Comparison and characterization of two different hemolysins from Aeromonas hydrophila

Abstract

Biochemical properties of purified hemolysins secreted by two different cultures of Aeromonas hydrophila were compared. The bacterial cultures have been isolated from infected fresh-water fishes, snakehead (Ophicephalus striatus, AH-1) and sand goby (Oxyeleotris mamoratus, GH-1). The two hemolysins have previously been demonstrated by other investigators to be unidentical. The toxins were purified in the present studies from stationary phase cultures grown in Luria-bertani medium at 37 degree C Preliminary purification involved sulfuric acid precipitation at pH 4.0 followed by QAE-Sephadex A-50 ion exchange chromatography. 7.1% and 18% of the total hemolytic activities were recovered at the final step of purification from AH-1 and GH-1 isolates, respectively. Both preparations were found to consist of a single major protein at 46 K and 53K, respectively with other minor protein bands following SDS-PAGE analysis. Pure hemolysin of GH-1 (53K) was obtained by further purification on SDS-PAGE. The toxin was free from other protein contamination and immunoreactive to homologous anti-hemolysin after transferring onto a nitrocellulose paper by Western blotting. In comparison, the AH-1 hemolysin (46 K) was always contaminated with the other protein which could not be completely removed by similar procedure. GH-1 hemolysin was more stable than AH-1 hemolysin on both heat treatment and storage. Hemolytic activity of GH-1 could be observed in situ following nondenaturing polyacrylamide gel electrophoresis. Oxidizing and alkylating agents of sulhydryl group partially inhibited the hemolytic activity of both GH-1 and AH-1 hemolysins. Surprisingly, the two hemolysins were affected oppositely by sulhydryl reducing compounds. GH-1 activity was significantly inhibited whereas AH-1 activity was markedly enhanced by 2-mercaptoethanol, dithiothreitol and reduced glutathione. Rabbit antiserum obtained from purified GH-1 hemolysin immunization was specific. Immunoperoxidase staining following SDS-PAGE and transferring onto nitrocellulose by Western boltting was positive to only GH-1 hemolysin but not to AH-1 hemolysin. The results clearly indicated immunochemical difference between the two hemolysins. In addition, GH-1 activity was completely insensitive to cell membrane components, phosphatidyl choline and phosphatidyl ethanolamine, but AH-1 activity was markedly inhibited by the two components suggesting possible different mechanism of the hemolysins in facilitating red cell membrane lysis. The present studies indicated that hemolysins secreted by A. hydrophila from different sources were not identical.