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Infection and Immunity, January 2009, p. 266-273, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00402-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Analysis of the Specificity of Panton-Valentine Leucocidin and Gamma-Hemolysin F Component Binding{triangledown}

Florent Meyer,{dagger} Raymonde Girardot, Yves Piémont, Gilles Prévost, and Didier A. Colin*

Laboratoire de Physiopathologie des Interactions Hôte-Bactérie, EA3432, Institut de Bactériologie, Université Louis-Pasteur, 3 rue Koeberlé, Strasbourg F-67000, France

Received 1 April 2008/ Returned for modification 11 May 2008/ Accepted 24 September 2008

In this study, the binding of F components of the staphylococcal bicomponent leukotoxins Panton-Valentine leucocidin (LukF-PV) and gamma-hemolysin (HlgB) on polymorphonuclear neutrophils (PMNs), monocytes, and lymphocytes was determined using labeled mutants and flow cytometry. Leukotoxin activity was evaluated by measuring Ca2+ entry or pore formation using spectrofluorometry or flow cytometry. Although HlgB had no affinity for cells in the absence of an S component, LukF-PV had high affinity for PMNs (dissociation constant [Kd], 6.2 ± 1.9 nM; n = 8), monocytes (Kd, 2.8 ± 0.8 nM; n = 7), and lymphocytes (Kd, 1.2 ± 0.2 nM; n = 7). Specific binding of HlgB was observed only after addition of LukS-PV on PMNs (Kd, 1.1 ± 0.2 nM; n = 4) and monocytes (Kd, 0.84 ± 0.31 nM; n = 4) or after addition of HlgC on PMNs, monocytes, and lymphocytes. Addition of LukS-PV or HlgC induced a second specific binding of LukF-PV on PMNs. HlgB and LukD competed only with LukF-PV molecules bound after addition of LukS-PV. LukF-PV and LukD competed with HlgB in the presence of LukS-PV on PMNs and monocytes. Use of antibodies and comparisons between binding and activity time courses showed that the LukF-PV molecules that bound to target cells before addition of LukS-PV were the only LukF-PV molecules responsible for Ca2+ entry and pore formation. In contrast, the active HlgB molecules were the HlgB molecules bound after addition of LukS-PV. In conclusion, LukF-PV must be linked to LukS-PV and to a binding site of the membrane to have toxin activity.

* Corresponding author. Mailing address: Laboratoire de Physiopathologie des Interactions Hôte-Bactérie, EA3432, Institut de Bactériologie, Université Louis-Pasteur, 3 rue Koeberlé, Strasbourg, F-67000 France. Phone: 33 390243754. Fax: 33 3900243808. E-mail: didier.colin{at}medecine.u-strasbg.fr

{triangledown} Published ahead of print on 6 October 2008.

Editor: S. R. Blanke

{dagger} Present address: Labortoire des Biomatériaux, Processus Biologiques et Biophysiques aux Interfaces, Faculté de Chirurgie Dentaire, Université Louis-Pasteur, 4 rue Kirschleger, Strasbourg F-67085, France.

Infection and Immunity, January 2009, p. 266-273, Vol. 77, No. 1
0019-9567/09/$08.00+0     doi:10.1128/IAI.00402-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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