Structural investigation of human S. aureus-targeting antibodies that bind wall teichoic acid

Abstract

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and β-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or β form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the β-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three β-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with β-WTA, fulfill two recognition principles: binding to the β-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.

Keywords: Staphylococcus aureus; WTA; Wall Teichoic Acid; antibody structure; antibody-carbohydrate interactions; monoclonal antibodies.

Figure 1.

Schematic of S. aureus Wall Teichoic Acid (WTA) molecular structure. WTA polymers are linked to N-acetylmuramic acid (MurNAc) residues in the peptidoglycan backbone. The WTA “linkage unit” is bound to MurNAc via a phosphodiester bond to a N-acetylglucosamine (GlcNAc)–N-acetylmannosamine (ManNAc) disaccharide, which is in turn linked by a second phosphodiester bond to up to three repeating units of glycerol phosphate. The WTA main chain is composed of up to 40 repeats of ribitol phosphate. The linear WTA ribitol sugars are decorated at the 2-position by GlcNAc and at the 4-position by D-Alanine. GlcNAc linkages can be in the equatorial (β, as depicted) or axial (α) anomeric conformation, and are catalyzed by the glycosyltransferases TarS and TarM, respectively.

Figure 2.

Antibody binding to S. aureus is dependent on WTA glycosyltransferases. Protein A-deficient USA300 S. aureus wild type, ΔtarM, and ΔtarS strains were incubated with anti-WTA (red) or isotype control (grey; human IgG1 anti-cytomegalovirus gD antigen) antibodies, followed by detection with a fluorescently-labeled (DyLight 649) secondary antibody. Stained cells were counted by flow cytometry and data visualized in the displayed histograms. The 4462 (A), 6078 (B), and 4497 (C) antibodies bound to wild type and ΔtarM strains, while 7578 (D) bound to wild type and ΔtarS strains.

Figure 3.

Analysis of anti-WTA antibody binding to S. aureus. Protein A-deficient USA300 S. aureus were incubated with ¹²⁵I-labelled anti-WTA antibodies and subjected to immunoradiometric assay and saturation binding analysis to determine antibody affinity. Titration curves are shown for 4462 (A), 6078 (B), 4497 (C) and 7578 (D), along with calculated K_D values and estimated number of sites/bacterium.

Figure 4.

Structure of the synthetic wall teichoic acid epitope. (A) Chemical structures of the synthetic 1-phosphate and 5-phosphate β-WTA epitopes used in crystallization studies. Isolated electron density (contoured at 1σ and shown in magenta) is shown for the 5-phosphate β-WTA epitopes bound to the CDRs of (B) 4462, (C) 6078, and (D) 4497. Light chain (colored shades of red and pink) and heavy chain (colored in shades of cyan) backbones are shown as cartoon representations.

Figure 5.

Structural analysis of the 4462 and 6078 antibodies. (A) Overview of the CDR interaction surface of the 4462 Fab bound to 5-phosphate β-WTA. The molecular surface is displayed as partially transparent to show CDR amino acids underneath. Light chain CDRs are labeled and colored in shades of pink, while heavy chain CDRs are labeled and shown in shades of cyan. The bound β-WTA is shown in yellow as a stick representation. (B) Close-up view of the interactions between the 5-phosphate β-WTA and 4462 CDRs. CDR loops are shown as sticks and colored as in (A) with polar contacts shown as red dashed lines. (C) Overview of the CDR interaction surface of the 6078 Fab. Surface is shown as in (A), with light chain CDRs colored in shades of red and heavy chain CDRs shown in shades of teal. (D) Close-up view of the interactions between the 5-phosphate β-WTA and 6078 CDRs. CDR loops are shown as sticks and colored as in (C) with polar contacts shown as red dashed lines.

Figure 6.

Structural analysis of the 4497 antibody. (A) Overview of the CDR interaction surface of the 4497 Fab in complex with 1-phosphate WTA (PDB ID 5D6C). The molecular surface is displayed as partially transparent to show CDR amino acids underneath. Light chain CDRs are labeled and colored in shades of purple, while heavy chain CDRs are labeled and shown in shades of cyan. The bound 1-phosphate β-WTA is shown in yellow as a stick representation. (B) Close-up view of the CDRs in the apo structure of 4497. CDR loops are shown as sticks and colored as in (A). (C, D) Close-up view of the interactions between the (C) 5-phosphate β-WTA or the (D) 1-phosphate β-WTA (PDB ID 5D6C) and the 4497 CDRs. CDR loops are shown as sticks and colored as in (A) with polar contacts shown as red dashed lines. CDR L1 residues Arg27d and 28, which contact the 1-phosphate, are shown in magenta to highlight changes in conformation between the apo- and WTA-bound structures.

Figure 7.

Comparison of anti-β-WTA antibodies to their germline sequences. Alignment of the (A) 4462, (B) 6078, and (C) 4497 light and heavy chain Fab sequences to their respective germline sequences. Antibody light chain CDRs are labeled in purple, and heavy chain CDRs labeled in blue. Amino acids changed from the germline are boxed in black with white lettering. CDR amino acids that contact the WTA epitope (4.5Å cutoff) are boxed in yellow. Germlines matches for antibodies 4462, 6078 and 4497 were determined using the IMGT/V-QUEST server (http://www.imgt.org).