables catalysis at significantly lower enzyme concentrations (0.4-1.0 molar equivalents relative to substrate)

• Substrate engineering:

  • Optimization of the LPXTG recognition motif sequence

  • Engineering nucleophile peptides (typically Gn) for enhanced reactivity

  • Development of bifunctional linkers optimized for specific applications

• Reaction condition optimization:

  • Buffer composition adjustments, particularly focusing on calcium concentration and pH

  • Temperature and time optimization based on specific SrtA variant properties

  • Addition of enhancers that stabilize the enzyme-substrate complex or prevent product inhibition

These advanced strategies have transformed SrtA from a relatively inefficient enzyme with limited practical utility to a powerful tool for diverse biotechnology applications, including antibody-drug conjugate preparation, protein cyclization, and site-specific PEGylation .

How might SrtA be utilized as a target for novel anti-virulence therapeutics?

Developing SrtA-targeted anti-virulence therapeutics represents an advanced research direction with several promising strategies:

• Small molecule inhibitor development:

  • Virtual screening approaches targeting the SrtA active site

  • Natural product screening has identified candidates like curcumin and morin that inhibit SrtA activity

  • Structure-activity relationship studies to enhance potency and specificity

  • Challenges include achieving bacterial cell penetration while maintaining SrtA specificity

• Peptide-based inhibitor strategies:

  • Development of non-cleavable LPXTG analogs that compete with natural substrates

  • Peptidomimetics that mimic the transition state of SrtA-catalyzed reactions

  • Advantages include high specificity but potential limitations in stability and delivery

• Combination therapy approaches:

  • SrtA inhibitors could sensitize bacteria to conventional antibiotics

  • Potential for synergistic effects when combined with host-directed immunomodulatory therapies

  • Research design should include checkerboard assays to identify optimal combinations and concentrations

• Therapeutic antibody development:

  • While SrtA is largely intracellular, engineered antibody fragments or alternative binding proteins might access SrtA

  • Antibodies targeting SrtA-dependent surface proteins represent an indirect approach

  • Experimental designs should include both in vitro and in vivo models to assess efficacy

• Anti-virulence vaccination strategies:

  • SrtA immunization could potentially generate both neutralizing antibodies and T cell responses

  • Experimental data shows that intranasal SrtA vaccination enhances neutrophil function, a potential target for prevention of GAS infections

  • This approach might be especially valuable for preventing recurrent infections

• Methodology considerations:

  • Target validation using conditional srtA knockdown systems to confirm effects on virulence

  • Development of high-throughput screening assays using fluorescence resonance energy transfer (FRET)-based SrtA activity assays

  • In vivo imaging to track the impact of SrtA inhibition on bacterial colonization and dissemination

These approaches represent promising alternatives to conventional antibiotics, potentially reducing selective pressure for resistance while effectively attenuating bacterial virulence. The conservation of SrtA across many Gram-positive pathogens suggests that effective SrtA inhibitors might have broad-spectrum activity against multiple clinically important bacteria .

What are the most significant technical challenges in using SrtA antibodies for research applications?

Researchers face several significant technical challenges when using SrtA antibodies for research applications:

• Specificity and cross-reactivity issues:

  • SrtA is conserved across many bacterial species but contains species-specific variations

  • Challenge: Ensuring antibodies specifically recognize the intended SrtA variant

  • Approach: Validation using multiple bacterial species and recombinant SrtA proteins as controls

  • Additional control: Testing on SrtA-knockout strains to confirm specificity

• Accessibility and sample preparation:

  • SrtA is predominantly located inside the bacterial cell wall

  • Challenge: Ensuring adequate cell lysis for detection while preserving antibody epitopes

  • Methodology: Optimized protocols using lysozyme treatment (10 mg/ml at 37°C for 2h) in specialized buffers (50 mM Tris-HCl, pH 7.5, 500 mM sucrose, 10 mM MgCl₂)

  • Protein precipitation: TCA precipitation may be necessary before immunoblotting

• Quantification challenges:

  • SrtA expression levels vary between species and growth conditions

  • Challenge: Accurate quantification across different experimental conditions

  • Approach: Using Bradford Protein Assay with standard curves having R² values of 0.98-0.99

  • Normalization: Using housekeeping proteins appropriate for the bacterial species being studied

• Detection sensitivity limitations:

  • Native SrtA expression can be relatively low in some bacterial species

  • Challenge: Achieving sufficient sensitivity for detection

  • Solution: Signal amplification methods or enhanced chemiluminescence detection systems

  • Alternative: Mass spectrometry-based approaches for detection and quantification

• Functional characterization constraints:

  • Antibody binding might interfere with SrtA enzymatic activity

  • Challenge: Distinguishing between antibody binding and functional inhibition

  • Control experiments: Comparing SrtA activity before and after antibody binding

  • Alternative approach: Using genetic approaches like CRISPR interference to complement antibody studies

How might SrtA antibodies be utilized to study the spatial and temporal dynamics of bacterial protein anchoring?

Advanced research into SrtA spatial and temporal dynamics using antibody-based approaches can employ several sophisticated methodologies:

• Super-resolution microscopy techniques:

  • Stimulated emission depletion (STED) microscopy with fluorescently labeled anti-SrtA antibodies

  • Single-molecule localization microscopy (PALM/STORM) to track individual SrtA molecules

  • Experimental design: Dual-color imaging with SrtA and substrate proteins to visualize co-localization events

  • Challenges: Achieving specific labeling without perturbing native SrtA function and location

• Live-cell imaging approaches:

  • Development of minimally disruptive anti-SrtA nanobodies fused to fluorescent proteins

  • FRET-based biosensors to detect SrtA-substrate interactions in real-time

  • Microfluidic systems to study SrtA dynamics during bacterial growth and division

  • Controls: Comparison with fixed-cell immunofluorescence to validate live-cell observations

• Time-resolved immunoprecipitation:

  • Sequential immunoprecipitation of SrtA at defined time points following stimuli

  • Mass spectrometry analysis of co-precipitated proteins to identify temporal changes in SrtA-substrate interactions

  • Quantitative proteomics to determine the kinetics of substrate processing

  • Validation: Confirmation with purified components in reconstituted systems

• Correlative light and electron microscopy (CLEM):

  • Immunogold labeling of SrtA combined with transmission electron microscopy

  • Correlation with fluorescence microscopy to provide context at different scales

  • Tomographic reconstruction to visualize 3D organization of SrtA relative to cell wall structures

  • Advantage: Nanometer-scale resolution of SrtA localization at the bacterial surface

• Proximity labeling technologies:

  • SrtA fusion with engineered peroxidases (APEX) or biotin ligases (TurboID)

  • Spatially-restricted labeling of proteins in proximity to SrtA

  • Mass spectrometry analysis of labeled proteins to map the SrtA interaction network

  • Controls: Comparison with SrtA catalytic mutants to distinguish enzymatic from structural interactions

These advanced approaches can provide unprecedented insights into fundamental questions about bacterial cell wall biogenesis, the spatial organization of virulence factor display, and the temporal regulation of surface protein anchoring during infection processes. Such information could inform new strategies for targeting the sortase system in antibacterial development.