Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic.

Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing the plasma membrane. These peptides generally do not act via specific receptors and are active in the micromolar range. Here it is shown that vancomycin and the antibacterial peptide nisin Z use the same target: the membrane-anchored cell wall precursor Lipid II. Nisin combines high affinity for Lipid II with its pore-forming ability, thus causing the peptide to be highly active (in the nanomolar range).

13C NMR studies on [4-13C] cholesterol incorporated in sonicated phosphatidylcholine vesicles.

1. 90.5 MHz 13C NMR linewidth measurements were performed on mixed sonicated [4-13C] cholesterol/phosphatidylcholine vesicles of different fatty acid composition. 2. From the Dy3+ -induced shift of the C4 resonance of cholesterol it suggested that this part of the molecule is localized in the ester bond region of the bilayer. 3. The local motion of the cholesterol ring system is restricted and independent of fatty acid composition. 4. At cholesterol concentrations below 30 mol percent the ring system becomes more immobilised when the fatty acids of the phosphatidylcholine molecules enter the gel state.

The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31P NMR study.

1. The polymorphic phase behaviour of aqueous dispersions of phosphatidylethanolamines isolated from human erythrocytes, hen egg yolk and Escherichia coli have been investigated employing 31P NMR techniques. All species exhibit well defined, reversible bilayer to hexagonal (H11) phase transitions as the temperature is increased. The temperatures at which these transition take place (10, 25--30 and 55--60 degrees C for erythrocyte, egg yolk and E. coli phosphatidylethanolamine, respectively) are sensitive to the fatty acid composition, occurring at a temperature up to 10 degrees C above the high temperature end of the hydrocarbon phase transition as detected by differential scanning calorimetry. In some cases the bilayer to hexagonal (H11) transitions may also be detected employing calorimetric techniques. 2. The addition of equimolar concentrations of cholesterol to these naturally occurring phosphatidylethanolamines does not dramatically affect the bilayer-hexagonal (H11) transition temperature, producing changes of up to 10 degrees C. 3. 18 : 1t/18 : 1t phosphatidylethanolamine undergoes the bilayer to hexagonal (H11) phase transition as the temperature is increased through the interval 50--55 degrees C. Alternatively, hydrated 12 : 0/12 : 0 phosphatidylethanolamine remains in the bilayer phase at temperatures up to 90 degrees C (50 degrees C above the hydrocarbon phase transition temperature). 4. The presence of 100 mM NaCl or 10 mM CaCl2 in aqueous dispersions of egg yolk phosphatidylethanolamine does not alter the temperature-dependent polymorphic phase behaviour significantly. However, at 40 degrees C, increasing the p2H above 8.0 results in progressive inhibition of the hexagonal (H11) phase and the appearance of a phase possibly of cubic structure at p2H 9.0. At p2H 10.0 the bilayer phase is preferred. 5. It is suggested that in biomembranes containing phosphatidylethanolamine as a majority species (such as that of E. coli) the fatty acid composition may primarily reflect the need to maintain bilayer structure. Alternatively, it is pointed out that in mammalian membranes such as that of the erythrocyte, phosphatidylethanolamine tends to destabilize bilayer structure. The resulting possibility that transitory non-bilayer lipid configurations may occur may be directly related to many important properties of biological membranes.

Rapid transbilayer movement of phospholipids induced by an asymmetrical perturbation of the bilayer.

Phospholipase D is used to convert egg phosphatidylcholine to phosphatidic acid in unilamellar vesicles. The transbilayer distribution of both lipids is determined by 31P NMR using paramagnetic ions. Phosphatidic acid formed in the outer monolayer is translocated to the inner monolayer with a halftime of 30-40 min or less. This is accompanied by an equally fast movement of part of the phosphatidylcholine from the inner to the outer monolayer. During these fast transbilayer movements the barrier properties of the vesicle bilayer are maintained.

Effect of amphotericin B on cholesterol-containing liposomes of egg phosphatidylcholine and didocosenoyl phosphatidylcholine. A refinement of the model for the formation of pores by amphotericin B in membranes.

(1) Binding and K+-permeability measurements were performed on egg and 22 : 1c/22 : 1c-phosphatidylcholine liposomes with or without cholesterol. (2) Amphotericin B binds specifically to cholesterol in both types of liposome despite the difference in bilayer thickness. (3) Addition of amphotericin B to one side of the cholesterol-containing egg phosphatidylcholine bilayers induces a fast K+ efflux from the outermost compartment of the liposomes. In contrast, the total K+ content of sonicated unilamellar cholesterol-containing egg phosphatidylcholine vesicles is released by amphotericin B. (4) Amphotericin B addition to one side of the cholesterol-containing 22 : 1c/22 : 1c-phosphatidylcholine liposomes does not cause a change in K+ permeability. The presence of amphotericin B on both sides of the bilayer, however, induces an increase in K+ permeability. (5) A model is proposed which accounts for the effect of bilayer thickness on the amphotericin B-induced permeability changes in membranes.

