The so-called Panton-Valentine leucocidin (PVL) was shown to differ from hemolysins secreted by strain V8, which was isolated from a patient with chronic furunculosis (16). Gladstone and Van Heyningen (9) have reported the nonhemolytic properties of PVL. Woodin (22, 23) characterized PVL as being composed of two protein components. Epidemiological studies have demonstrated that PVL is secreted by clinical strains associated with abscesses, furuncles (5, 8), and community-acquired pneumonia (13). Genes encoding PVL were cloned and sequenced, and proteins were named LukS-PV (32,317 Da) and 386 Da). They belong to the staphylococcal bicomponent pore-forming leukotoxin family (18). PVL induces the opening of Ca 2ϩ channels responsible for an influx of Ca 2ϩ (19) and the formation of pores through the membrane of target cells (7).Previous work (4) showed that the binding of LukS-PV is a prerequisite for the binding of LukF-PV and subsequent activation of polymorphonuclear neutrophils (PMNs). Binding studies by Colin et al. (4) indicated that LukS-PV had a K d of 6 nM and showed a maximal binding capacity (B m ) of 39,000 molecules per PMN using an iodinated toxin. Several reasons prompted us to reevaluate this determination and to use a simpler, nonradioactive technique. First, the radioiodination of LukS-PV had altered its biological activity to some extent, and second, a very high PMN concentration was used (3 ϫ 10 6 PMNs/ml). In addition, we chose flow cytometry, which allows both the analysis of low cell concentrations and fluorescence determinations. Furthermore, since LukS-PV does not possess any cysteine, substitution of a cysteine for a glycine was carried out by site-directed mutagenesis in order to label one leukotoxin molecule with one fluorescein. In these conditions, we could accurately analyze the binding of very low concentrations of leukotoxin to measure its apparent affinity and to characterize some of its binding properties. MATERIALS AND METHODSChemical reagents. H-89, phorbol 12-myristate 13-acetate (PMA), staurosporine, wortmannin, and salts were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France); yeast extract was purchased from Oxoid (Dardilly, France); and Bacto-Casamino Acids were purchased from Difco (Becton Dickinson, Le Pont de Claix, France).Leukotoxin purification. Leukotoxins were produced from cultures of Staphylococcus aureus strain V8 (ATCC 49775) harvested at the stationary growth phase, as described previously (17). Briefly, the strain was grown for 17 h in YCP medium (3% [wt/vol] .0) at 37°C with vigorous shaking (10). The exoproteins were concentrated after precipitation with 80% (wt/vol) ammonium sulfate and dialysis against 30 mM Na-phosphate (pH 6.5). A bulk of positively charged proteins was selected on a Sepharose SP Fast Flow chromatography plate (Pharmacia, Uppsala, Sweden) after elution in 0.5 M NaCl. The resulting proteins were then subjected to cation-exchange MonoS fast-performance liquid chromatography (Pharmacia) and to further alkyl-Superose fast-perfo...

Staphylococcus aureus strains causing human pathologies produce several toxins, including a pore-forming protein family formed by the single-component K K-hemolysin and the bicomponent leukocidins and Q Q-hemolysins. The last comprise two protein elements, S and F, that co-operatively form the active toxin. K K-Hemolysin is always expressed by S. aureus strains, whereas bicomponent leukotoxins are more speci¢cally involved in a few diseases. X-ray crystallography of the K K-hemolysin pore has shown it is a mushroom-shaped, hollow heptamer, almost entirely consisting of L L-structure. Monomeric F subunits have a very similar core structure, except for the transmembrane stem domain which has to refold during pore formation. Large deletions in this domain abolished activity, whereas shorter deletions sometimes improved it, possibly by removing some of the interactions stabilizing the folded structure. Even before stem extension is completed, the formation of an oligomeric pre-pore can trigger Ca 2+ -mediated activation of some white cells, initiating an in£ammatory response. Within the bicomponent toxins, Q Q-hemolysins de¢ne three proteins (HlgA, HlgB, HlgC) that can generate two toxins: HlgA+HlgB and HlgC+HlgB. Like K K-hemolysin they form pores in planar bilayers with similar conductance, but opposite selectivity (cation instead of anion) for the presence of negative charges in the ion pathway. Q Q-Hemolysin pores seem to be organized as K K-hemolysin, but should contain an even number of each component, alternating in a 1:1 stoichiometry.

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