Structural Basis for Binding of Neutralizing Antibodies to Clostridioides difficile Binary Toxin

Abstract

Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes 15,000 deaths annually in the United States, prompting a need for vaccine development. In addition to the important toxins TcdA and TcdB, binary toxin (CDT) plays a significant role in the pathogenesis of certain C. difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells. However, the mechanisms of CDT neutralization by antibodies have not been studied, limiting our understanding of key epitopes for CDT antigen design. Therefore, we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically. Here, 2.5-Å and 2.6-Å resolution X-ray crystal structures of the antibodies BINTOXB/22 and BINTOXB/9, respectively, in complex with CDTb-the CDT subunit that forms a heptameric pore for the delivery of toxic CDTa enzyme into the host cytosol-showed that both antibodies sterically clash with adjacent protomers in the assembled heptamer. Assessment of trypsin-induced oligomerization of the purified CDTb protoxin in vitro showed that BINTOXB/22 and BINTOXB/9 prevented the assembly of di-heptamers upon prodomain cleavage. This work suggests that the CDT oligomerization process can be effectively targeted by antibodies, which will aid in the development of C. difficile vaccines and therapeutics. IMPORTANCE Clostridioides difficile strains associated with worse clinical outcomes have been found to secrete a toxin called CDT (or binary toxin). As blocking the function of this toxin could help mitigate C. difficile infections, we sought to determine the molecular basis for the inhibition of CDT by monoclonal antibodies. We isolated monoclonal antibodies targeting the B-component of CDT (CDTb) and selected two with neutralizing activity for detailed structural and biochemical characterization. High-resolution crystal structures of each antibody bound to CDTb showed that their presence would preclude the assembly of a CDTb oligomer required for activity. Oligomerization of CDTb in vitro was shown to be blocked in the presence of the neutralizing antibodies, but not a control antibody.

Keywords: Clostridioides difficile; X-ray crystallography; neutralizing antibodies; pore-forming toxins.

FIG 1

Monoclonal CDTb antibodies neutralize CDT. (A) CDT neutralization assay scheme based on cell survival. (B) Neutralization curves for BINTOXB/9 IgG (orange circles), BINTOXB/22 IgG (green circles), and BINTOXB/19 IgG (pink circles). (C) Surface plasmon resonance sensorgrams for BINTOXB/9 Fab and BINTOXB/22 Fab binding to proCDTb. Black traces show measured response and red traces show the kinetic fit to a 1:1 binding model. The association phase was 180 s and the dissociation phase was 600 s. CDT, C. difficile transferase toxin; K_D, equilibrium dissociation constant; k_a, association rate constant; k_d, dissociation rate constant.

FIG 2

BINTOXB/9 binds to the flexible D4. (A) Negative-stain electron microscopy (nsEM) micrograph of proCDTb+BINTOXB/9 Fab. (B) Representative 2D class averages of proCDTb+BINTOXB/9 particles. (C) nsEM 3D reconstruction of CDTb+BINTOXB/9 Fab. CDTb prodomain and D1-D3′ are colored blue, proCDTb D4 is colored pink, and BINTOXB/9 Fab is colored green. (D) 3D reconstruction from panel C with a transparent surface and fitted models. The mature CDTb model (D1-D4; blue and pink) was obtained from the oligomer structure (PDB ID: 6UWT) and is shown as ribbons. The prodomain model (purple) was obtained by aligning the Bacillus anthracis protective antigen (PA) protoxin structure (PDB ID: 1ACC) to mature CDTb. (E) Cleaved, mature CDTb prepore heptamer (PDB ID: 6UWT) with each protomer shown as a molecular surface. Right: cleaved, mature CDTb prepore heptamer with 1 protomer hidden. One protomer is shown as both ribbon and molecular surface, with D1-D3′ colored blue and D4 colored pink.

FIG 3

BINTOXB/9 binds to the D4 oligomerization interface. (A) Domain organization of full-length proCDTb and the D4 fragment used for crystallization are shown in the top left, with the secretion signal shown in gray, the CDTb prodomain and D1-D3′ in blue, and D4 in pink. The crystal structure of BINTOXB/9 in complex with the D4 fragment is shown on the bottom left as ribbon representation, with D4 colored pink, BINTOXB/9 Fab heavy chain colored green, and BINTOXB/9 Fab light chain colored pale green. For reference, a model of the CDTb prepore heptamer (PDB ID: 6UWR) is shown on the right, with each protomer shown as a molecular surface. One protomer is shown as both ribbon and molecular surface, with D1-D3′ colored blue and D4 colored pink. (B and C) Zoomed-in views of the BINTOXB/9-D4 interface. BINTOXB/9 heavy chain (green), BINTOXB/9 light chain (pale green) and D4 (pink) are shown as ribbons, with interface residues shown as sticks. Oxygens are colored red and nitrogens are colored blue.

FIG 4

BINTOXB/22 binds to proCDTb D3 and D3′. (A) Crystal structure of BINTOXB/22 in complex with proCDTbΔD4 is shown on the top left as ribbons, with proCDTbΔD4 colored blue, BINTOXB/22 Fab heavy chain colored magenta, and BINTOXB/22 Fab light chain colored pink. Bottom left: domain organization of full-length proCDTb and the proCDTbΔD4 construct used for crystallization, with the secretion signal shown in gray, the CDTb prodomain and D1-D3′ colored blue, and D4 colored pink. For reference, a model of the cleaved, mature CDTb prepore heptamer (PDB ID: 6UWR) is shown on the right, with each protomer shown as a molecular surface. One protomer is shown as both ribbon and molecular surface, with D1-D3′ colored blue and D4 colored pink. (B and C) Zoomed-in views of the BINTOXB/22-proCDTb interface. BINTOXB/22 heavy chain (magenta), BINTOXB/22 light chain (pink), and proCDTbΔD4 (blue) are shown as ribbons, with interface residues shown as sticks. Oxygens are colored red and nitrogens are colored blue.

FIG 5

BINTOXB/9 and BINTOXB/22 clash with adjacent protomers in the cleaved, mature CDTb heptamer. The cleaved, mature CDTb prepore heptamer (PDB ID: 6UWT) is shown with 6 protomers shown as molecular surfaces and one protomer as a ribbon. BINTOXB/9 (green) and BINTOXB/22 (purple) are modeled bound to the CDTb protomer, shown as ribbons, based on the Fab-antigen crystal structures.

FIG 6

BINTOXB/9 and BINTOXB/22 prevent CDTb oligomerization in vitro. (A) Assay for the assessment of CDTb oligomerization in vitro. (B) Size exclusion chromatography (SEC) profiles of untreated proCDTb, and proCDTb trypsinized with or without Fabs, showing the elution volume range that contains the expected peak for the cleaved CDTb double heptamer. The trace for the untreated proCDTb is shown as a dotted line. The trace for proCDTb trypsinized with no Fab is shown in green, with BINTOXB/19 Fab in pink, with BINTOXB/9 Fab in orange, and BINTOXB/22 in teal. (C) Quantification of the area under the curve of the oligomer peaks obtained after trypsinization in the presence or absence of Fabs.