Be. Menzies et Ds. Kernodle, SITE-DIRECTED MUTAGENESIS OF THE ALPHA-TOXIN GENE OF STAPHYLOCOCCUS-AUREUS - ROLE OF HISTIDINES IN TOXIN ACTIVITY IN-VITRO AND IN A MURINE MODEL, Infection and immunity, 62(5), 1994, pp. 1843-1847
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.