sdrE Antibody

What is SdrE and what role does it play in Staphylococcus aureus pathogenicity?

SdrE is a surface-associated protein of Staphylococcus aureus belonging to the family of surface proteins with serine-aspartate repeats (Sdr), which includes other members like SdrC, SdrD, and clumping factors (ClfA and ClfB) . SdrE plays a critical role in S. aureus pathogenicity by binding to the complement regulatory protein factor H (fH), which inhibits the alternative pathway of complement activation . This binding represents a crucial immune evasion mechanism that allows S. aureus to evade host defenses.

SdrE recruitment of factor H provides a survival advantage for S. aureus by negatively affecting the formation of complement activating complexes, thereby dampening the host immune response . Studies have shown that 90% of 497 S. aureus isolates tested were sdrE positive, with SdrE-expressing strains typically associated with invasive infections .

How does SdrE contribute to S. aureus immune evasion mechanisms?

SdrE contributes to S. aureus immune evasion through a specific molecular interaction with the host complement regulatory protein factor H (fH). This process occurs through several documented mechanisms:

• SdrE binds fH in both a time- and dose-dependent manner, whether fH is purified or present in serum .

• SdrE-bound fH maintains its cofactor functionality for factor I (fI)-mediated cleavage of C3b to iC3b .

• The generation of iC3b positively correlates with increasing amounts of fH bound to SdrE .

• Cleaved C3b (iC3b) can no longer participate in the formation of C3- and C5-convertases, which negatively affects amplification of the complement cascade .

• Surface expression of SdrE leads to reduced C3-fragment deposition, decreased C5a generation, and reduced killing by polymorphonuclear cells .

Experimental evidence using a gain-of-function Lactococcus lactis model demonstrated that SdrE expression on the bacterial surface significantly enhances fH recruitment, which confirms the rSdrE-fH binding functionality . This recruitment ultimately results in decreased phagocytosis and enhanced bacterial survival.

What are effective methods for developing antibodies targeting specific epitopes of SdrE?

Developing antibodies targeting specific epitopes of SdrE can be approached through both traditional and rational design methods. Based on current research, the following methodological approaches are recommended:

• Rational Design Approach: Although not specifically described for SdrE in the literature, rational design methods that have been successful for other targets can be applied. This involves:

  • Identification of a peptide complementary to a target region within SdrE

  • Grafting this peptide onto the complementarity-determining region (CDR) of an antibody scaffold

  • Using computational tools such as homology modeling with PIGS server or the AbPredict algorithm to create 3D structures

  • Refining the structure through molecular dynamics simulations

• Stable Antibody Scaffold Selection: Choose a stable antibody scaffold, such as a human heavy chain variable (VH) domain that:

  • Is soluble and stable without a light chain partner

  • Has folding that is insensitive to mutations in its CDR loops, particularly CDR3

  • Can tolerate insertions in its CDR3 region

• Validation Methods:

  • Test binding affinity using techniques like ELISA or surface plasmon resonance

  • Confirm specificity through western blotting or immunoprecipitation

  • Evaluate functional impact in relevant biological assays (e.g., complement activation assays)

The successful antibody should demonstrate good expression in bacteria (>5 mg/L), high purity after a single chromatography step (>95%), and stability in its folded state .

How should researchers evaluate SdrE antibody specificity and cross-reactivity?

Evaluating SdrE antibody specificity and cross-reactivity requires a multi-faceted approach to ensure reliable research outcomes:

Recommended Experimental Protocol:

• Primary Specificity Testing:

  • Western blot analysis against purified SdrE, related Sdr proteins (SdrC, SdrD), and unrelated S. aureus surface proteins

  • Immunoprecipitation of SdrE from S. aureus cell lysates

  • ELISA assays with purified target proteins

• Cross-reactivity Assessment:

  • Test antibody binding against a panel of related staphylococcal surface proteins, particularly other members of the Sdr family

  • Evaluate potential binding to homologous proteins from different bacterial species

  • Perform dot blot analysis with various bacterial cell wall fractions

• Advanced Specificity Characterization:

  • Saturation transfer difference NMR (STD-NMR) to define the antigen contact surface

  • Quantitative glycan microarray screening if SdrE glycosylation is relevant

  • Site-directed mutagenesis to identify key residues in the antibody combining site

• Computational Validation:

  • Molecular docking and dynamics simulations to predict potential cross-reactivity

  • Screening of the selected antibody 3D-model against related bacterial proteins

When interpreting results, it's important to establish clear threshold criteria for specific versus non-specific binding based on appropriate controls and statistical analysis of binding curves.

