ISOLATION AND CHARACTERIZATION OF A CLOSTRIDIUM-BOTULINUM C2 TOXIN-RESISTANT CELL-LINE - EVIDENCE FOR POSSIBLE INVOLVEMENT OF THE CELLULAR C2II RECEPTOR IN GROWTH-REGULATION

Clostridium botulinum C2 toxin, which consists of the binding componen t C2II and the enzyme component C2I, acts on eukaryotic cells by selec tive ADP-ribosylation of G-actin. To obtain C2 toxin-resistant cells, we mutagenized CHO-K1 cells with N-nitroso-N-methylurea and selected f or C2 resistance. Cells which survived the selection procedure with 50 ng of C2I and 100 ng of C2II per ml were obtained with a frequency of 30 x 10(-6). The colony-forming ability of CHO wild-type cells was re duced to 50% with 10 ng of C2I and 20 ng of C2II per ml. In contrast, the colony-forming ability of the isolated CHO mutant cells was not in fluenced by up to 200 ng of C2I and 400 ng of C2II per ml. Toxin-induc ed ADP-ribosylation of G-actin was not impaired in lysates of mutant c ells. The C2 toxin-resistant phenotype remained sensitive to the cell- rounding activities of cytotoxins from C. perfringens (iota-toxin), C. novyi, C. difficile, and C. botulinum (C3) and to cytochalasin D. Bin ding of component C2II was impaired in resistant CHO cells, suggesting mutation of the toxin cell surface receptor. Serum factors protected wild-type cells against the cytotoxic effect of C2 toxin. Furthermore, the C2-resistant phenotype correlated with an increased serum depende ncy. The data suggest that the action of C, botulinum C2 toxin is medi ated by its binding and uptake via a cell surface receptor which might be involved in growth regulation.