Flow cytometric determination of Panton-Valentine leucocidin S component binding

The binding of the S component (LukS-PV) from the bicomponent staphylococcal Panton-Valentine leucocidin to human polymorphonuclear neutrophils (PMNs) and monocytes was determined using flow cytometry and a single-cysteine substitution mutant of LukS-PV, The mutant was engineered by replacing a glycine at position 10 with a cysteine and was labeled with a fluorescein moiety, The biological activity of the mutant was identical to that of the native protein. It has been shown that LukS-PV has a high affinity for PMNs (K-d = 0.07 +/- 0.02 nM, n = 5) and monocytes (K-d = 0.020 +/- 0.003 nM, n = 3) with maximal binding capacities of 197,000 and 80,000 LukS-PV molecules per cell, respectively. The nonspecifically bound molecules of LukS-PV do not form pores in the presence of the F component (LukF-PV) of leucocidin, LukS-PV and HlgC share the same receptor on PMNs, but the S components of other staphylococcal leukotoxins, HlgA, LukE, and LukM, do not compete with LukS-PV for its receptor. Extracellular Ca2+ at physiological concentrations (1 to 2 nM) has only a slight influence on the LukS-PV binding, in contrast to its complete inhibition by Zn2+, The down-regulation by phorbol 12-myristate 13-acetate (PMA) of the binding of LukS-PV was blocked by staurosporine, suggesting that the regulatory effect of PMA depends on protein kinase C activation, The labeled mutant form of LukS-PV has proved very useful for detailed binding studies of circulating white cells by how cytometry, LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.