Skip to main content
Article thumbnail
Location of Repository

Site-directed mutagenesis of the alpha-toxin gene of Staphylococcus aureus: role of histidines in toxin activity in vitro and in a murine model.

By B E Menzies and D S Kernodle


Staphylococcus aureus alpha-toxin is a membrane-damaging exoprotein that oligomerizes to form transmembrane pores. Chemical modification of histidines with diethylpyrocarbonate has been shown to reduce the hemolytic activity of alpha-toxin, suggesting that one or more of the histidine residues is important for toxin function. To individually assess the functional importance of each of the four histidine residues (residues 35, 48, 144, and 259), we used oligonucleotide-directed mutagenesis of the cloned alpha-toxin gene to replace each histidine with leucine. The mutant toxins were expressed in S. aureus and evaluated for hemolytic activity in vitro and for lethality in an intraperitoneal murine 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 hemolytic activity of wild-type toxin. Immunoblotting of purified H35L toxin incubated with liposomal membranes demonstrated intact membrane binding and hexamer formation that was clearly detectable but reduced compared with that of the wild-type toxin. This suggests that hexamer formation alone is not sufficient for the expression of alpha-toxin activity. The nature of the defect underlying the lack of activity of the H35L mutant toxin remains to be elucidated but may involve failure of the hexamer to span the lipid bilayer to form a transmembrane pore or a change in the internal surface and permeability characteristics of the pore

Topics: Research Article
Year: 1994
OAI identifier:
Provided by: PubMed Central
Sorry, our data provider has not provided any external links therefore we are unable to provide a link to the full text.

Suggested articles

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.