Functional properties of the carboxy-terminal host cell-binding domains of the two toxins, TcdA and TcdB, expressed by Clostridium difficile.
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)betaGal(1,4)beta Glc 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)betaGal(1,4)beta Glc-derivatized Sepharose, the TcdB CBD was inactive in these functional assays. Moreover, retention by alpha Gal(1,3)betaGal(1,4)beta Glc-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)betaGal(1,4)beta Glc sequences with similar avidities. Additional ES-MS experiments using chemically altered alpha Gal(1,3)betaGal(1,4)beta Glc 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.
Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB T2N 1N4, Canada.
Tanis
T
Dingle
Stefanie
S
Wee
George L
GL
Mulvey
Antonio
A
Greco
Elena N
EN
Kitova
Jiangxiao
J
Sun
Shuangjun
S
Lin
John S
JS
Klassen
Monica M
MM
Palcic
Kenneth K S
KK
Ng
Glen D
GD
Armstrong
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000378:metabolism; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D054884:Host-Pathogen Interactions / Q000502:physiology; D008954:Models, Biological; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D018675:Toxicity Tests; D014709:Vero Cells
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D010446:Peptide Fragments; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile