Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer. In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the fluorescent dye N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water. PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD. Each purified NBD-labeled PFO was then bound to membranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers. Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment. This pattern indicates that this portion of the membrane-bound PFO spans the membrane in an antiparallel beta-sheet conformation. The alternating exposure of these residues to the hydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains. Residues K189-N218 therefore form a two-stranded, amphipathic beta-sheet in the membrane-bound PFO that creates a stable interface between the pore and the membrane. This same region packs as three short alpha-helices in the soluble, monomeric form of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition in which three alpha-helices unfold to form a membrane-spanning amphipathic beta-sheet.

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 beta 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 beta hairpins completely spans the membrane. In the soluble monomer, these transmembrane segments are folded into six alpha 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.

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of membrane-penetrating toxins. The CDCs form large homooligomers (estimated to be comprised of up to 50 CDC monomers) that are responsible for generating a large pore in cholesterol-containing membranes of eukaryotic cells. The assembly of the PFO cytolytic complex was examined to determine whether it forms an oligomeric prepore complex on the membrane prior to the insertion of its membrane-spanning β-sheet. A PFO oligomeric complex was formed on liposomes at both 4 °C and 37 °C and shown by SDS-agarose gel electrophoresis to be comprised of a large, comparatively homogeneous complex instead of a distribution of oligomer sizes. At low temperature, the processes of oligomerization and membrane insertion could be resolved, and PFO was found to form an oligomer without significant membrane insertion of its β-hairpins. Furthermore, PFO was found to increase the ion conductivity through a planar bilayer by large and discrete stepwise changes in conductance that are consistent with the insertion of a preassembled pore complex into the bilayer. The combined results of these analyses strongly support the hypothesis that PFO forms a large oligomeric prepore complex on the membrane surface prior to the insertion of its transmembrane β-sheet.