Coupling of Cholesterol and Cone-shaped Lipids in Bilayers Augments Membrane Permeabilization by the Cholesterol-specific Toxins Streptolysin O and Vibrio cholerae Cytolysin

Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective asd-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal α-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins. Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective asd-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal α-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins. Vibrio cholerae cytolysin streptolysin O phosphatidylcholine phosphatidylethanolamine phosphatidylglycerol phosphatidylserine d-erythro-N-acetylsphingosine d-erythro-N-dodecylsphingosine d-erythro-N-hexadecylsphingosine d-erythro-N-arachidylsphingosine egg yolk PC egg yolk PG large unilamellar vesicles cytolysin dosage required for release of 25% (50%) of calcein from vesicles To bacterial pore-forming cytolysins, cholesterol is a logical choice as a target molecule, because it confers specificity for animal as opposed to bacterial cell membranes. The specificity for cholesterol is shared between Vibrio cholerae cytolysin (VCC)1 (1Ikigai H. Akatsuka A. Tsujiyama H. Nakae T. Shimamura T. Infect. Immun. 1996; 64: 2968-2973Crossref PubMed Google Scholar) and streptolysin O (SLO) (2Prigent D. Alouf J.E. Biochim. Biophys. Acta. 1976; 443: 288-300Crossref PubMed Scopus (60) Google Scholar). Otherwise, these toxins are not related, and the oligomeric pores they form are very different in size and morphology (1Ikigai H. Akatsuka A. Tsujiyama H. Nakae T. Shimamura T. Infect. Immun. 1996; 64: 2968-2973Crossref PubMed Google Scholar, 3Bhakdi S. Tranum-Jensen J. Sziegoleit A. Infect. Immun. 1985; 47: 52-60Crossref PubMed Google Scholar). Although with SLO the sterol is already required in the initial event of membrane binding of the monomeric toxin (4Alouf J.E. Geoffroy C. Alouf J.E. Freer J.H. Sourcebook of Bacterial Protein Toxins. Academic Press, London1991: 147-186Google Scholar), it only comes into play at the stage of oligomerization in the case of VCC (5Zitzer A. Palmer M. Weller U. Wassenaar T. Biermann C. Tranum-Jensen J. Bhakdi S. Eur. J. Biochem. 1997; 247: 209-216Crossref PubMed Scopus (56) Google Scholar, 6Zitzer A. Harris R. Kemminer S.E. Zitzer O. Bhakdi S. Muething J. Palmer M. Biochim. Biophys. Acta. 2000; 1509: 264-274Crossref PubMed Scopus (29) Google Scholar). When the sterol is incorporated into phosphatidylcholine (PC) bilayers at physiologically realistic concentrations (i.e. up to 40% by mol), these membranes do not become significantly sensitive to VCC. However, it was previously found that membrane susceptibility toward the cytolysin was greatly enhanced by inclusion of ceramide; free ceramide and monohexosyl ceramides proved similarly effective (7Zitzer A. Zitzer O. Bhakdi S. Palmer M. J. Biol. Chem. 1999; 274: 1375-1380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). A combined specificity for cholesterol and sphingolipids has previously been shown for the fusion protein of Semliki Forest virus. In that instance, the interaction with ceramide proved to be highly stereoselective (8Moesby L. Corver J. Erukulla R.K. Bittman R. Wilschut J. Biochemistry. 1995; 34: 10319-10324Crossref PubMed Scopus (48) Google Scholar, 9Wilschut J. Corver J. Nieva J.L. Bron R. Moesby L. Reddy K.C. Bittman R. Mol. Membr. Biol. 1995; 12: 143-149Crossref PubMed Scopus (49) Google Scholar, 10Corver J. Moesby L. Erukulla R.K. Reddy K.C. Bittman R. Wilschut J. J. Virol. 1995; 69: 3220-3223Crossref PubMed Google Scholar). Accordingly, we have examined the structural properties of the ceramide molecule responsible for the sensitization of membranes to VCC. To our surprise, no dependence on stereospecific features of ceramide could be detected. Membrane sensitization was readily achieved with a variety of synthetic ceramides and even with 1,2-diacyl-sn-glycerol, which is not closely related in structure to ceramide. However, with both sphingo- and glycerolipids, the presence of a phosphocholine headgroup led to a decrease in membrane susceptibility toward VCC. In glycerolipids other than PC and diacylglycerol, headgroups smaller in size than phosphocholine were associated with higher membrane susceptibility to the cytolysin. Among lipid species sharing the same headgroup, a complementary trend was generally apparent in which acyl chains having a large cross-section were associated with higher VCC activity, although there were significant exceptions to this correlation. Remarkably, despite its lack of a structural homology with VCC, SLO was affected in its activity toward cholesterol-containing bilayers in a very similar way by the incorporation of sphingo- and glycerolipids into the bilayer. We propose that the effects of sphingo- and glycerolipids on membrane susceptibility to VCC and SLO do not arise because of any direct, specific effect upon the toxins but instead are mediated by their ability to enhance the interaction of cholesterol with the toxins. VCC and α-hemolysin were purified from bacterial culture supernatants as described previously (5Zitzer A. Palmer M. Weller U. Wassenaar T. Biermann C. Tranum-Jensen J. Bhakdi S. Eur. J. Biochem. 1997; 247: 209-216Crossref PubMed Scopus (56) Google Scholar, 11Palmer M. Weller U. Messner M. Bhakdi S. J. Biol. Chem. 1993; 268: 11963-11967Abstract Full Text PDF PubMed Google Scholar). SLO was expressed recombinantly inEscherichia coli as a maltose-binding protein fusion protein and purified from bacterial cell lysates (12Weller U. Mueller L. Messner M. Palmer M. Valeva A. Tranum-Jensen J. Agrawal P. Biermann C. Doebereiner A. Kehoe M.A. Bhakdi S. Eur. J. Biochem. 1996; 236: 34-39Crossref PubMed Scopus (61) Google Scholar). 1-(16-Indolyl-palmitoyl)-2-oleoyl-sn-glycero-3-phosphocholine and 1-(16-bromo-palmitoyl)-2-oleoyl-sn-glycero-3-phosphocholine were kindly supplied by Dr. John Silvius, McGill University. The synthesis of the four stereoisomers of n-C8:0-ceramide (see Fig. 1) were described previously (13Karasavvas N. Erukulla R.K. Bittman R. Lockshin R. Zakeri Z. Eur. J. Biochem. 1996; 236: 729-737Crossref PubMed Scopus (82) Google Scholar) and characterized as theN-biphenylcarboxamido derivatives of sphingosine by high pressure liquid chromatography (14Jarvis W.D. Fornari F.A. Traylor R.S. Martin H.A. Kramer L.B. Erukulla R.K. Bittman R. Grant S. J. Biol. Chem. 1996; 271: 8275-8284Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar).d-erythro-Triple bond-C16-Cer (see Fig. 1) was synthesized as described previously (15Karasavvas N. Erukulla R.K. Bittman R. Lockshin R. Hockenbery D. Zakeri Z. Cell Death Differ. 1996; 3: 149-151PubMed Google Scholar).d-erythro-Aryl-C4-Cer (see Fig. 1) was synthesized as described recently (16Chun J. He L. Byun H.-S. Bittman R. J. Org. Chem. 2000; 65: 7634-7640Crossref PubMed Scopus (55) Google Scholar). The other ceramide analogs shown in Fig. 1 were prepared by using the synthetic sequences outlined below. EYPC, EYPG, bovine brain β-galactosylceramide, bovine brain non-hydroxy and α-hydroxy fatty acyl ceramides, and cholesterol acetate were purchased from Sigma. All other lipids were obtained from Avanti Polar Lipids (Alabaster, AL). Into a round-bottomed flask were combined 25 mg (0.08 mmol) ofd-erythro-sphingosine, 24 mg (0.09 mmol) ofp-nitrophenyl caprylate, and 5 ml of anhydrous tetrahydrofuran. The reaction mixture was stirred at room temperature for 24 h, at which time TLC analysis indicated the consumption of sphingosine (silica gel-coated aluminum plates, eluted with ethyl acetate (Rf = 0.73) and visualized with 10% sulfuric acid in methanol). The reaction mixture was concentrated on a rotary evaporator. The residue was dissolved in 1 ml of a 25% solution of ethyl acetate in hexanes, which was loaded onto a silica gel column (10 × 100 mm). The column was eluted with 100 ml of 25% ethyl acetate in hexanes and then with 200 ml of ethyl acetate. The fractions containing the product were collected and concentrated to afford 33 mg (97%) of the product as a colorless solid. The film was lyophilized from 5 ml of anhydrous benzene to afford a colorless powder.1H NMR (400 MHz, CDCl3) δ 6.35 (NH, d,J = 7.3 Hz), 5.77 (1H, dt, J = 14.5, 6.7 Hz), 4.30 (1H, m), 3.91 (2H, m), 3.68 (1H, dd, J = 10.7, 2.7 Hz), 2.22 (2H, t, J = 7.4 Hz), 2.05 (2H, dt,J = 14.0, 6.9 Hz), 1.63 (2H, m), 1.26 (30H, m), 0.88 (6H, t, J = 6.5 Hz); 13C NMR (100 MHz, CDCl3) δ 174.1, 134.2, 128.8, 74.4, 62.4, 54.6, 36.8, 32.3, 31.9, 31.7, 29.7 (3Bhakdi S. Tranum-Jensen J. Sziegoleit A. Infect. Immun. 1985; 47: 52-60Crossref PubMed Google Scholar), 29.68, 29.67, 29.5, 29.4, 29.3, 29.28, 29.26, 29.18, 29.06, 25.8, 22.7, 14.1. Into a 25-ml round-bottomed flask 1.3 ml of a 15% (by weight) solution of diethylzinc (1.16 mmol) in n-hexane was added to a stirring solution of 110 mmol) of in 5 ml of at room temperature the reaction mixture was stirred for 33 mg of was added as a solution in ml of 1 the reaction was by the of 5 ml of The was and with 25-ml of ethyl acetate. The combined were and concentrated The residue was on silica to mg of the product (Rf = and mg of its (Rf = as colorless of the two was lyophilized from 5 ml of benzene to afford colorless NMR (400 MHz, CDCl3) δ (NH, d,J = Hz), (1H, dd, J = Hz), (1H, m), (1H, dd, J = Hz), (1H, dd, J = Hz), 2.22 (2H, = Hz), m), 1.26 (30H, m), 0.88 (6H, m), (2H, m), (1H, m), (1H, 13C NMR (100 MHz, CDCl3) δ 36.8, 31.9, 31.7, (4Alouf J.E. Geoffroy C. Alouf J.E. Freer J.H. Sourcebook of Bacterial Protein Toxins. Academic Press, London1991: 147-186Google Scholar), 29.28, 22.7, 14.0, NMR (400 MHz, CDCl3) δ (NH, d,J = Hz), (1H, m), (1H, = Hz), (1H, dd, J = 2.7 Hz), (1H, m), (2H, m), m), 1.26 (30H, m), (6H, m), (2H, m), (1H, m), (1H, 13C NMR (100 MHz, CDCl3) δ 31.9, 31.7, (4Alouf J.E. Geoffroy C. Alouf J.E. Freer J.H. Sourcebook of Bacterial Protein Toxins. Academic Press, London1991: 147-186Google Scholar), 29.68, (2Prigent D. Alouf J.E. Biochim. Biophys. Acta. 1976; 443: 288-300Crossref PubMed Scopus (60) Google Scholar), 22.7, Into a round-bottomed flask was mg mmol) of and ml of A of anhydrous was and the solution was to for 5 The was by The residue was dissolved in 5 ml of anhydrous and the solution was The solution was concentrated pressure to as a A of the was dissolved in ml of and was added to a solution containing 25 mg (0.08 mmol) and of The solution was stirred for at room temperature The reaction mixture was concentrated and the residue was on silica = to mg of the product as solid. The was lyophilized from 5 ml of benzene to the product as powder.1H NMR (400 MHz, CDCl3) δ (1H, dt, J = Hz), (1H, = Hz), (NH, J = Hz), 3.91 (1H, dd, J = Hz), (1H, m), (1H, m), (2H, m), (2H, dt, J = Hz), (2H, m), m), 1.26 0.88 (6H, 13C NMR (100 MHz, CDCl3) δ 32.3, 31.9, 29.68, 29.5, 29.26, 22.7, The lipids were dissolved in and at the indicated 5 and with in a flask to form a The lipids were in calcein by if and The were and and then membranes size 100 using a The calcein was by gel on in The lipid of the was by using a of cholesterol a solution of the cytolysin in in with bovine were To of the of calcein lipid was for at the were into of and the calcein was in a The of calcein release was from the of a of the to permeabilization was on a with mm). The of toxin required for 25 release of calcein were from of toxin dosage by from the two data were prepared from and cholesterol and and the of 200 the lipid instead of The were for were as in of was dissolved with of the of this was to the of the The decrease in by the lipid with and was then from the The was with and in of and were up to (by with 1 were for at with were then with the and by to VCC was added to was time by cell In a it was found that of ceramide to PC bilayers containing cholesterol by enhanced permeabilization (7Zitzer A. Zitzer O. Bhakdi S. Palmer M. J. Biol. Chem. 1999; 274: 1375-1380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). To the structural features of the ceramide molecule responsible for this we a variety of synthetic ceramide Fig. 1 the of the synthetic ceramide analogs we The ceramides were added to to a mixture of lipids cholesterol and up the to 100 these lipid loaded with the calcein were prepared as described previously (7Zitzer A. Zitzer O. Bhakdi S. Palmer M. J. Biol. Chem. 1999; 274: 1375-1380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and the release of calcein was as a of VCC Fig. that of the by does not a change in membrane that there is no stereospecific interaction between ceramide and VCC. The of VCC required to the release of calcein to 25% were to a of the effect of lipids on membrane susceptibility to VCC. Fig. the obtained with molecular in ceramide is that the of the of the from its by a a even a benzene does not significantly change the activity of VCC. is in to the of the in the of fusion J. Moesby L. Erukulla R.K. Reddy K.C. Bittman R. Wilschut J. J. Virol. 