Effects of lipid composition on membrane permeabilization by sticholysin Iand II, two cytolysins of the sea anemone Stichodactyla helianthus

Sticholysin I and II (St I and St II), two basic cytolysins purified from t he Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid Vesicles by forming pores in their membranes. A general characterist ic of these toxins is their preference for membranes containing sphingomyel in (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composi tion of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized fast er and to a higher extent than vesicles of PC and SM. As in the case of PC/ SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was similar to1. The preference for membranes containing PA was confirmed by i nhibition experiments in which the hemolytic activity of St I was diminishe d by pre-incubation with vesicles of different composition. The inclusion o f even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at s imilar to5 mol % of PA. Inclusion of other negatively charged lipids (phosp hatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), o r cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein r elease, the potency being in the order CL approximate to PA much greater th an PG approximate to PI approximate to PS. However, some boosting effect wa s also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stear ylamine (SA). This indicated that the effect was not mediated by electrosta tic interactions between the cytolysin and the negative surface of the vesi cles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with un charged vesicles than with negatively charged vesicles. A study of the flui dity of the different vesicles, probed by the environment-sensitive fluores cent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the inser tion of the toxin channel could imply the formation in the bilayer of a non lamellar structure, a toroidal lipid pore. In this case, the presence of li pids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the p ore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecule s, which indicates local disruption of the lameliar structure.