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Infection and Immunity, April 2001, p. 2390-2395, Vol. 69, No. 4
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.4.2390-2395.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Flow Cytometric Determination of Panton-Valentine Leucocidin S Component Binding

Valérie Gauduchon,dagger Sandra Werner, Gilles Prévost, Henri Monteil, and Didier A. Colin*

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

Received 17 October 2000/Returned for modification 28 November 2000/Accepted 17 January 2001

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.

* Corresponding author. Mailing address: LTAB, Institut de Bactériologie, 3 rue Koeberlé, 67000 Strasbourg, France. Phone: 33 (0) 3 90 24 37 54. Fax: 33 (0) 3 88 25 11 13. E-mail: didier.colin{at}medecine.u-strasbg.fr.

dagger Present address: Centre National de Référence des Toxémies Staphylococciques, Faculté de Médecine, Lyon, France.

Infection and Immunity, April 2001, p. 2390-2395, Vol. 69, No. 4
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.4.2390-2395.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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Copyright © 2001 by the American Society for Microbiology. All rights reserved.