Crystallization and Some Properties of Leukocidin from Pseudomonas aeruginosa

Abstract

A leukocidin was isolated and purified from autolysates of Pseudomonas aeruginosa strain 158 by a combination of procedures such as column chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-100, and zone electrophoresis on pevikon. The purified preparation showed a single band in each experiment using electrophoreses in the presence or absence of sodium dodecyl sulfate (SDS), and the agar-gel Ouchterlony immunodiffusion test. The purified pseudomonal leukocidin was crystallized by salting out with saturated ammonium sulfate at pH 7.0 in a needle-leaf like form. The molecular weight of the leukocidin was estimated to be 42,500 by SDS-polyacrylamide gel electrophoresis, 40,000 by gel filtration, and 44,700 (3.3 S20,w) by sucrose density gradient centrifugation. The isoelectric point of the leukocidin was estimated to be at pH 6.3 by isoelectrofocusing. Morphological studies of a leukocidin-treated leukocyte showed that the formation of vacuoles of cytosolic granules and the swelling of the lobulated nuclei occurred prior to leukocyte enlargement.In a slide adhesion test with rabbit polymorphonuclear leukocytes (I X 10 6 ) , the minimum cytotoxic dose for the destruction of all leukocytes was 13-20 ng of the crystallized toxin. Rabbit lymphocytes were one-thirtieth as sensitive as rabbit leukocytes. Leukocidin did not act on rabbit erythrocytes or on platelets.The leukocidin of Pseudomonas aeruginosa was firstly described in 1976 by Scharmann (18) and showed a striking resemblance to the biological activity ofstaphylococcal leukocidin (13-16, 26) inflicting a series of morphological changes on rabbit leukocytes but not on rabbit erythrocytes. However, the differences of the mode of toxic actions of these leukocidins were enormous.Although most of our knowledge concerning the leukocidin of P. aeruginosa comes from the studies of Scharmann (18-22) and Lutz (11), the mode of cytotoxic action at the molecular level is still not understood. In the present paper, we described a procedure for purification and crystallization of pseudomonal leukocidin with good reproducibility differing from the method of Scharmann (18) and Lutz (11) based on solubilization of the toxin from its ammonium sulfate precipi ta te. Considerable information concerning the cytotoxic action of pseudomonal leukocidin on rabbit leukocytes is given.

Pseudomonas aeruginosa cytotoxin: the nucleotide sequence of the gene and the mechanism of activation of the protoxin

“…A very small amount of the 60 kD protein can be detected on SDS-PAGE or immunoblot if a large amount of the purified preparation is loaded on SDS-PAGE gel after boiling in the sample buffer (data not shown). Hirayama et al (1983;1984a;1984b) have characterized a cytotoxic protein, leucocidin. isolated from the autolysate cf PAl 58 which has a leucocytctoxic activity similar to that of cyctotoxin but a different moieoular weight (42.5 kD).…”

Binding of pseudomonal leukocidin to rabbit polymorphonuclear leukocytes

“…Preparation of pseudomonal leukocidin. Purification and crystallization of pseudomonal leukocidin were carried out as described previously (3,6).…”

“…counted by phase-contrast microscopy, and "leukocyte de-struction" was calculated as described previously (6).…”

“…Pseudomonal leukocidin is a 42,500-dalton protein that is produced by most strains of pathogenic Pseudomonas aeruginosa (6). Its biological significance is that it damages granulocytes and lymphocytes of various animal species (6,9,13).…”

“…Pseudomonal leukocidin is a 42,500-dalton protein that is produced by most strains of pathogenic Pseudomonas aeruginosa (6). Its biological significance is that it damages granulocytes and lymphocytes of various animal species (6,9,13). We recently reported that the cytotoxic action of pseudomonal leukocidin on rabbit polymorphonuclear leukocytes is essentially mediated by an increase in cytosolicfree Ca>2 concentration resulting from rapid metabolic conversion of phosphatidylinositol to phosphatidic acid (4,5).…”