Use of a neutralizing antibody helps identify structural features critical for binding of toxin TcdA to the host cell surface.

Kroh HK, Chandrasekaran R, Rosenthal K, Woods R, Jin X, Ohi MD, Nyborg AC, Rainey GJ, Warrener P, Spiller BW, Lacy DB
J Biol Chem. 2017 292 (35): 14401-14412

PMID: 28705932 · PMCID: PMC5582835 · DOI:10.1074/jbc.M117.781112

is a clinically significant pathogen that causes mild-to-severe (and often recurrent) colon infections. Disease symptoms stem from the activities of two large, multidomain toxins known as TcdA and TcdB. The toxins can bind, enter, and perturb host cell function through a multistep mechanism of receptor binding, endocytosis, pore formation, autoproteolysis, and glucosyltransferase-mediated modification of host substrates. Monoclonal antibodies that neutralize toxin activity provide a survival benefit in preclinical animal models and prevent recurrent infections in human clinical trials. However, the molecular mechanisms involved in these neutralizing activities are unclear. To this end, we performed structural studies on a neutralizing monoclonal antibody, PA50, a humanized mAb with both potent and broad-spectrum neutralizing activity, in complex with TcdA. Electron microscopy imaging and multiangle light-scattering analysis revealed that PA50 binds multiple sites on the TcdA C-terminal combined repetitive oligopeptides (CROPs) domain. A crystal structure of two PA50 Fabs bound to a segment of the TcdA CROPs helped define a conserved epitope that is distinct from previously identified carbohydrate-binding sites. Binding of TcdA to the host cell surface was directly blocked by either PA50 mAb or Fab and suggested that receptor blockade is the mechanism by which PA50 neutralizes TcdA. These findings highlight the importance of the CROPs C terminus in cell-surface binding and a role for neutralizing antibodies in defining structural features critical to a pathogen's mechanism of action. We conclude that PA50 protects host cells by blocking the binding of TcdA to cell surfaces.

MeSH Terms (23)

Amino Acid Sequence Anti-Bacterial Agents Antibodies, Monoclonal, Humanized Antibodies, Neutralizing Bacterial Proteins Bacterial Toxins Binding Sites, Antibody Caco-2 Cells Clostridium difficile Conserved Sequence Crystallography, X-Ray Enterocytes Enterotoxins Epitope Mapping Glucosyltransferases Humans Immunoglobulin Fab Fragments Models, Molecular Peptide Fragments Protein Conformation Protein Interaction Domains and Motifs Recombinant Proteins Repetitive Sequences, Amino Acid

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