The mechanism of membrane insertion for a cholesterol-dependent cytolysin: A novel paradigm for pore-forming toxins

Perfringolysin O (PFO), a water-soluble monomeric cytolysin secreted by pathogenic Clostridium perfringens, oligomerizes and forms large pores upon encountering cholesterol-containing membranes. Whereas all pore-forming bacterial toxins examined previously have been shown to penetrate the membrane using a single amphipathic β hairpin per polypeptide, cysteine- scanning mutagenesis and multiple independent fluorescence techniques here reveal that each PFO monomer contains a second domain involved in pore formation, and that each of the two amphipathic β hairpins completely spans the membrane. In the soluble monomer, these transmembrane segments are folded into six α helices. The insertion of two transmembrane hairpins per toxin monomer and the major change in secondary structure are striking and define a novel paradigm for the mechanism of membrane insertion by a cytolytic toxin.