Chimeric clostridial cytotoxins: Identification of the N-terminal region involved in protein substrate recognition

Clostridium sordellii lethal toxin is a member of the family of large clostridial cytotoxins that glucosylate small GTPases, In contrast to Clostridium difficile toxins A and B, which exclusively modify Rho subfamily proteins, C.sordellii lethal toxin also glucosylates Ras subfamily proteins. By deletion analysis and construction of chimeric fusion proteins of C.sordellii lethal toxin and C.difficile toxin IB, Ive localized the enzyme activity of the lethal toxin to the N terminus of the holotoxin and identified the region involved in protein substrate specificity. The to?;in fragment of the N-terminal 546 amino acid residues of C. sordellii lethal toxin glucosylated. Rho and Ras subfamily proteins, as the holotoxin did. Deletion of a further 30 amino acid residues from the C terminus of this active fragment drastically reduced glucotransferase activity and blocked glucohydrolase activity. Exchange of amino acid residues 364 through 516 of lethal toxin for those in the active toxin B: fragment (1 to 546) allowed glucosylation of Ras subfamily proteins. In contrast, the chimera with amino acids 1 to 364 ham toxin B, 365 eo 468 from lethal toxin, and. 469 to 546 from toxin B exhibited markedly reduced modification of Res subfamily proteins, whereas modification of Rac and Cdc42 was hardly changed. The data indicate that the region of amino acid residues 364 through 516 primarily defines the substrate specificity: of C. sordellii lethal toxin.