How can SdrE antibodies be used to study S. aureus virulence mechanisms?

SdrE antibodies serve as powerful tools for investigating S. aureus virulence mechanisms through multiple experimental approaches:

• Visualizing SdrE Expression and Localization:

  • Immunofluorescence microscopy to detect SdrE on the bacterial surface

  • Flow cytometry to quantify SdrE expression levels across different S. aureus strains

  • Immunoelectron microscopy for precise subcellular localization

• Functional Blocking Studies:

  • Inhibition of factor H recruitment to assess complement evasion

  • Neutralization experiments to determine the impact on C3b cleavage and iC3b generation

  • Phagocytosis assays to measure the effect on neutrophil-mediated killing

• Pathogenesis Investigations:

  • In vivo tracking of SdrE-expressing bacteria during infection

  • Correlation of SdrE expression with disease severity in clinical isolates

  • Assessment of SdrE contribution to biofilm formation

• Vaccine Development Support:

  • Epitope mapping to identify immunodominant regions

  • Evaluation of antibody-mediated protection in animal models

  • Analysis of opsonophagocytic antibodies generated against SdrE

Research findings indicate that SdrE-specific antibodies can provide valuable insights into S. aureus immune evasion strategies. For instance, studies using a gain-of-function L. lactis model demonstrated that SdrE expression led to decreased C3-fragment deposition, reduced C5a generation, and diminished neutrophil-mediated killing—all of which could be monitored and potentially blocked using SdrE-specific antibodies .

What role does SdrE play in potential vaccine development against S. aureus?

SdrE has emerged as a promising vaccine antigen candidate based on several key findings:

• Protective Immunity Evidence:

  • SdrE was identified as one of four antigens (along with IsdA, IsdB, and SdrD) that generated significant protective immunity in a murine model of abscess formation

  • Protection correlated with the induction of opsonophagocytic antibodies

• Vaccine Formulation Potential:

  • When combined with IsdA, IsdB, and SdrD in a quadrivalent vaccine, SdrE contributed to high levels of protection against invasive disease or lethal challenge with human clinical S. aureus isolates

  • The combined vaccine elicited immune responses with greater protective immunity than immunization with individual components

• Human Immunogenicity:

  • SdrE is already known to be immunogenic in humans, as antibodies against SdrE can be found in healthy individuals or in patients with S. aureus disease

  • This natural immunogenicity suggests potential efficacy in human vaccination

Research suggests a functional correlation between antibody titers for these surface antigens, the opsonophagocytic properties of these antibodies, and protective immunity . This correlation provides a rational basis for developing vaccines that can protect humans at high risk for invasive S. aureus infection.

How can computational approaches improve SdrE antibody design and characterization?

Computational approaches offer powerful methods to enhance SdrE antibody design and characterization:

• Antibody Structure Modeling:

  • Homology modeling using specialized servers like PIGS (http://circe.med.uniroma1.it/pigs) provides a rapid method for generating initial antibody structures

  • The AbPredict algorithm combines segments from various antibodies and samples large conformational space to identify low-energy homology models

  • Molecular dynamics simulations can refine these models by exploring the conformational flexibility of the antibody-antigen complex

• Epitope Prediction and Analysis:

  • Computational tools can identify potentially immunogenic regions within SdrE

  • Molecular docking simulations can predict antibody-antigen interactions at atomic resolution

  • Energy calculations can evaluate the strength of these interactions and help prioritize candidate antibodies

• Optimization of Antibody Properties:

  • In silico mutagenesis can identify specificity-enhancing mutations

  • Virtual screening of modified antibodies against structural libraries of related antigens can predict cross-reactivity

  • Pharmacokinetic modeling can help optimize antibody stability and half-life

• Validation Metrics:

  • Key residues identified by computational approaches can be experimentally validated through site-directed mutagenesis

  • The glycan-antigen contact surface predicted computationally can be compared with results from saturation transfer difference NMR (STD-NMR)

  • Binding affinity predictions can be correlated with experimental measurements

The integration of computational approaches with experimental validation creates a powerful iterative process for antibody design. For example, after generating thousands of plausible antibody-glycan complex models through automated docking and molecular dynamics, experimental data like STD-NMR results can be used as selection metrics to identify the optimal 3D model .

What techniques are most effective for analyzing the kinetics of SdrE-antibody interactions?

