Purification,cloning and characterization of variant LukE-LukD with strong leukocidal activity of staphylococcal bi-component leukotoxin family

Staphylococcus aureus produces bi-component leukotoxins composed of non-associated soluble proteins, S and F. Neither S nor F component alone is cytotoxic, but components together are active. These include Panton-Valentine leukocidin (PVL), gamma-hemolysin, LukE-LukD and others. Purification of leukotoxin from Staphylococcus aureus V8 strain (ATCC 27733) which does not have PVL genes, identified an F component with 100% identical to that of PVL in the first twenty-five N-terminal amino acids. Molecular cloning of this toxin obtained 2,595 nucleotides sequences containing two novel open reading frames for S and F. Deduced amino acid sequences of the S and F were respectively 91 and 94% identical to those of LukE and LukD. These were named variant of LukE-LukD (LukEv-LukDv). The activity of the recombinant LukEv-LukDv to rabbit leukocytes was similar to that of recombinant PVL. LukEv-LukDv was hemolytic to rabbit red blood cells although the activity was only 8% of gamma-hemolysin, but PVL was not. These activities were quite different from the LukE-LukD which was reported no hemolytic and poorly cytotoxic to leukocytes compared to PVL. The lukEv-IukDv was found in 87% of clinical isolates of Staphylococcus aureus but lukE-lukD was not detected. These data demonstrate the existence of variant LukE-LukD in V8 strain (ATCC 27733).