Production and properties of extracellular factors from Aeromonas salmonicida
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The production of extracellular products by Aeromonas salmonicida, in vitro, has been investigated. The results indicated that the bacterium produces at least two haemolytic activities in vitro. Unshaken cultural conditions favoured the production of a haemolysin with a broad spectrum of activity against various erythrocyte types (H-lysin), whilst shaken cultural conditions favoured the production of a haemolysin active against trout erythrocytes (T-lysin). The effects of growth medium type and culture conditions on the production of these haemolytic activities has been investigated. The activity of the T-lysin appeared to be attributable to the combined effects of an activity which caused incomplete lysis of the erythrocytes (T1 activity) and caseinase. The T1 activity appears to be found in culture supernate associated with fragments of the bacterial cell wall or membrane resulting in apparent molecular heterogeneity. H-lysin activity appeared to be due to a single protein, which did not require a divalent cation for the expression of activity. The haemolysin was synthesised by the bacterium as an inactive precursor molecule (pro-H-lysin) which was cleaved by the bacterial protease to give the active haemolysin; other commercially available proteases were also able to effect this activation. An unidentified component of a variety of animal sera was also able to effect conversion of the pro-H-lysin to the active form, however, this conversion only occurred after the serum component had entered the bacterial cell. The H-lysin was purified 1770 fold using freeze fractionation, salt fractionation, ion exchange chromatography and gel filtration chromatography. The partially purified protein possessed erythrocyte lysing and glycerophospholipid:cholesterol acyltransferase activities, however it was not clear whether these activities were attributable to the same molecule. Investigation of the kinetics of erythrocyte lysis by the partially purified H-lysin suggested that the haemolysin possessed an enzymatic mode of action. In vitro the haemolysin was active against both rainbow trout leucocytes and tissue culture cells. However, in vivo the haemolysin had no obvious effect on rainbow trout.
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