Mode of Action of Staphylococcal Leukocidin: Relationship between Binding of ¹²⁵ I-Labeled S and F Components of Leukocidin to Rabbit Polymorphonuclear Leukocytes and Leukocidin Activity

Abstract

The binding of "2'I-labeled S component to rabbit polymorphonuclear leukocytes was found to be concentration dependent and saturable at 37°C. Scatchard analysis of the binding curve gave a straight line, indicating that S component binds to a single population of sites. The dissociation constant, KD, derived from the Scatchard plot was 5.57 x 10-9 M, and the number of binding sites per leukocyte was calculated to be approximately 5,300. Unlabeled S component (10-8 M) or subunit B of cholera toxin (10-7 M) readily competed with 1 I-labeled S component binding, and the labeled S component, preincubated with ganglioside GM1 at equimolar proportions for 5 min, lost the binding capacity to the leukocyte membranes. The binding number of '25I-labeled F component to leukocidinsensitive cells, such as rabbit polymorphonuclear leukocytes and the established human myelocytic leukemia cells, in the absence and in the presence of the unlabeled S component (2.1 nM), was calculated to be 50 and 1,300 molecules per cell, respectively. This increased binding of the labeled F component was time and temperature dependent. The binding number of labeled F component to other cell types comparatively insensitive to leukocidin, such as erythrocytes, adipocytes, intestinal cells, and HeLa cells, was calculated to be less than 50 molecules per cell in spite of the sufficient amount of unlabeled S component bound to their cells. These observations are consistent with the view that in rabbit leukocyte the S component, preferentially bound to the cell surface at 5,300 molecules per cell, contributes to enhance the F component binding up to about 1,300 molecules per cell and may thus play a role of synergistic action of both leukocidin components on the cell membranes in the leukocytolysis.

Changes in the Susceptibility of 12‐O‐Tetradecanoylphorbol 13‐Acetate (TPA)‐Treated HL‐60 Cells to Staphylococcal Leukocidin

“…Cytotoxic action of leukocidin to rabbit polymorphonuclear leukocytes (8)(9)(10)(11)(12) and HL-60 cells (3)(4)(5)(6) has been reported. The mode of action was thought to be as follows: After binding of leukocidin to specific receptors, the activation of membrane-associated enzymes just like phospholipase A2 and phospholipase C occurred, resulting in membrane perturbation including changes in ion permeability and leukocytolysis.…”

“…phocytes and other cells (2,9,11,14). S and F components specifically bind to ganglioside GMl and phosphatidylcholine, respectively (9,10). In the previous paper, it was reported that the specific binding of leukocidin to rabbit polymorphonuclear leukocytes induced activation of phospholipase A2 (11) and phospholipase C in leukocyte membrane (17) and it was suggested that those activities are responsible for the primary action of leukocidin leading to calcium and potassium ion permeability changes in membrane (11).…”

An early effect of the S component of staphylococcal leukocidin on methylation of phospholipid in various leukocytes

“…Preparations of the labeled S and F components had specific activities of 9 x 105 to 2 x 106 cpm/ILg of protein. Binding of 125I-labeled leukocidin to polymorphonuclear leukocytes was assayed as described previously (12).…”

“…Staphylococcal leukocidin consists of two protein components (S and F) that act synergistically to induce cytotoxic changes in human and rabbit polymorphonuclear leukocytes. Previously (7,10,12,14) we reported that the S and F components of leukocidin were preferentially bound and inactivated by GM1 ganglioside and phosphatidylcholine, respectively, in rabbit polymorphonuclear leukocyte membranes. The specific binding of the S component to GM1 ganglioside induces activation of cell membrane-associated phospholipase A2, resulting in the release of prostaglandins and leukotrienes from the cells and then in enhancement of chemotaxis of polymorphonuclear leukocytes (11,13).…”

Crystallization and Some Properties of Leukocidin from Pseudomonas aeruginosa

“…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)(14)(15)(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.…”