Several sophisticated techniques can effectively analyze the kinetics of SdrE-antibody interactions:

• Surface Plasmon Resonance (SPR):

  • Provides real-time, label-free measurement of binding kinetics

  • Determines association (kon) and dissociation (koff) rate constants

  • Calculates equilibrium dissociation constant (KD)

  • Allows temperature-dependent analysis to determine thermodynamic parameters

• Bio-Layer Interferometry (BLI):

  • Alternative optical technique for real-time binding analysis

  • Requires less sample than SPR

  • Useful for high-throughput screening of antibody variants

• Isothermal Titration Calorimetry (ITC):

  • Measures heat released or absorbed during binding

  • Provides complete thermodynamic profile (ΔH, ΔS, ΔG)

  • Can determine stoichiometry of the interaction

• Microscale Thermophoresis (MST):

  • Detects changes in the movement of molecules along temperature gradients

  • Requires minimal sample amounts

  • Works well with crude sample preparations

• Advanced NMR Techniques:

  • Saturation Transfer Difference NMR (STD-NMR) for epitope mapping

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to identify binding interfaces

  • NMR relaxation measurements to characterize binding dynamics

Data Interpretation Guidance:

• For accurate kinetic analysis, use global fitting of data at multiple concentrations

• Include control surfaces/samples to account for non-specific binding

• Consider mass transport limitations that may affect kinetic parameters

• Validate results across multiple techniques when possible

Understanding the kinetics of SdrE-antibody interactions is crucial for developing therapeutic antibodies and evaluating their potential efficacy in neutralizing SdrE's immune evasion functions.

What are common challenges in SdrE antibody experiments and how can they be addressed?

Researchers working with SdrE antibodies may encounter several technical challenges that require specific troubleshooting strategies:

• Inconsistent Antibody Binding

  • Potential Causes: Conformational changes in SdrE, interference from other S. aureus surface proteins, or variation in SdrE expression levels

  • Solutions:

    • Use multiple antibody clones targeting different epitopes

    • Optimize sample preparation to preserve native protein conformation

    • Standardize bacterial growth conditions to ensure consistent SdrE expression

    • Consider using recombinant SdrE as a positive control

• Cross-Reactivity with Other Sdr Family Proteins

  • Potential Causes: Structural similarity between SdrE and other Sdr proteins (SdrC, SdrD)

  • Solutions:

    • Perform detailed cross-reactivity testing against all Sdr family proteins

    • Use computational approaches to identify unique epitopes within SdrE

    • Create antibodies targeting the most divergent regions of SdrE

    • Validate specificity using sdrE knockout mutants as negative controls

• Variable Results in Functional Assays

  • Potential Causes: Inconsistent factor H levels in serum samples, variation in complement activity

  • Solutions:

    • Use standardized serum sources or purified factor H

    • Include appropriate positive and negative controls in each experiment

    • Develop quantitative assays for factor H binding and C3b cleavage

    • Perform time-course experiments to capture the dynamic range of the interaction

• Poor Antibody Performance in Complex Biological Samples

  • Potential Causes: Matrix effects, competitive binding from other proteins

  • Solutions:

    • Optimize sample preparation methods to reduce interference

    • Use more sensitive detection methods (e.g., amplified ELISA systems)

    • Consider using antibody pairs targeting different epitopes for capture/detection

How should researchers design experiments to study the contribution of SdrE to S. aureus virulence in animal models?

Designing robust experiments to study SdrE's contribution to S. aureus virulence requires careful planning:

Recommended Experimental Design Framework:

• Bacterial Strain Selection and Preparation:

  • Include wild-type S. aureus (SdrE+), isogenic sdrE deletion mutant, and complemented strain

  • Consider using surrogate models like L. lactis expressing SdrE for gain-of-function studies

  • Verify SdrE expression/absence by Western blot before animal experiments

  • Standardize bacterial inoculum preparation for consistency

• Animal Model Selection:

  • Acute Infection Models:

    • Murine abscess formation model (validated for SdrE studies)

    • Bacteremia model to assess systemic spread

    • Pneumonia model for respiratory infection

  • Chronic Infection Models:

    • Endocarditis model (relevant given SdrE's role in immune evasion)

    • Osteomyelitis model for bone infections

    • Implant-associated biofilm model

• Intervention Strategies:

  • Passive immunization with anti-SdrE antibodies at different timepoints

  • Active immunization with purified SdrE before challenge

  • Treatment with antibody-antibiotic combinations

• Outcome Measurements:

  • Bacterial load in tissues (CFU counting)

  • Histopathological assessment of inflammation and tissue damage

  • Flow cytometry analysis of immune cell infiltration

  • Cytokine/chemokine profiling

  • Survival rates and clinical scoring

• Mechanistic Studies:

  • Ex vivo analysis of bacterial samples recovered from animals

  • Assessment of SdrE expression levels during infection

  • Measurement of factor H recruitment in vivo

  • Complement deposition analysis on bacterial surfaces

  • Neutrophil phagocytosis assays with bacteria recovered from animals

• Controls and Variables to Consider:

  • Include multiple S. aureus clinical isolates to ensure findings are not strain-specific

  • Control for potential compensatory mechanisms in sdrE mutants

  • Consider sex as a biological variable in animal studies

  • Include appropriate sample sizes based on power calculations

This comprehensive approach will help researchers generate robust data on SdrE's contribution to virulence while addressing potential confounding factors.

