A neutralizing antibody that blocks delivery of the enzymatic cargo of Clostridium difficile toxin TcdB into host cells

Abstract

infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect against infection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array of strains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism of toxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.

Phylogenomics of 8,839Clostridioides difficilegenomes reveals recombination-driven evolution and diversification of toxin A and B

“…binding 55 , and E3 binding 54 were then analyzed in terms of their variation across subtypes. E3 binding residues were identified by analysis of PDB structure 6OQ5 54…”

Cellular microbiology: Bacterial toxin interference drives understanding of eukaryotic cell function

“…Knowledge of bacterial toxin entry mechanisms has led to ideas to inhibit their toxic action during acute infection, for example, by injecting soluble anthrax toxin receptor to competitively bind toxin as a treatment for anthrax (Thomas et al, ). Humanized monoclonal antibodies have been isolated that target toxin regions essential to enter cells, an approach that is looking promising for treatment of Clostridioides difficile associated infections (Kroh et al, ). Another promising approach for therapy leverages is the understanding of how toxins utilise host cell vesicular trafficking to translocate into the cytosol.…”

Application of Antibody-Mediated Therapy for Treatment and Prevention of Clostridium difficile Infection

“…A camelid single domain antibody against TcdA could be easily optimized by an affinity maturation platform and could serve as an alternative therapeutic modality in the future (Sulea et al, 2018 ). A novel humanized monoclonal antibody recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and neutralizes TcdB from various CD strains (Kroh et al, 2018 ).…”