Cytosolic delivery and characterization of the TcdB glucosylating domain by using a heterologous protein fusion

TcdB from Clostridium difficile glucosylates small GTPases (Rho, pac, and C dc42) and is an important virulence factor in the human disease pseudomembr anous colitis. In these experiments, in-frame genetic fusions between the g enes for the 255 amino-terminal residues of anthrax toxin lethal factor (LF n) and the TcdB(1-556) coding region were constructed, expressed, and purif ied from Escherichia call. LFnTcdB(1-556) was enzymatically active and gluc osylated recombinant RhoA, pac, Cdc42, and substrates from cell extracts. L FnTcdB(1-556) plus anthrax toxin protective antigen intoxicated cultured ma mmalian cells and caused actin reorganization and mouse lethality, all simi lar to those caused by wild-type TcdB.