How should researchers interpret contradictory results between serological and microbiological detection of S. aureus involving SdrE?

Interpreting contradictory results between serological and microbiological detection of S. aureus requires a systematic analytical approach:

• Understanding Common Discrepancies:
  Research has documented several patterns of discrepancy between serological and microbiological detection:

  • Serology confirming microbiological diagnosis in only a minority of cases (2/13 in one study)

  • Serology contradicting microbiological diagnosis in some cases (3/13)

  • Serology identifying pathogens where microbiological diagnosis failed (3/13)

  • Negative serology with positive microbiological diagnosis (3/13)

  • Both serology and microbiological diagnosis being negative (2/13)

• Analytical Framework for Resolving Contradictions:

  Scenario	Possible Explanations	Recommended Actions
  Positive microbiology, Negative serology	Early infection stage before antibody response; Immunosuppression; Antigenic variation in SdrE	Collect follow-up samples; Test for additional antigens; Sequence sdrE gene from isolate
  Negative microbiology, Positive serology	Prior exposure without active infection; Cross-reactive antibodies; Non-cultivable bacteria	Use molecular detection methods; Assess antibody specificity; Evaluate for persistent infection
  Conflicting microbiology and serology	Mixed infection; Contamination; Technical issues	Use multiple detection methods; Sequence isolates; Employ species-specific PCR

• Temporal Considerations:

  • Antibody kinetics during infection progression should be considered

  • In some cases, increases in antibody levels were observed to be directed against gut microbes, supporting the leaky gut hypothesis

  • Analysis of back-up and follow-up plasma samples can provide valuable information about antibody response kinetics

• Technical Validation Steps:

  • Confirm specificity of serological assays for SdrE

  • Verify growth conditions for optimal SdrE expression in microbiological cultures

  • Consider the impact of antibiotic treatment prior to sampling

  • Examine whether the SdrE variant in the infecting strain matches the antigen used in serological tests

When faced with contradictory results, researchers should not automatically privilege one method over the other, but rather consider the biological and technical factors that could explain the discrepancy. Integration of multiple detection methods and longitudinal sampling can help resolve these contradictions.

What statistical approaches are most appropriate for analyzing SdrE antibody data in experimental and clinical studies?

Selecting appropriate statistical approaches for analyzing SdrE antibody data requires consideration of the specific experimental design and data characteristics:

• For Binding and Affinity Studies:

  • Nonlinear regression to fit binding curves and determine KD values

  • Scatchard analysis for multiple binding site characterization

  • Statistical moments analysis for heterogeneous binding populations

  • Bootstrap methods to generate confidence intervals for binding parameters

• For Comparative Studies (e.g., wild-type vs. mutant SdrE):

  • Student's t-test (paired or unpaired) for comparing two groups

  • ANOVA with post-hoc tests (e.g., Tukey's, Bonferroni) for multiple group comparisons

  • Non-parametric alternatives (Mann-Whitney U, Kruskal-Wallis) when normality assumptions are violated

  • Mixed-effects models for repeated measures designs

• For Clinical Sample Analysis:

  • Receiver Operating Characteristic (ROC) analysis to determine optimal cutoff values

  • Sensitivity and specificity calculations for diagnostic applications

  • Survival analysis methods (Kaplan-Meier, Cox proportional hazards) for outcome studies

  • Correlation analyses (Pearson's, Spearman's) to assess relationships between antibody levels and clinical parameters

• For Complex Datasets:

  • Principal Component Analysis (PCA) for dimensionality reduction in multiplex antibody data

  • Cluster analysis to identify patterns in antibody responses

  • Machine learning approaches for predicting outcomes based on antibody profiles

  • Bayesian methods for incorporating prior knowledge into analysis

• Sample Size and Power Considerations:

  • A priori power analysis to determine adequate sample sizes

  • Sequential analysis for adaptive study designs

  • Effect size calculations to interpret the biological significance of statistical findings

• Reporting Recommendations:

  • Always report exact p-values rather than threshold significance

  • Include measures of effect size alongside p-values

  • Report confidence intervals to indicate precision of estimates

  • Consider multiple testing corrections (e.g., Bonferroni, False Discovery Rate) when appropriate

When analyzing antibody response kinetics, time-series statistical methods may be particularly valuable. Additionally, when interpreting serological data against microbiological findings, approaches such as concordance analysis (e.g., Cohen's kappa) can quantify the level of agreement between methods.

