Analysis of the specificity of Panton-Valentine leucocidin and gamma-hemolysin F component binding.
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 Ca(2+) 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 [K(d)], 6.2 +/- 1.9 nM; n = 8), monocytes (K(d), 2.8 +/- 0.8 nM; n = 7), and lymphocytes (K(d), 1.2 +/- 0.2 nM; n = 7). Specific binding of HlgB was observed only after addition of LukS-PV on PMNs (K(d), 1.1 +/- 0.2 nM; n = 4) and monocytes (K(d), 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 Ca(2+) 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.
Meyer
Florent
F
Laboratoire de Physiopathologie des Interactions Hôte-Bactérie, EA3432, Institut de Bactériologie, Université Louis-Pasteur, 3 rue Koeberlé, Strasbourg, F-67000 France.
Girardot
Raymonde
R
Piémont
Yves
Y
Prévost
Gilles
G
Colin
Didier A
DA
D001426:Bacterial Proteins / Q000378:metabolism* / Q000633:toxicity; D001427:Bacterial Toxins / Q000378:metabolism* / Q000633:toxicity; D002118:Calcium / Q000378:metabolism; D005098:Exotoxins / Q000378:metabolism* / Q000633:toxicity; D006460:Hemolysin Proteins / Q000378:metabolism* / Q000633:toxicity; D007956:Leukocidins / Q000378:metabolism* / Q000633:toxicity; D008214:Lymphocytes / Q000187:drug effects*; D009000:Monocytes / Q000187:drug effects*; D009504:Neutrophils / Q000187:drug effects*; D052899:Pore Forming Cytotoxic Proteins / Q000378:metabolism / Q000633:toxicity; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D013211:Staphylococcus aureus / Q000502:physiology*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D005098:Exotoxins; D006460:Hemolysin Proteins; D007956:Leukocidins; C078284:Panton-Valentine leukocidin; D052899:Pore Forming Cytotoxic Proteins; D021122:Protein Subunits; C037956:gamma-hemolysin, Staphylococcus aureus; D002118:Calcium