Secretion of extracellular proteins by Aeromonas salmonicida and related species

Abstract

This thesis describes the study of extracellular enzyme activities secreted by the Gram- negative bacterium Aeromonas salmonicida and related species. Initially, cell fractionation criteria were used to define operationally the cellular and extracellular location of proteins. It was demonstrated that proteins detectable in the extracellular medium of A. salmonicida were selectively secreted beyond the outer membrane and not present as a result of general non-specific release due to cell lysis. Several extracellular enzyme activities were detected in the culture filtrate of A. salmonicida, of which a number of protease and amylase activities were partially characterised. The principal extracellular protease of M, 70000 was purified to homogeneity by a combination of ion-exchange and gel filtration chromatography, while another major extracellular protein of M, 56000 was purified by preparative-SDS-polyacrylamide gel electrophoresis. Rabbit polyclonal antibodies were then raised against these purified extracellular proteins. In assays with trout erythrocytes, haemolytic activity in the culture filtrate was inhibited by pre incubating with antibodies raised against the 56kDa protein, suggesting that this protein is in fact a haemolysin. After the preliminary studies on the production of extracellular proteins by this organism, a series of experiments was carried out to compare the extracellular protein production of A. salmonicida and related species. The study of strain variation at species and sub-species level, with respect to extracellular protein production, was approached in several ways. Firstly, the strains were examined for the production of extracellular enzyme activity; secondly, polyclonal antibodies raised against the purified extracellular protease and haemolysin enzymes were used in Western blotting experiment to screen strains for cross-reactivity; and lastly, culture filtrates of all strains studied were applied to substrate-SDS-polyacrylamide gels to detect hydrolytic enzyme activity.

It was found that the extracellular protease and haemolysin enzymes were common to the majority of A. salmonicida ssp. salmonicida strains studied. Positive cross- reactivity was also observed with the extracellular fraction of A. hydrophila when probed with A. salmonicida antibodies against the protease and haemolysin activities.

On the evidence of Western blotting, extracellular protein production by other Aeromonas species does not appear to be related to A. salmonicida. Detection of protease and amylase activity in substrate-SDS-polyacrylamide gels allowed a detailed comparison of the hydrolytic enzyme production by members of the Aeromonas genus. The results obtained in this way demonstrated striking honmgeneity of protease and amylase secretion within A. salmonicida ssp. Salmonicida but substantial variation between species and subspecies of the Arromonas genus.