Differences in the carboxy-terminal (putative phospholipid binding) domains of Clostridium perfringens and Clostridium bifermentans phospholipases C influence the hemolytic and lethal properties of these enzymes

Abstract

The phospholipases C of C. perfringens (alpha-toxin) and C. bifermentans (Cbp) show >50% amino acid homology but differ in their hemolytic and toxic properties. We report here the purification and characterisation of alpha- toxin and Cbp. The phospholipase C activity of alpha-toxin and Cbp was similar when tested with phosphatidylcholine in egg yolk or in liposomes. However, the hemolytic activity of alpha-toxin was more than 100-fold that of Cbp. To investigate whether differences in the carboxy-terminal domains of these proteins were responsible for differences in the hemolytic and toxic properties, a hybrid protein (N(bi)C(α)) was constructed comprising the N domain of Cbp and the C domain of alpha-toxin. The hemolytic activity of N(bi)C(α) was 10-fold that of Cbp, and the hybrid enzyme was toxic. These results confirm that the C-terminal domain of these proteins confers different properties on the enzymatically active N-terminal domain of these proteins.