Loss of virulence in a protease-deficient mutant of Aeromonas salmonicida

Abstract

The importance of extracellular protease production by Aeromonas salmonicida, the bacterial pathogen of fish furunculosis, was investigated with four virulent strains (which were autoagglutinative, hemagglutinative, resistant to fish serum, adhesive to fish tissue culture, protease positive, hemolysin positive, and leukocytolysin positive) and three avirulent strains (which were nonagglutinative, nonhemagglutinative, sensitive to serum, nonadhesive, protease positive, hemolysin positive, and leukocytolysin positive). A protease-deficient mutant (NTG-1) was induced by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine from virulent strain A-7301, showing protease production, which was common to the two strain groups. Strain NTG-1 showed loss of virulence in determinations of 50% lethal doses, although it remained autoagglutinative, hemagglutinative, serum resistant, adhesive, hemolysin positive, and leukocytolysin positive. A protease fraction separated from extracellular products of strain A-7301 by DEAE-cellulose column chromatography had the capacity to produce skin lesions (furuncles) and high mortality in sockeye salmon. A comparable protein fraction from extracellular products of NTG-1 resulted in no protease activity and no pathological effects on the fish. The avirulent strains were eliminated from rainbow trout in a short time, whereas the virulent strains (including A-7301) were highly infective and proliferated in hosts. NTG-1 preserved its infectivity, but fish showed no signs of disease and no mortality. These findings indicate that extracellular protease is a major virulence factor and that protease production in the host is closely implicated in the pathogenesis of fish furunculosis.