Isolation of mutant toxins of Vibrio parahaemolyticus hemolysin by in vitro mutagenesis.

Thermostable direct hemolysin produced by Vibrio parahaemolyticus is a major virulence factor of the organism. The hemolysin has a variety of biological activities such as lethality to mice, cytotoxicity to cultured cells, cardiotoxicity, and fluid accumulating activity in rabbit ileal loop test. In this study, we attempted to isolate less hemolytic mutant toxins of the thermostable direct hemolysin to use them for analysis of mode of action of the hemolysin. Six mutant toxins were obtained by in vitro mutagenesis of the cloned gene for the hemolysin. Characterization of the mutant toxins demonstrated that single amino acid substitutions at Gly62, Trp65, Thr67, Gly86, Glu116 and Glu138 resulted in a loss or lowering of the hemolytic activity. Two of the mutant toxins inhibited hemolysis by wild-type toxin on rabbit blood agar plates, while their hemolytic activity was below the detectable level. These mutant toxins would be useful for identifying the as yet unknown receptor for the hemolysin on the target cell membrane.