B-154. Exchange of Differential Amino Acids within a Functionally Important Region of CNF1 to CNF2, Alters Toxin Activity and Recognition by the CNF1-specific Monoclonal Antibody, NG8

K. K. Grande1, S. B. Rasmussen1, K. C. Meysick2, A. D. O'Brien1;
1Uniformed Services Univ. of the Hlth. Sci., Rockville, MD, 2FDA/CBER, Bethesda, MD.

Cytotoxic Necrotizing Factor type 1 (CNF1) and type 2 (CNF2) are produced by pathogenic Escherichia coli and share 85% identity over 1014 amino acids. Both are members of a toxin family that modify Rho GTPases. (i.e., RhoA, Rac1, and Cdc42). CNF1 is a more potent inducer of multinucleation in HEp-2 cells, binds more efficiently to HEp-2 cells, and despite the conservation of amino acids (C866 and H881) required for enzymatic activity of the toxins, deamidates RhoA and Cdc42 better than does CNF2. To delineate regions of CNF1 and CNF2 that confer these differential toxin characteristics, fifteen CNF1/CNF2 site directed mutants were generated to exchange differential amino acids within a functionally important region of CNF1 between amino acids 546 and 869. This region is recognized by the CNF1-specific neutralizing monoclonal antibody (mAb) NG8 and is important for interaction with HEp-2 cells. Toxins were evaluated for multinucleation, RhoA substrate deamidation, binding to HEp-2 cells, and recognition by mAb NG8. All of the mutants retained the capacity to multinucleate HEp-2 cells. However, the CNF1 double mutant D591E F593L, and single mutant H661Q displayed drastically reduced reactivity with NG8 as assessed by Western blot analyses and ELISA. The reverse chimeric triple mutant, E591D L593F Q661H, imparted mAb NG8 recognition to CNF2. CNF1 mutants G585R S588N, D776N, and N780D had reduced HEp-2-cell-binding capacities and/or RhoA-modifying activities. CNF1 mutants D591E F593L, S610R, and H781F also showed reduced deamidation of RhoA in vitro. These results indicate that in addition to the catalytic domain, important functional amino acids within CNF1 are located within a loop region near the catalytic pocket and in a portion of the toxin that has not been well-characterized.