What emerging technologies might revolutionize SdrE antibody research?

Several cutting-edge technologies are poised to transform SdrE antibody research in the coming years:

• Single B Cell Sequencing and Antibody Repertoire Analysis:

  • Enables identification of naturally occurring anti-SdrE antibodies from infected or vaccinated subjects

  • Allows tracking of B cell clonal evolution during S. aureus infection

  • Provides insights into antibody affinity maturation against SdrE

• CRISPR-Based Antibody Engineering:

  • Facilitates rapid generation of antibody variants with enhanced specificity or affinity

  • Enables high-throughput screening of antibody libraries

  • Allows precise epitope targeting through directed mutagenesis

• Advanced Structural Biology Techniques:

  • Cryo-electron microscopy for high-resolution structure determination of SdrE-antibody complexes

  • Hydrogen-deuterium exchange mass spectrometry for conformational analysis

  • Integrative structural biology approaches combining multiple experimental techniques

• Synthetic Biology and Antibody Mimetics:

  • Development of non-antibody scaffolds targeting SdrE

  • Creation of synthetic binding proteins with improved stability and tissue penetration

  • Design of multispecific molecules targeting SdrE and other virulence factors simultaneously

• Advanced Imaging Technologies:

  • Super-resolution microscopy for visualizing SdrE distribution on bacterial surfaces

  • Intravital imaging to track antibody-mediated clearance of S. aureus in real-time

  • Correlative light and electron microscopy for multimodal visualization of host-pathogen interactions

• Artificial Intelligence in Antibody Design:

  • Deep learning approaches for optimizing antibody sequences

  • AI-driven prediction of antibody stability and manufacturability

  • Automated design of antibody libraries for directed evolution

• Microfluidic and Organ-on-Chip Systems:

  • High-throughput screening of antibody functionality

  • Creation of physiologically relevant infection models

  • Real-time analysis of antibody-mediated bacterial clearance

These emerging technologies promise to accelerate the development of highly specific and effective anti-SdrE antibodies, potentially leading to new diagnostic and therapeutic applications for S. aureus infections.

What are the key unresolved questions about SdrE function that antibody research could help address?

Despite significant advances in understanding SdrE, several critical questions remain that targeted antibody research could help resolve:

• Structural Determinants of fH Binding:

  • Which specific domains or residues within SdrE are essential for factor H binding?

  • How does the three-dimensional structure of SdrE facilitate this interaction?

  • Development of domain-specific antibodies could map the functional regions through epitope blocking studies

• Regulatory Mechanisms:

  • How is SdrE expression regulated during different phases of infection?

  • What environmental signals modulate SdrE levels?

  • Antibodies could be used to quantify SdrE expression under various conditions, particularly in vivo

• Potential Additional Functions:

  • Does SdrE have functions beyond factor H binding and complement evasion?

  • Could it interact with other host proteins or contribute to biofilm formation?

  • Antibody inhibition studies could reveal previously unrecognized SdrE functions

• In Vivo Relevance:

  • How important is SdrE-mediated immune evasion in different types of S. aureus infections?

  • Does its contribution vary by infection site or patient population?

  • Passive immunization studies with anti-SdrE antibodies could assess its importance in various infection models

• Interplay with Other Virulence Factors:

  • How does SdrE complement other S. aureus immune evasion mechanisms?

  • Is there functional redundancy or synergy with other complement inhibitors?

  • Combinatorial antibody approaches targeting multiple virulence factors could elucidate these relationships

• Strain Variation:

  • How conserved is SdrE structure and function across diverse S. aureus lineages?

  • Do clinical isolates show variations that affect antibody binding or function?

  • Pan-SdrE antibodies versus strain-specific antibodies could address questions of conservation

• Therapeutic Potential:

  • Can SdrE neutralization provide protection against S. aureus infection?

  • What antibody properties (isotype, affinity, epitope) correlate with protective efficacy?

  • Therapeutic antibody development and testing would directly address these questions

Strategic development of antibody tools, particularly those targeting specific functional domains or with defined blocking capabilities, would provide valuable reagents for answering these fundamental questions about SdrE biology and its role in S. aureus pathogenesis.