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 (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.
Laboratoire de Toxinologie et d'Antibiologie Bactériennes (UPRES-EA 1318), Institut de Bactériologie de la Faculté de Médecine, Université Louis Pasteur-Hôpitaux Universitaires de Strasbourg, 67000 Strasbourg, France.
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Gauduchon
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Werner
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Prévost
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Monteil
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Colin
D001427:Bacterial Toxins; D001667:Binding, Competitive; D002118:Calcium / Q000494:pharmacology; D005098:Exotoxins; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007956:Leukocidins / Q000378:metabolism*; D008297:Male; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology; D013211:Staphylococcus aureus; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001427:Bacterial Toxins; D005098:Exotoxins; D007956:Leukocidins; C078284:Panton-Valentine leukocidin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium