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

Abstract

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 findingsmore » 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.« less

Authors:

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]

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+ Show Author Affiliations

1. Vanderbilt Univ. Medical Center, Nashville, TN (United States)
2. MedImmune LLC, Gaithersburg, MD (United States)
3. Vanderbilt Univ., Nashville, TN (United States)
4. MedImmune LLC, Gaithersburg, MD (United States); MabVax Therapeutics Holdings, Inc., San Diego, CA (United States)
5. Vanderbilt Univ. Medical Center, Nashville, TN (United States); Vanderbilt Univ., Nashville, TN (United States)
6. 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)

Publication Date:

Thu Jul 13 00:00:00 EDT 2017

Research Org.:

Argonne National Lab. (ANL), Argonne, IL (United States)

Sponsoring Org.:

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

OSTI Identifier:

1439612

Grant/Contract Number:  

AC02–06CH11357; BX002943; AI095755; AI108778; S10 RR026915; 085P1000817

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Biological Chemistry

Additional Journal Information:

Journal Volume: 292; Journal Issue: 35; Journal ID: ISSN 0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Country of Publication:

United States

Language:

ENGLISH

Subject:

59 BASIC BIOLOGICAL SCIENCES; bacterial toxin; cell surface receptor; crystal structure; electron microscopy (EM); monoclonal antibody; Clostridium difficile; SEC-MALS