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The cytotoxic action of leukocidin from Pseudomonas aeruginosa was supported by the following observations. (i) The destruction of rabbit leukocytes by the toxin was reduced in the absence of Ca2+ and stimulated by the addition of calcium ionophore A23187 but inhibited by EDTA, EGTA, and TMB-8, an antagonist of intracellular Ca2+ transport. (ii) Uptake of 45Ca into leukocytes exposed to the toxin was enhanced about threefold the rate of uptake into untreated cells. The increased 45Ca uptake into the cells was slightly inhibited by trifluoperazine, an inhibitor of Ca2+-calmodulin activity, but not by ruthenium red. (iii) Pseudomonal leukocidin enhanced rapidly the labeling of phosphatidylinositol, polyphosphoinositides, phosphatidic acid, and lysophosphatidic acid from [32P]phosphate. The time course experiments of the labeling and breakdown of these phospholipids suggested that the initial action of this toxin was to stimulate phosphatidic acid production, presumably causing a rapid metabolic change of phosphatidylinositol correlating with the activities of phosphatidylinositol-specific phospholipase C and 1,2-diacylglycerol kinase. It was considered that a rapid formation of phosphatidic acid and degradation of polyphosphoinositides might be related to a Ca2+ movement from extra- and intracellular space. (iv) In leukocytes exposed to the toxin, acid phosphatase activity as a marker enzyme of lysosome was activated up to 75% of the lysosomal enzyme before cell destruction. The leakage of lysosomal enzyme from the cells occurred at the almost same time as leukocyte destruction. The mode of cytotoxic action of pseudomonal leukocidin is discussed.

Mode of cytotoxic action of pseudomonal leukocidin on phosphatidylinositol metabolism and activation of lysosomal enzyme in rabbit leukocytes