Flow cytometric determination of Panton-Valentine leucocidin S component binding

Abstract

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 (Kd = 0.07 +/- 0.02 nM, n = 5) and monocytes (Kd = 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 flow cytometry. LukS-PV possesses a high specific affinity for a unique receptor on PMNs and monocytes.

FIG. 1

Flow cytometry analysis of the time course of free intracellular Ca²⁺ variations (A) and of pore formation (B) in human PMNs after the simultaneous addition of LukF-PV and of the wild-type or mutated LukS-PV. PMNs are either loaded with Fluo3 (A) or suspended in the presence of 4 μM ethidium bromide (B). LukS-PV, LukSG10C, and LukSG10C* were each added at a 1 nM concentration (arrow) to PMNs (5 × 10⁵ PMNs/ml) in the presence of 30 nM LukF-PV.

FIG. 2

Flow cytometry analysis of the binding of LukSG10C* to increasing concentrations of human PMNs. PMNs were incubated with 0.1 and 1 nM LukSG10C* for 60 and 15 min, respectively. The binding was not modified by PMN concentrations of up to 10⁵ PMNs/ml for 0.1 nM LukSG10C* and to 7 × 10⁵ PMNs/ml for 1 nM LukSG10C*.

FIG. 3

Example of a flow cytometry analysis of kinetics of binding of increasing concentrations of LukSG10C* to human PMNs in the absence of Ca²⁺. PMN concentrations were 5 × 10⁵ PMNs/ml for 0.5 to 10 nM LukSG10C* and 8 × 10⁴ PMNs/ml for 0.01 to 0.2 nM LukSG10C*.

FIG. 4

Calculation by nonlinear regression of the equilibrium constants of LukSG10C* binding from values generated from the experiment whose results are shown in Fig. 3. non spec. coef., nonspecific coefficient.

FIG. 5

Flow cytometry determination of the time course of the pore formation induced in human PMNs by increasing LukS-PV concentrations in the presence of LukF-PV. The pore formation is determined by the ethidium influx stimulated in 5 × 10⁴ PMNs/ml by the addition of 0.01 to 100 nM LukS-PV and 30 nM LukF-PV.

FIG. 6

Determination by flow cytometry of the competitive binding between LukSG10C* and S components (LukS-PV, HlgC, HlgA, LukM, and LukE) in human PMNs. LukSG10C* concentration, 1 nM; PMN concentration, 5 × 10⁴ PMNs/ml; incubation time, 30 min; n = 4.

FIG. 7

Determination by flow cytometry of the effect of PMA preincubation on the binding of LukSG10C* on human PMNs. LukSG10C* concentration, 1 nM. PMNs (5 × 10⁵ PMNs/ml) were preincubated at each PMA concentration and sampled every 10 min. Then LukSG10C* was added 9 min before analysis (100% = binding of LukSG10C* in the absence of PMA).

FIG. 8

Determination by flow cytometry of the inhibition by staurosporine (STA) of the effect of PMA on the binding of LukSG10C* on human PMNs. Measurements were made as described for Fig. 7. LukSG10C* concentration, 1 nM; PMA concentration, 10⁻⁸ M. PMNs (5 × 10⁵ PMNs/ml) were preincubated for 60 min with 10⁻⁶ M staurosporine.