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Glycobiology Advance Access originally published online on May 28, 2008
Glycobiology 2008 18(9):698-706; doi:10.1093/glycob/cwn048
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile

Tanis Dingle2, Stefanie Wee2, George L Mulvey2, Antonio Greco3, Elena N Kitova4, Jiangxiao Sun4, Shuangjun Lin4, John S Klassen4, Monica M Palcic4, Kenneth K S Ng3 and Glen D Armstrong1,2

2 Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB T2N 1N4, Canada
3 Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada
4 Department of Chemistry, University of Alberta, Edmonton, AB T6G 2R3, Canada

1 To whom correspondence should be addressed: Tel: 1+403-220-6885; Fax: 1-403-270-2772; e-mail: glen.armstrong{at}ucalgary.ca

Received on March 20, 2008; revised on May 22, 2008; accepted on May 25, 2008

The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBDs) and subdomains of the two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) expressed by Clostridium difficile were examined in the hemagglutination and Verocytotoxicity neutralization assays and by qualitative affinity chromatography using Sepharose-linked {alpha}Gal(1,3)βGal(1,4)βGlc as well as the direct electrospray ionization mass spectrometry (ES-MS) assay. These studies revealed that, whereas the full-length TcdA CBD agglutinated rabbit erythrocytes, neutralized TcdA-mediated Vero cell death and bound to {alpha}Gal(1,3)βGal(1,4)βGlc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by {alpha}Gal(1,3)βGal(1,4)βGlc-derivatized Sepharose corresponded to the number of available TcdA subdomain ligand-binding sites. By contrast, the ES-MS assays revealed that both the TcdA and TcdB CBD bind to 8-methoxycarbonyloctyl-{alpha}Gal(1,3)βGal(1,4)βGlc sequences with similar avidities. Additional ES-MS experiments using chemically altered {alpha}Gal(1,3)βGal(1,4)βGlc sequences also revealed that the TcdA and TcdB CBD will tolerate a fair amount of structural variation in their complementary glycan ligands. Although the studies are consistent with the known ligand-binding properties of the TcdA and TcdB holotoxins, they also revealed subtle heretofore unrecognized functional differences in their receptor recognition properties.

Key words: affinity / Clostridium difficile / ligand binding / mass spectroscopy / toxin


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