Retrieving biological activity from LukF-PV mutants combined with different S components implies compatibility between the stem domains of these staphylococcal bicomponent leucotoxins.
Bicomponent leucotoxins, such as Panton-Valentine leucocidin, are composed of two classes of proteins, a class S protein such as LukS-PV, which bears the cell membrane binding function, and a class F protein such as LukF-PV, which interacts to form a bipartite hexameric pore. These leucotoxins induce cell activation, linked to a Ca(2+) influx, and pore formation as two consecutive and independently inhibitable events. Knowledge of the LukF-PV monomer structure has indicated that the stem domain is folded into three antiparallel beta-strands in the water-soluble form and has to refold into a transmembrane beta-hairpin during pore formation. To investigate the requirements for the cooperative assembly of the stems of the S and F components to produce biological activity, we introduced multiple deletions or single point mutations into the stem domains of LukF-PV and HlgB. While the binding of the mutated proteins was weakly dependent on these changes, Ca(2+) influx and pore formation were affected differently, confirming that they are independent events. Ca(2+) entry into human polymorphonuclear cells requires oligomerization and may follow the formation of a prepore. The activity of some of the LukF-PV mutants, carrying the shorter deletions, was actually improved. This demonstrated that a crucial event in the action of these toxins is the transition of the prefolded stem into the extended beta-hairpins and that this step may be facilitated by small deletions that remove some of the interactions stabilizing the folded structure.
Werner
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Laboratoire de Physiopathologie et d'Antibiologie des Infections Bactériennes Emergentes et Nosocomiales, Institut de Bactériologie de la Faculté de Médecine, Université Louis Pasteur, F-67000 Strasbourg, France.
Colin
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Coraiola
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Menestrina
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Monteil
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Prévost
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics / Q000494:pharmacology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002463:Cell Membrane Permeability; D004996:Ethidium / Q000378:metabolism; D006460:Hemolysin Proteins; D006801:Humans; D007956:Leukocidins / Q000235:genetics / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009504:Neutrophils / Q000187:drug effects; D011485:Protein Binding; D011487:Protein Conformation; D013210:Staphylococcus / Q000737:chemistry*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002413:Cations, Divalent; D006460:Hemolysin Proteins; D007956:Leukocidins; C037956:gamma-hemolysin, Staphylococcus aureus; C108483:leukocidin S-component protein, Staphylococcus; C074300:LukF protein, Staphylococcus aureus; D004996:Ethidium; D002118:Calcium