Purification and characterization of salmolysin, an extracellular hemolytic toxin from Aeromonas salmonicida

Abstract

An extracellular hemolytic toxin of Aeromonas salmonicida, termed salmolysin, was purified 945-fold by ammonium sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and gel filtration chromatography on Sephadex G-100 and Sepharose 2B. Salmolysin appeared homogeneous upon cellulose acetate membrane electrophoresis and immunodiffusion analysis. The molecular weight of the toxin was estimated to be approximately 200,000 by the sedimentation equilibrium method. The UV absorption spectrum showed a maximum at 275 nm and a minimum at 262 nm. The isoelectric point was found to be at pl 5.4. Carbohydrate and protein analyses and other biochemical data indicated that salmolysin is a glycoprotein, containing approximately 62% carbohydrates. The toxin is a heat-labile substance and is stable at a neutral pH value. Ferrous ion inhibited the activity, whereas metal-chelating agents did not affect the activity. Sulfhydryl reagents did not inhibit the toxin, whereas reducing agents, such as L-cysteine and reduced glutathione, inhibited the toxin to a certain extent. Salmolysin was inactivated by a nonionic detergent but was stimulated by an anionic detergent, sodium deoxycholate, at a low concentration. The toxin was also inactivated by subtilisin and trypsin but was not inhibited by papain and pepsin. Salmolysin, with a remarkable hemolytic activity against salmonid erythrocytes, was lethal to rainbow trout when it was injected intramuscularly.

Characterisation of haemolytic activity fromAeromonas caviae

“…The present study demonstrates that BPD significantly inhibits the growth of A. caviae possibly by iron limitation. Similar to aerolysin of A. hydrophila [29] and salmolysin of A. salmonicida [24] the ,3-haemolysin of A. caviae is inhibited by iron compounds; however, BPD enhances the haemolytic activity of A. caviae by several fold. This supports the hypothesis that the ,3-haemolysin of A. caviae might be regulated by iron limitation.…”

“…Aerolysin of A. hydrophila [21,23], salmolysin of A. salmonicida [24] and fihaemolysin of A. sorbia [22] are also produced extracellularly during the growth of the bacteria. The fl-haemolysin of A. caviae resembles that of Porphyromonas gingivalis [25] as they are stimulated by both Mg2" and Ca2+ ions and inhibited by EDTA whereas an exotoxin of Proteus vulgaris, P. mirabilis and P. morganii [26] and lysolipoid endotoxin of Pseudomonas pseudomallei [27] required only Ca2+ for their enhanced activity.…”

“…hydrophila [23] salmolysin of A. samonicida [24] and ,-haemolysin of P. gingivalis [25] displayed similar properties.…”

Lethal toxicity and immunogenicity of salmolysin, Aeromonas salmonicida hemolysin to white-spotted char, Salvelinus leucomaenis.

“…Salmolysin purification and hemolytic activity assay All procedures for salmolysin purification and hemolytic activity assay were performed as previously described by NOMURA et al (1988). For assay of hemolytic activity, yearling white spotted chars weighing about 120 g were used to supply erythrocytes.…”

Development of a Vaccine for Bacterial Kidney Disease in Salmon, 1988 Final Report.

“…salmoninarum and their putative functions Turaga et al, 1987a & b;Turaga and Kaattri, in preparation;Wiens and Kaattari, in preparation (Nottage and Birkbeck, 1987); isolation of immunosuppressor substance produced by Strantococcus mutans (Santarem et al, 1987), purification of hemolytic toxin from a fish pathogen, Aeromonas salmonicida (Nomura et al, 1988) and purification of an extracellular-protease from Pseudomas cenacia (McKevitt et al, 1988 Initially, a sample is subjected to electrophoresis for separation of antigens according to their charge and size, or size alone. A second electrophoretic step is conducted to transfer the antigens from the gel to an immobilizing surface, such as niuocellulose paper as originally described by Towbin et al (1983).…”