Characterization of Diphtheria Toxin-induced Lesions in Liposomal Membranes

Abstract

Diphtheria toxin interaction with membranes has been studied by following the release of a fluorescent dye (calcein) encapsulated within large unilamellar vesicles. Results showed that diphtheria toxin induced temperature- as well as pH-dependent permeability changes in these model membranes. Interestingly, insertion of the "channel-forming" B domain was not sufficient for calcein release, since dye release from vesicles composed of dimyristoyllecithin:cholesterol:dicetylphosphate 4:3:0.8) was completely inhibited at low temperatures which permitted B insertion. Rather, the temperature dependence of calcein release from and A domain insertion into dimyristoyllecithin:cholesterol:dicetyl phosphate vesicles suggest some relationship between "channel formation" and fragment A translocation across membranes. However, the nature of the toxin channel is called into question by our observations that channel size, in addition to activity, was pH-dependent. On the basis of these experiments, it is proposed that the toxin "channel" is the result of localized perturbations in the lipid bilayer at the interface between lipids and inserted toxin molecules that are sufficiently large in fluid membranes at low pH to allow the translocation of fragment A across the bilayer.