Interaction of .theta.-Toxin (Perfringolysin O), a Cholesterol-Binding Cytolysin, with Liposomal Membranes: Change in the Aromatic Side Chains upon Binding and Insertion

Abstract

To understand the mechanism of membrane lysis by theta-toxin (perfringolysin O) from Clostridium perfringens, a cholesterol-binding, pore-forming cytolysin, we undertook a spectroscopic analysis of the structural changes that occur during the lytic process using lipid vesicles. In particular, the spectra were compared with those obtained using a modified theta-toxin, MC theta, that binds membrane cholesterol without forming oligomeric pores, thus bypassing the oligomerization step. The interaction of theta-toxin with liposomes composed of cholesterol and phosphatidylcholine but not with cholesterol-free liposomes caused a remarkable increase in the intensity of the tryptophan fluorescence emission spectra and ellipticity changes in the near- and far-UV CD peaks. A CD peak shift from 292 to 300 nm was specific for theta-toxin, suggesting oligomerization-specific changes occurring around tryptophan residues. Structural changes in the aromatic side chains were detected in the near-UV CD and fluorescence spectra upon MC theta-liposome interaction, although the far-UV CD spectra indicate that the beta-rich secondary structure of MC theta is well-conserved after membrane binding. Quenching of the intrinsic tryptophan fluorescence of MC theta by brominated lecithin/cholesterol liposomes suggests that theta-toxin inserts at least partly into membranes in the absence of oligomerization. These results indicate that regardless of oligomerization, the binding of theta-toxin to cholesterol induces partial membrane insertion and triggers conformational changes accompanied by aromatic side chain rearrangement with retention of secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

C-terminal Amino Acid Residues Are Required for the Folding and Cholesterol Binding Property of Perfringolysin O, a Pore-forming Cytolysin

“…This is consistent with our previous findings that a C-terminal tryptic fragment that contains predominantly domain 4 binds to cholesterol and to cholesterol-containing membrane (18). Our findings that the toxin binding to cholesterol in liposomal membrane triggers a conformational change around tryptophan residues in domain 4 also support this view (19,20). Recently, possible roles of the C-terminal region in cell binding were suggested by a report that a monoclonal antibody thought to bind near the C terminus specifically blocks cell binding, although the exact epitope was not identified (21).…”

“…When -toxin interacts with cholesterol on dioleoylphosphatidyl choline/cholesterol liposomes, there is an increase in the intensity of the tryptophan fluorescence (19). The two truncated toxins, ⌬471 and ⌬470, also showed increases in the intensity when incubated with dioleoylphosphatidyl choline/ cholesterol liposomes.…”

Redefining cholesterol's role in the mechanism of the cholesterol-dependent cytolysins

“…2 and 3) based on several lines of evidence including one that CDCs are not cytolytically active on cholesterol-deficient membranes and another that the depletion of membrane cholesterol reduces the extent of membrane binding to various eukaryotic cell types (4). Many studies, primarily performed with perfringolysin O (PFO), have shown that membrane binding is sensitive to the loss of cholesterol (4)(5)(6)(7)(8)(9)(10)(11) and that derivatives of PFO have been used as probes for membrane cholesterol (12)(13)(14)(15).…”

Biotinylated θ-toxin derivative as a probe to examine intracellular cholesterol-rich domains in normal and Niemann-Pick type C1 cells

“…The probe is derived from a pore-forming cytolysin produced by the pathogenic bacterium Clostridium perfringens (28). This thiol-activated cytolysin, called perfringolysin O ( -toxin), specifically binds to free (unesterified) cholesterol (29)(30)(31)(32) and forms oligomeric pores in the membranes (33). The probe BC was prepared by a twostep procedure.…”