Separation and in vivo analysis of two extracellular proteases and the T-hemolysm from Aeromonas salmonicida

Abstract

A procedure using DEAE cellulose and hydroxylapetite was developed for the separation of Aeromonas salmonicida P1 protease, P2 protease, and trout cell-specific hemolysin (T-lysin) from supernatants of broth cultures. The Mferent proteases were demonstrated using protease inhibitors, substrate specificities, and polyacrylamide gel electrophoresis with protease substrates in the gels. Isolated P1 protease and T-lysin were shown to act independently in the complete lysis of rainbow trout erythrocytes in vitro. The T-lysin acted on the outer membrane and P1 protease acted on the nuclear membrane. Analysis of cell-free exudate from lesions of trout infected with A. salmonicida by injection demonstrated that more than one protease was also present in vivo. P1 protease was present in both lesions and in culture supernatants, but P2 protease was detected only in culture supernatants. No T-lysin activity was detected in cell-free exudate from lesions caused by A. salmonicida infection.