MOLECULAR LOCALIZATION OF THE ESCHERICHIA-COLI CYTOTOXIC NECROTIZING FACTOR CNF1 CELL-BINDING AND CATALYTIC DOMAINS

Cytotoxic necrotizing factor type 1 (CNF1) induces, in epithelial cell s, the development of stress fibres via the GTPase Rho pathway. We sho wed that CNF1 is able to modify Rho both in vitro and in vivo. Recombi nant N-terminal 33 kDa (CNF1Nter) and C-terminal 14.8-31.5 kDa (CNF1Ct er) regions of the CNF1 protein allowed us to demonstrate that the N-t erminal region contains the cell-binding domain of the toxin and that the C-terminal region is responsible for its catalytic activity. CNF1N ter lowered the activity of CNF1 when provided to cells before the tox in whereas CNF1Cter had no effect on CNF1 cell toxicity. CNF1Cter was sufficient to induce a typical CNF1 phenotype when microinjected into African green monkey kidney cells (Vero cells), and was able to modify Rho as previously reported for CNF1. The C-terminal domain lost its c atalytic activity when deleted of various subdomains, suggesting a sca ttered distribution of catalytic-site amino acids, Elucidation of the CNF1 functional organization and analysis of amino acid homologies bet ween CNFs (CNF1, CNF2), Pasteurella multocida toxin (PMT) and dermonec rotic toxin of Bordetella pertussis (DNT) allowed us to postulate that CNFs and DNT act on Rho via the same enzymatic activity located in th eir C-terminus, and that CNFs and PMT probably bind to analogous cell receptors.