Discoupling the Ca2+-activation from the pore-forming function of the bi-component Panton-Valentine leucocidin in human PMNs

The consecutive cell activation, including Ca2+-channel opening, and pore f ormation leading to human neutrophil lysis were the two functions of the st aphylococcal Panton-Valentine leucocidin attempted to be discoupled by site -directed mutagenesis, In a first approach consisting in deletions of the c ytoplasmic extremity of the transmembranous domain, we produced a LukF-PV D elta Ser125-Leu128 with a slightly reduced Ca2+ induction but with a signif icantly lowered lytic activity when combined with its synergistic protein L ukS-PV, The second approach consisted in the modification of charges and/or introduction of a steric hindrance inside the pore, which also led to inte resting mutated proteins: LukF-PV G131D, G131W and G130D, The latter had an intact Ca2+ induction ability while the lytic one was 20-fold diminished. Binding properties and intrinsic pore diameters of these discoupled toxins remained comparable to the wild-type protein, The mutated proteins promoted interleukin-8 secretion, but they were rather inactive in an experimental model. New insights are brought concerning the role of the two functions in the virulence of this bi-component leucotoxin. (C) 1999 Federation of Euro pean Biochemical Societies.