Effects of lipid composition on membrane permeabilization by sticholysin I and II, two cytolysins of the sea anemone Stichodactyla helianthus.
Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (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 composition 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 faster 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 ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of 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 ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA >> PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.
Valcarcel
C A
CA
CNR-ITC, Centro di Fisica degli Stati Aggregati, I-38050 Povo, Italy.
Dalla Serra
M
M
Potrich
C
C
Bernhart
I
I
Tejuca
M
M
Martinez
D
D
Pazos
F
F
Lanio
M E
ME
Menestrina
G
G
D000818:Animals; D002308:Cardiolipins / Q000378:metabolism; D002463:Cell Membrane Permeability; D003064:Cnidarian Venoms / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D006460:Hemolysin Proteins / Q000378:metabolism*; D006461:Hemolysis; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008954:Models, Biological; D009930:Organic Chemicals; D009994:Osmolar Concentration; D010712:Phosphatidic Acids / Q000378:metabolism; D010715:Phosphatidylglycerols / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D012615:Sea Anemones; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
D002308:Cardiolipins; D003064:Cnidarian Venoms; D005452:Fluoresceins; D006460:Hemolysin Proteins; D008081:Liposomes; D008563:Membrane Lipids; D009930:Organic Chemicals; D010712:Phosphatidic Acids; D010715:Phosphatidylglycerols; D010716:Phosphatidylinositols; D010718:Phosphatidylserines; C404794:sticholysin II; C121348:stycholysin I; C007740:fluorexon