Structural basis for antibody targeting of the broadly expressed microbial polysaccharide poly- N-acetylglucosamine

Abstract

In response to the widespread emergence of antibiotic-resistant microbes, new therapeutic agents are required for many human pathogens. A non-mammalian polysaccharide, poly-N-acetyl-d-glucosamine (PNAG), is produced by bacteria, fungi, and protozoan parasites. Antibodies that bind to PNAG and its deacetylated form (dPNAG) exhibit promising in vitro and in vivo activities against many microbes. A human IgG1 mAb (F598) that binds both PNAG and dPNAG has opsonic and protective activities against multiple microbial pathogens and is undergoing preclinical and clinical assessments as a broad-spectrum antimicrobial therapy. Here, to understand how F598 targets PNAG, we determined crystal structures of the unliganded F598 antigen-binding fragment (Fab) and its complexes with N-acetyl-d-glucosamine (GlcNAc) and a PNAG oligosaccharide. We found that F598 recognizes PNAG through a large groove-shaped binding site that traverses the entire light- and heavy-chain interface and accommodates at least five GlcNAc residues. The Fab-GlcNAc complex revealed a deep binding pocket in which the monosaccharide and a core GlcNAc of the oligosaccharide were almost identically positioned, suggesting an anchored binding mechanism of PNAG by F598. The Fab used in our structural analyses retained binding to PNAG on the surface of an antibiotic-resistant, biofilm-forming strain of Staphylococcus aureus Additionally, a model of intact F598 binding to two pentasaccharide epitopes indicates that the Fab arms can span at least 40 GlcNAc residues on an extended PNAG chain. Our findings unravel the structural basis for F598 binding to PNAG on microbial surfaces and biofilms.

Keywords: Staphylococcus aureus (S. aureus); antibiotic resistance; antibody structure; biofilm; carbohydrate-binding protein; crystal structure; monoclonal antibody; poly-N-acetyl-D-glucosamine; vaccine development.

Figure 1.

Fluorescence microscopy showing F598 IgG and Fab binding to S. aureus and PNAG in biofilms. A, S. aureus was targeted by F598 IgG, F598 Fab, positive control (rabbit anti-S. aureus IgG and a goat anti-rabbit IgG-AF488 conjugate), and a negative control (goat anti-rabbit IgG-AF488 conjugate). B, S. aureus biofilm cultures at 48 h. Biofilms were stained for bacterial DNA with DAPI (blue) and AF488-labeled F598 IgG or Fab and a negative control. For these experiments, F598 IgG and Fab were directly labeled with AF488 and used at a concentration of 5 μg/ml. Scale bars, 10 μm.

Figure 2.

Electron density maps for the F598 Fab and carbohydrate complexes. A, the free Fab at 1.7-Å resolution. B, Fab–GlcNAc complex at 1.6-Å resolution. C, Fab–9NAc complex at 1.9-Å resolution. D, additional density was observed at both ends of the ordered pentasaccharide, emerging from the O1 position (left) and O6 position (right). Composite omit 2F_o − F_c maps (displayed at 1.5σ level) are shown for each structure with carbon atoms of the L chain in blue, H chain in purple, GlcNAc in green, and 9NAc in yellow. Non-carbon atoms are by type (oxygen, red; nitrogen, dark blue).

Figure 3.

Crystal structure of F598 Fab in its free and ligand-bound forms. Ribbons side views are shown for the free F598 Fab (A), the F598 Fab–GlcNAc complex (B), and the F598 Fab–9NAc (C). Carbohydrates are shown as stick representations with carbon atoms for GlcNAc monosaccharide in green and for 9NAc in yellow (other carbohydrate atoms: nitrogen, blue; oxygen, red). End-on solvent-accessible views are also depicted for the free F598 Fab (D), the F598 Fab–GlcNAc complex (E), and the Fab–9NAc complex (F). The antibody L chains (blue) and H chains (magenta) have the CDRs in lighter shades.

Figure 4.

Detailed views of GlcNAc and PNAG recognition by the F598 antibody. A, overlay of the F598 Fab–GlcNAc and Fab–9NAc crystal structures. Details of the interactions between F598 Fab and the cocrystallized carbohydrate ligands are shown for GlcNAc (B) and 9NAc (C). Fabs are depicted as thin sticks for the L (blue) and H (magenta) chains. Carbohydrate ligands are shown as thicker sticks with carbon atoms of GlcNAc in green and 9NAc in yellow. Ordered water molecules involved in interactions with the carbohydrate are shown as light blue spheres. Antibody residues are numbered sequentially (see Fig. S1). Hydrogen bonds are shown as black dashed lines.

Figure 5.

Protein-bound conformations of PNAG oligosaccharides. A, PNAG pentasaccharide from the structure of F598 Fab in complex with the nonamer 9NAc. B, PNAG tetrasaccharide from the structure of E. coli PgaB C-terminal domain in complex with a PNAG hexamer (Protein Data Bank code 4P7R). C, overlay of the PNAG ligand from each structure. Ligands are shown as sticks with carbon atoms of 9NAc in yellow and the PNAG from the PgaB complex in light blue.

Figure 6.

Comparison of F598 Fab–9NAc with previously determined bacterial saccharide complexes with antibody fragments. A, F598 Fab with the PNAG pentasaccharide epitope (this study). B, SYA/J-6 with a pentasaccharide from Shigella flexneri Y lipopolysaccharide (Protein Data Bank code 1M7I). C, mAb735 with half of octasialic acid, a homopolymer of group B meningitidis polysaccharide (Protein Data Bank code 3WBD). D, S24-2 in complex with a α2–8-linked keto-deoxyoctulosonate trisaccharide from Chlamydia (Protein Data Bank code 3SY0). E, CS-35 in complex with the hexasaccharide oligoarabinofuranosyl from mycobacteria (Protein Data Bank code 3HNS). F, NVS-1-19-5 in complex with the DP2 oligosaccharide from group B Streptococcus type III (Protein Data Bank code 5M63). Each Fv (VL-VH) complex is shown as a ribbon representation (side views) and as a solvent-accessible surface depiction (end-on views). All saccharide ligands are shown as space-filling Corey-Pauling-Koltun spheres with contact atoms in yellow (using a 4-Å cutoff from any antibody atom).

Figure 7.

Three-dimensional model of intact F598 IgG with two bound PNAG epitopes. In the context of IgG, the two Fab arms are tethered to an Fc region with a distance between the 9NAc ligands of ∼150 Å. A chain of at least 40 GlcNAc units would be required to span the distance between the bound PNAG epitopes (orange).