Membrane potential-driven translocation of a lipid-conjugated rhodamine.

The present study demonstrates that the permanently positively charged, lipid-conjugated rhodamine, R18, can be transported from the outer to the inner leaflet of lipid bilayers in response of a transmembrane potential (negative inside). This conclusion was based on the following observations. (i) A fast decrease of the R18 fluorescence, when present at self-quenching concentrations in DOPC large unilamellar vesicles, was revealed upon induction of a valinomycin-induced K(+)-diffusion potential. (ii) Iodide quenching experiments demonstrated that R18 was no longer accessible to externally added aqueous quencher after application of a transmembrane potential. (iii) 2H-NMR measurements, using DOPC, specifically deuterated at the alpha-position of the phosphocholine head group, revealed a massive transbilayer movement of R18 upon induction of a membrane potential. The extent of the fluorescence changes were found to be dependent on the magnitude of the applied transmembrane potential, which opens possibilities for novel applications of R18 as an internal lipid-conjugated membrane potential probe.

Cytochrome c specifically induces non-bilayer structures in cardiolipin-containing model membranes.

(1) The effect of cytochrome c addition on the phospholipid structure of liposomes composed of cardiolipin, phosphatidylserine, phosphatidylglycerol, phosphatidylcholine or phosphatidylethanolamine in a pure form or in mixtures was investigated by 31P-NMR and freeze-fracture techniques. (2) Cytochrome c specifically induces the hexagonal Hii phase and possibly an inverted micellar structure of part of the phospholipids in cardiolipin-containing model membranes. (3) These results are compared with the effect of Ca2+ on cardiolipin and are discussed in relation to the structure and function of the inner mitochondrial membrane.

The influence of poly(L-lysine) on phospholipid polymorphism. Evidence that electrostatic polypeptide-phospholipid interactions can modulate bilayer/non-bilayer transitions.

31P-NMR shows that poly(L-lysine) binding to cardiolipin, phosphatidylserine or phosphatidylglycerol does not affect the macroscopic structure or local order (in the phosphate region) of the phospholipids. In the case of cardiolipin poly(L-lysine) inhibits the ability of Ca2+ to induce the hexagonal HII phase. Alternatively, poly(L-lysine) induces the hexagonal HII phase for a fraction of the phospholipids in phosphatidylethanolamine-cardiolipin (2:1) dispersions.

Induction of a relatively fast transbilayer movement of phosphatidylcholine in vesicles. A 13CNMR study.

[N-13CH3]Phosphatidylcholines are introduced into the outer monolayer of phosphatidylcholine vesicles with the phosphatidylcholine exchange protein from bovine liver. The transbilayer distribution of the [N-13CH3]phosphatidyl-choline is measured with 13C NMR. The transbilayer movements of [N-13CH3]-dioleoyl phosphatidylcholine and [N-13CH3]dimyristoyl phosphatidylcholine at 30 degrees C in vesicles composed of these phosphatidylcholines are extremely slow processes with estimated half-times of days. [N-13CH3]Dioleoyl phosphatidyl-choline introduced into dimyristoyl phosphatidylcholine vesicles migrates from the outer to the inner monolayer with a half-time of less than 12 h. The data suggest that differential changes in the lateral packing of the two monolayers might be a driving force for transbilayer transport of phospholipids.

Effects of adriamycin on respiratory chain activities in mitochondria from rat liver, rat heart and bovine heart. Evidence for a preferential inhibition of complex III and IV.

The inhibition of respiratory chain activities in rat liver, rat heart and bovine heart mitochondria by the anthracycline antibiotic adriamycin was measured in order to determine the adriamycin-sensitive sites. It appeared that complex III and IV are efficiently affected such that their activities were reduced to 50% of control values at 175 +/- 25 microM adriamycin. Complex I displayed a minor sensitivity to the drug. Of the complex-I-related activities tested, only duroquinone oxidation appeared sensitive (50% inhibition at approx. 450 microM adriamycin). Electron-transfer activities catalyzed by complex II remained essentially unaltered up to high drug concentrations. Of the activities measured for this complex, only duroquinone oxidation was significantly affected. However, the adriamycin concentration required to reduce this activity to 50% exceeded 1 mM. Mitochondria isolated from rat liver, rat heart and bovine heart behaved essentially identical in their response to adriamycin. These data support the conclusion that, in these three mitochondrial systems, the major drug-sensitive sites lie in complex III and IV. Cytochrome c oxidase and succinate oxidase activity in whole mitochondria exhibited a similar sensitivity towards adriamycin, as inner membrane ghosts, suggesting that the drug has direct access to its inner membrane target sites irrespective of the presence of the outer membrane. By measuring NADH and succinate oxidase activities in the presence of exogenously added cytochrome c, it appeared that adriamycin was less inhibitory under these conditions. This suggests that adriamycin competes with cytochrome c for binding to the same site on the inner membrane, presumably cardiolipin.