1995; 69: 3220-3223Crossref PubMed Google Scholar, L. Byun J. Wilschut J. Bittman R. J. Chem. 2000; Scopus Google Scholar), as as in by and A. Bittman R. S. Mol. 1996; Google Scholar) and in the of in (14Jarvis W.D. Fornari F.A. Traylor R.S. Martin H.A. Kramer L.B. Erukulla R.K. Bittman R. Grant S. J. Biol. Chem. 1996; 271: 8275-8284Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, W.D. Fornari F.A. Traylor R.S. Grant S. U. S. A. PubMed Scopus Google Scholar, A. D. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). the of the and of the with a do not have effects on the activity of VCC Fig. also that even a similarly sensitization as do the if ceramide and share a of the features of molecule are not was previously found that was less effective than ceramide in membranes to VCC, suggesting of the phosphocholine headgroup (7Zitzer A. Zitzer O. Bhakdi S. Palmer M. J. Biol. Chem. 1999; 274: 1375-1380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The that is effective than PC that the phosphocholine is not only in sphingolipids but also in In lipid bilayers with cholesterol, the headgroups of are to the of cholesterol from J. Biophys. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). from then also be from VCC, which for the effect of the phosphocholine In of cholesterol in PC and it was that because of its headgroup, a for the sterol than J. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). The effect of headgroup size on the activity of VCC was examined with the species of the A of the of VCC with these lipids their headgroup a between headgroup and cytolysin dosage (i.e. of headgroup and membrane which is with the way to the to the sterol be to decrease the of the fatty acyl The of membrane susceptibility to VCC on the acyl cross-section was with synthetic The are shown in Fig. and have a effect than which in turn is by a synthetic lipid with and acyl chains of acyl cross-section and membrane susceptibility to VCC is also with the However, as not also have acyl chains than are less effective than the The effect of acyl was also with ceramides A between acyl and membrane susceptibility was to the However, the was membrane susceptibility to VCC A between the fatty acyl and membrane susceptibility only be if is to for all of the lipid species in the membrane bilayer. However, both glycerolipids S. H. Biochemistry. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar) and ceramides M. Biochemistry. PubMed Scopus Google Scholar, Chem. 1997; PubMed Scopus Google Scholar) highly acyl chains are to within the which the decrease of membrane susceptibility with ceramide acyl containing of were examined using a of lipid also two PC the and the species In similar this of was found to enhanced on which was to their into the Biochemistry. PubMed Scopus Google Scholar). The was with the with respect to that does not in the and readily than of into the effective of this lipid that is for with other membrane and for the of membrane sensitization by to readily than in this which for the membrane susceptibility with of a lipid in the of the membrane is less to its is Fig. and are than at 5 The of significant membrane susceptibility to VCC with such of ceramide is in its similar were with the effective of the glycerolipids not The data the that the molecular shape of the lipid the activity of VCC on the target Lipids that have a conical molecular a cross-section than does the headgroup, enhance membrane susceptibility to VCC. this effect be significant not only with synthetic but also with membranes is in Fig. D. of to ceramide We