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.
Dingle
Tanis
T
Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, AB T2N 1N4, Canada.
Wee
Stefanie
S
Mulvey
George L
GL
Greco
Antonio
A
Kitova
Elena N
EN
Sun
Jiangxiao
J
Lin
Shuangjun
S
Klassen
John S
JS
Palcic
Monica M
MM
Ng
Kenneth K S
KK
Armstrong
Glen D
GD
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