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

Kroh, Heather K.; Chandrasekaran, Ramyavardhanee; Rosenthal, Kim; Woods, Rob; Jin, Xiaofang; Ohi, Melanie D.; Nyborg, Andrew C.; Rainey, G. Jonah; Warrener, Paul; Spiller, Benjamin W.; Lacy, D. Borden

doi:10.1074/jbc.M117.781112

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

Journal Article · Thu Jul 13 00:00:00 EDT 2017 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M117.781112· OSTI ID:1439612

Kroh, Heather K. ^[1]; Chandrasekaran, Ramyavardhanee ^[1]; Rosenthal, Kim ^[2]; Woods, Rob ^[2]; Jin, Xiaofang ^[2]; Ohi, Melanie D. ^[3]; Nyborg, Andrew C. ^[2]; Rainey, G. Jonah ^[4]; Warrener, Paul ^[2]; Spiller, Benjamin W. ^[5]; Lacy, D. Borden ^[6]

Vanderbilt Univ. Medical Center, Nashville, TN (United States)
MedImmune LLC, Gaithersburg, MD (United States)
Vanderbilt Univ., Nashville, TN (United States)
MedImmune LLC, Gaithersburg, MD (United States); MabVax Therapeutics Holdings, Inc., San Diego, CA (United States)
Vanderbilt Univ. Medical Center, Nashville, TN (United States); Vanderbilt Univ., Nashville, TN (United States)
Vanderbilt Univ. Medical Center, Nashville, TN (United States); Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN (United States); Vanderbilt Univ. School of Medicine, Nashville, TN (United States)

Clostridium difficile 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.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22). Scientific User Facilities Division; United States Dept. of Veterans Affairs; Public Health Service; Burroughs Wellcome Fund; Michigan Economic Development Corp.; Michigan Technology Tri-Corridor

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1439612

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 35 Vol. 292; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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The role of toxins in Clostridium difficile infection Chandrasekaran, Ramyavardhanee; Lacy, D. Borden FEMS Microbiology Reviews, Vol. 41, Issue 6 https://doi.org/10.1093/femsre/fux048	journal	October 2017
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59 BASIC BIOLOGICAL SCIENCES
Clostridium difficile
SEC-MALS
bacterial toxin
cell surface receptor
crystal structure
electron microscopy (EM)
monoclonal antibody

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

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Cited By (3)

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