MECHANISM OF ACTION OF CYTOTOXIC NECROTIZING FACTOR(CNF)PRODUCED BY PATHOGENIC ESCHERICHIA COLI

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The aim of this project was to undcrstand the molccular mcchanism how the cytotoxic necrotizing factor (CNF) produced by pathogccin Eschcrichia coli activate small (G-protein Rho and its relatcd families in culture cells. We utilized E.coli coexpression system to know the effect of the toxin on the small GTPascs. Therefore we gencrated E.coli carrying cnf2 and cDNA of rhoA, cdc42, or rac1. Since these GTPases possesed FLAG-Lag at thte N-terminal, they could be purified by using Sepharose beads with monoclonal antibody against FLAG-tag as ligand. Accordingly we purified CNF-modificd and -unmodified small GTPascs, and measured their intrinsic GTPasc activity, CAP (GTPase activating protein) stimulated GTPase activity, GTPrS binding activity. The data indicated that CNF2 inhibited GTPase activity of RhoA and Rac1. On the other hand, CNF1 inhiibited GTPasc activity of RhoA and Cdc42. Analysis of overproduced RhoA modified by CNF2 indicated that CNF2 deamidated RhoA at 63rd Q and converted it to U.We also used polyclonal antibody raised against Switch H domain of Rho family GTPascs with substitution of 63rd (or 61st) Q as E.This antibody presumably recognizes CNF-modificd small GTPascs. As expected. Western analysis of CNF2-modificd small GTPascs revealed that RhoA and Racl were immunolabeled with this antibody but not was Cdc42. On the other hand, Western analysis of CNF1-modified small GTPascs showed that RhoA and Cdc42 were positive while Raci was negative. These results clearly indicated that CNF2 possess deamidase activity like CNF1. but their substrate specificitics arc distinct.