Purification and characterization of leucocidin from Pseudomonas aeruginosa

Abstract

Leucocidin from Pseudomonas aeruginosa strain 158 was released from bacteria by autolysis and purified 19-fold by ammonium sulphate precipitation (20% saturation) and combined 'tandem' gel filtration on Sephadex G-100 superfine and Bio Gel P-100. The product gave a single band (mol. wt. 27000) after poly-acrylamide gel electrophoresis with sodium dodecyl sulphate (SDS). However, it was separated into two active peaks (pI 5-0 and 5-2) by isoelectric focusing, and into five bands by disc electrophoresis without SDS. All bands contained leucocidic activity of about the same specific activity and retained their homogeneity. The purified toxin was thermolabile and was inactivated by pronase, but not by several other proteases. The ultraviolet light absorbancy was typical of proteins. Antibodies directed against leucocidin were detected by passive haemagglutination and by toxin-neutralization. These antibodies inhibited the cytotoxic action of leucocidin bound to granulocytes. The toxin damaged all tested leucocytes (granulocytes of various animal species and lymphocytes of humans) and a number of tissue cultures, but was ineffective against erythrocytes, thrombocytes and isolated granules from polymorphonuclear leucocytes. The intravenous lethal dose for mice was about 1 mug.