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Glycobiology Advance Access published online on May 28, 2008
Glycobiology, 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 Dingle1, Stefanie Wee1, George L. Mulvey1, Antonio Greco2, Elena N. Kitova3, Jiangxiao Sun3, Shuangjun Lin3, John S. Klassen3, Monica M. Palcic3, Kenneth K.S. Ng2 and Glen D. Armstrong1,*

1 Department of Microbiology & amp, Infectious Diseases, University of Calgary, Calgary, AB T2N 4N1, Canada
2 Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada
3 Department of Chemistry, University of Alberta, Edmonton, AB T6G 2R3, Canada

* Corresponding Author's address: Department of Microbiology & Infectious Diseases, Faculty of Medicine, University of Calgary 3330 Hospital drive, NW Calgary, AB, CANADA T2N 4N1, Email: glen.armstrong{at}ucalgary.ca, Telephone: (403) 220 6885, Fax: (403) 270 2772

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

The biological and ligand-binding properties of recombinant C-terminal cell-binding domains (CBD) and sub-domains 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 sub-domain 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: Clostridium difficile / ligand binding / toxin / affinity / mass spectroscopy


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