SITE-DIRECTED MUTAGENESIS OF THE ALPHA-TOXIN GENE OF STAPHYLOCOCCUS-AUREUS - ROLE OF HISTIDINES IN TOXIN ACTIVITY IN-VITRO AND IN A MURINE MODEL

Staphylococcus aureus alpha-toxin is a membrane-damaging exoprotein th at oligomerizes to form transmembrane pores. Chemical modification of histidines with diethylpyrocarbonate has been shown to reduce the hemo lytic activity of alpha-toxin, suggesting that one or more of the hist idine residues is important for toxin function. To individually assess the functional importance of each of the four histidine residues (res idues 35, 48, 144, and 259), we used oligonucleotide-directed mutagene sis of the cloned alpha-toxin gene to replace each histidine with leuc ine. The mutant toxins were expressed in S. aureus and evaluated for h emolytic activity in vitro and for lethality in an intraperitoneal mur ine model. Substitution of histidine 35 with leucine produced a mutant toxin (H35L) without hemolytic or lethal activity. Mutant toxins H48L , H144L, and H259L exhibited 7, 16, and 46%, respectively, of the hemo lytic activity of wild-type toxin. Immunoblotting of purified H35L tox in incubated with liposomal membranes demonstrated intact membrane bin ding and hexamer formation that was clearly detectable but reduced com pared with that of the wild-type toxin. This suggests that hexamer for mation alone is not sufficient for the expression of alpha-toxin activ ity. The nature of the defect underlying the lack of activity of the H 35L mutant toxin remains to be elucidated but may involve failure of t he hexamer to span the lipid bilayer to form a transmembrane pore or a change in the internal surface and permeability characteristics of th e pore.