The complete receptor-binding domain of Clostridium difficile toxin A is required for endocytosis.
Clostridium difficile toxin A, the chief pathogenicity factor of the antibiotic-associated pseudomembranous colitis, is an intracellular acting cytotoxin that reaches its targets, the Rho GTPases, after receptor-mediated endocytosis. The C-terminal part, constructed of repetitive peptide elements, is thought to bind to a lot of carbohydrate containing receptor molecules to induce clustering and endocytosis. To study which part of the receptor-binding domain is in charge of addressing toxin A into the target cells, we studied the functional, i.e., endocytosis-inducing, binding of toxin A. By a competition assay between various receptor-binding fragments of toxin A and the holotoxin A we found that the complete receptor-binding domain, encompassing the entire repetitive elements, but not parts of it, is necessary for binding-induced endocytosis. The receptor binding domain itself shows weaker competition with holotoxin A than the fragment consisting of receptor-binding domain plus intermediary part of the toxin. All toxin A fragments that compete with holotoxin A are capable of inducing their own endocytosis. Thus, the entire receptor-binding domain, covering the C-terminal third of the toxin A molecule, is responsible for cell uptake of toxin A and the intermediary part contributes to the correct folding and assembly of the repetitive domains.
Frisch
Cornelia
C
Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Universität Freiburg, Albertstr. 25, 79104 Freiberg, Germany.
Gerhard
Ralf
R
Aktories
Klaus
K
Hofmann
Fred
F
Just
Ingo
I
D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D001667:Binding, Competitive / Q000502:physiology; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004768:Enterotoxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D019073:HT29 Cells; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013696:Temperature
D001427:Bacterial Toxins; D004768:Enterotoxins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; C041348:tcdA protein, Clostridium difficile