propose that the of action of ceramides and glycerolipids is they change the state of membrane cholesterol and only the activity of VCC, which on cholesterol. To this we examined the effects of ceramides and of glycerolipids on another cholesterol-specific SLO to a of cholesterol-specific toxins (4Alouf J.E. Geoffroy C. Alouf J.E. Freer J.H. Sourcebook of Bacterial Protein Toxins. Academic Press, London1991: 147-186Google Scholar) that are not at all structurally similar to VCC. A SLO is affected by the presence of ceramides in the target membranes in a that closely VCC. is also found with glycerolipids, with glycerolipids the is different Fig. the of the two toxins with all the that were with both VCC and the lipids we in structure ceramides, and glycerolipids, and even the of is we examined a staphylococcal is that in this case the with VCC is which that lipid molecular shape is not as a of the activity of staphylococcal In the of any ceramides glycerolipids, both VCC and SLO are highly the of membrane cholesterol is from to not A very similar effect be obtained by of cholesterol with acetate. However, acetate is a very of cholesterol as a specific for SLO VCC not because the is of with both VCC (1Ikigai H. Akatsuka A. Tsujiyama H. Nakae T. Shimamura T. Infect. Immun. 1996; 64: 2968-2973Crossref PubMed Google Scholar) and SLO (2Prigent D. Alouf J.E. Biochim. Biophys. Acta. 1976; 443: 288-300Crossref PubMed Scopus (60) Google Scholar, K.C. Biochem. J. PubMed Scopus Google Scholar). that the effect of of the cholesterol does not stereospecific interaction between the toxins and the The do not our that VCC a specificity for cholesterol and ceramide (7Zitzer A. Zitzer O. Bhakdi S. Palmer M. J. Biol. Chem. 1999; 274: 1375-1380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In to the fusion protein of Semliki Forest which such specificity (8Moesby L. Corver J. Erukulla R.K. Bittman R. Wilschut J. Biochemistry. 1995; 34: 10319-10324Crossref PubMed Scopus (48) Google Scholar, J.L. Bron R. Corver J. Wilschut J. J. PubMed Scopus Google Scholar), VCC is not significantly affected even by structural in the ceramide ceramides and glycerolipids augment the activity of VCC, up to in the presence of membrane concentrations of cholesterol. N. Biophys. J. 1993; 65: Full Text PDF PubMed Scopus (48) Google Scholar) the of headgroups on in of and the in the membranes were for is to their with the effects of the same lipids on cytolysin activity all cytolysins there is a between the ability of lipids to the and cytolysin α-hemolysin is not on cholesterol, there be a in the effect of on pore-forming toxins. a be in with on α-hemolysin, does not membrane cholesterol, although are not in A. Biochemistry. 2000; PubMed Scopus Google Scholar). However, the between the temperature and toxin activity is and with the two cholesterol-specific toxins than with α-hemolysin that the sterol in the of toxin is cholesterol to all of the cholesterol is a cone-shaped it in the but does not for headgroup which it from cholesterol within a with all other cone-shaped for headgroup no these are glycerolipids, different other cholesterol The case in membranes with high cholesterol of headgroup the sterol to as a the free of cholesterol is to with its membrane J. Biophys. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). cholesterol from the and which readily with than with PC bilayers J. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar), in with the that PC a for the sterol J. Biophys. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The binding of cytolysins another for cholesterol to from the with To the that the sterol is closely by the cholesterol in effect the and of the associated the the interaction between the sterol and the the sensitive the protein to the cholesterol free in to the of the bilayer. The the effects of a wide variety of lipids and of in the cholesterol M. S. S. Biochim. Biophys. Acta. PubMed Scopus Google Scholar) a also the at lipid VCC is in small unilamellar vesicles than in (7Zitzer A. Zitzer O. Bhakdi S. Palmer M. J. Biol. 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