Antibody Recognition of Different Staphylococcus aureus Wall Teichoic Acid Glycoforms

Abstract

Wall teichoic acids (WTAs) are glycopolymers decorating the surface of Gram-positive bacteria and potential targets for antibodymediated treatments against Staphylococcus aureus, including methicillinresistant (MRSA) strains. Through a combination of glycan microarray, synthetic chemistry, crystallography, NMR, and computational studies, we unraveled the molecular and structural details of fully defined synthetic WTA fragments recognized by previously described monoclonal antibodies (mAbs 4461 and 4497). Our results unveiled the structural requirements for the discriminatory recognition of αand β-GlcNAcmodified WTA glycoforms by the complementarity-determining regions (CDRs) of the heavy and light chains of the mAbs. Both mAbs interacted not only with the sugar moiety but also with the phosphate groups as well as residues in the ribitol phosphate (RboP) units of the WTA backbone, highlighting their significant role in ligand specificity. Using elongated WTA fragments, containing two sugar modifications, we also demonstrated that the internal carbohydrate moiety of α-GlcNAc-modified WTA is preferentially accommodated in the binding pocket of mAb 4461 with respect to the terminal moiety. Our results also explained the recently documented cross-reactivity of mAb 4497 for β-1,3/β-1,4-GlcNAc-modified WTA, revealing that the flexibility of the RboP backbone is crucial to allow positioning of both glycans in the antibody binding pocket.

Serum antibody screening using glycan arrays

“…The elegant approach of using glycan microarrays-coupled with X-ray crystallography, NMR spectroscopy, and computational studies-revealed that both mAbs recognized the RboP and the GlcNAc residues, and that mAb 4461 specifically recognized a1-4-GlcNAc-modified WTA, while mAb 4497 recognized b1-3-/b1-4-GlcNAc-modified WTAs. 150 Complementary studies have investigated GroP-based WTAs, which are also found in the cell walls of S. aureus and other enterococcus and staphylococcus species. A recent study used microarrays comprised of various synthetic WTA glycans to identify the recognition motif of a mAb (WH7.01) generated by immunization of mice with WH7, a synthetic version of a GroP WTA hexamer.…”

“…The elegant approach of using glycan microarrays—coupled with X-ray crystallography, NMR spectroscopy, and computational studies—revealed that both mAbs recognized the RboP and the GlcNAc residues, and that mAb 4461 specifically recognized α1-4-GlcNAc-modified WTA, while mAb 4497 recognized β1-3-/β1-4-GlcNAc-modified WTAs. 150…”

A Far-Red Fluorescent Probe to VisualizeStaphylococcus aureusin Patient Samples

“…An antibody against wall teichoic acids (anti-WTA) detects S. aureus more comprehensively but is expensive and synthesis is protected by patent. 7,8 Alternatively, antibiotics conjugated with fluorophores can be used as bacterial detection tools. 9 These small-molecule probes can have significant advantages over antibodies, including better tissue penetration, higher chemical stability, compatibility with antibody-based detection of other biomolecules, and lower cost.…”

A Far-Red Fluorescent Probe to Visualize Gram-Positive Bacteria in Patient Samples

“…An antibody against S. aureus surface proteins (anti-SA) is commonly applied to detect S. aureus , but exhibits staining heterogeneity due to varying levels of protein expression. An antibody against wall teichoic acids (anti-WTA) detects Gram-positive bacteria more comprehensively, but scaling up experiments using this expensive antibody is rather costly. , Alternatively, antibiotics conjugated with fluorophores can be used as bacterial detection tools . These small-molecule probes can have significant advantages over antibodies, including better tissue penetration, higher chemical stability, compatibility with antibody-based detection of other biomolecules, and lower cost.…”