PORE FORMATION BY A 2-COMPONENT LEUKOCIDIN FROM STAPHYLOCOCCUS-AUREUSWITHIN THE MEMBRANE OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES

The effects of the Staphylococcus aureus leukocidin (PVL), a two-compo nent non-hemolytic toxin, were investigated on the membrane permeabili ty of human polymorphonuclear leukocytes (PMNs). In the absence of ext racellular Ca2+, the fluorescence of ethidium bromide added to the ext racellular medium increased after PVL application in a concentration-d ependent manner and no variations in the free intracellular [Ca2+] of Fura2-loaded PMNs were detected. In the presence of extracellular Ca2, the fluorescence of ethidium was not modified but the free intracell ular [Ca2+] of PMNs increased after application of PVL in a concentrat ion-dependent manner. The time lag observed before an increase in the ethidium fluorescence was longer than the time lag observed before a F ura2 fluorescence increase. Simultaneous recordings of the two probes fluorescence variations have shown the protective effect of Ca2+ and Z n2+ and the closing of the pore by 50 mM Ca2+ or 2 mM Zn2+. Moreover, the effect of Ca2+ could be reversed by the addition of EGTA. In the p resence of 1 mM extracellular Ca2+ or 0.8 mM extracellular Zn2+, the p ore induced by PVL had an ionic size allowing Ca2+, Mn2+, Zn2+ and Mg2 + fluxes. The additon of antibodies against either component of PVL in hibits the permeabilization provoked by the toxin even after it was in itiated. It is concluded that leukocidin from S. aureus is a pore-form ing toxin which, under physiological conditions ([Ca2+] = 1 to 1.5 mM) , provokes the formation of an ion-sized pore inducing an increase in the free intracellular Ca2+ which may activate PMN functions.