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 o f biological activities such as lethality to mice, cytotoxicity to cul tured cells, cardiotoxicity, and fluid accumulating activity in rabbit ileal loop test. In this study, we attempted to isolate less hemolyti c mutant toxins of the thermostable direct hemolysin to use them for a nalysis of mode of action of the hemolysin. Six mutant toxins were obt ained by in vitro mutagenesis of the cloned gene for the hemolysin. Ch aracterization of the mutant toxins demonstrated that single amino aci d substitutions at Gly(62), Trp(65), Thr(67), Gly(86), Glu(116) and Gl u(138) resulted in a loss or lowering of the hemolytic activity. Two o f the mutant toxins inhibited hemolysis by wild-type toxin on rabbit b lood agar plates, while their hemolytic activity was below the detecta ble level. These mutant toxins would be useful for identifying the as yet unknown receptor for the hemolysin on the target cell membrane.