The lantibiotic nisin, a special case or not?

Nisin is a 34-residue-long peptide belonging to the group A lantibiotics with antimicrobial activity against Gram-positive bacteria. The presence of dehydrated residues and lanthionine rings (thioether bonds) in nisin, imposing structural restrains on the peptide, make it an interesting case for studying the mode of action. In addition, the relatively high activity (nM range) of nisin against Gram-positive bacteria indicates that nisin may be a special case in the large family of pore-forming peptides antibiotics. In this review, we attempted to dissect the mode of action of nisin concentrating on studies that used model membranes or biological membranes. The picture that emerges suggests that in model membrane systems, composed of only phospholipids, nisin behaves similar to the antimicrobial peptide magainin, albeit with an activity that is much lower as compared to its activity towards biological membranes. This difference can be contributed to a missing factor which nisin needs for its high activity. Novel results have identified the factor as Lipid II, a precursor in the bacterial cell wall synthesis. The special high affinity interaction of nisin with Lipid II resulting in high activity and the active role of Lipid II in the pore-formation process make nisin a special case.

Interrelationships between tyrocidine and gramicidin A' in their interaction with phospholipids in model membranes.

(1) The interaction of tyrocidine with different lipids is studied in model membranes and the results are compared to the gramicinid-lipid interaction. (2) The tyrocidine-dielaidoylphosphatidylethanolamine interaction gives rise to a population of phospholipids with a lower gel to liquid-crystalline transition temperature and to an abolition of the bilayer to HII phase transition, resulting in a macroscopic organization with dynamic and structural properties different from those of the pure lipid. (3) Tyrocidine has a strong fluidizing effect on the acyl chains of phosphatidylcholines, manifested by a decrease in enthalpy of the main thermotropic transition. (4) No evidence of a gramicidin A'-like lipid-structure modulating activity was found. However, tyrocidine inhibits the formation by gramicidin of an HII phase in dioleoylphosphatidylcholine model membranes. Instead, a cubic type of lipid organization is observed. (5) Tyrocidine greatly perturbs the barrier properties of dioleoylphosphatidylcholine model membrane. (6) Gramicidin A' reverses the effect of tyrocidine on membrane permeability by forming a complex in the model membrane with an apparent 1:1 stoichiometry. (7) The results suggest that both peptide antibiotics, which are produced by Bacillus brevis ATC 8185 prior to sporulation, show antagonism in their effect on membrane structure similar to their effect on superhelical DNA (Bogh, A. and Ristow, H. (1986) Eur. J. Biochem. 160, 587-591. The possible underlying basic mechanism is indicated.

Polymorphic phase behaviour of cardiolipin from bovine heart and from Bacillus subtilis as detected by 31P-NMR and freeze-fracture techniques. Effects of Ca2+, Mg2+, Ba2+ and temperature.

The structures formed by aqueous dispersions of cardiolipin isolated from bovine heart and B. subtilis have been studied by 31P-NMR and freeze-fracture electron microscopy. The sodium salts of both cardiolipins form bilayers. The Ca2+, Mg2+ and Ba2+ salts undergo well-defined bilayer leads to hexagonal (HII) transitions, the temperature of which is dependent on the cation involved and the fatty acid composition of the cardiolipin.

31P-NMR studies on membrane phospholipids in microsomes, rat liver slices and intact perfused rat liver.

1. The 36.4 and 81 MHz 31P-NMR spectra of isolated rat liver microsomes, rat liver slices and perfused rat liver have been recorded in the 4-40 degree C temperature range. 2. In isolated microsomes at 37 degrees C the majority of the phospholipids undergo isotropic motion, whereas at 4 degrees C most of the phospholipids give rise to typical 'bilayer' spectra. 3. Isolated hydrated rat liver microsomal phosphatidylethanolamine is organised in the hexagonal HII phase above 7 degrees C. 4. The Mn2+ permeability of the microsomal membrane is strongly temperature dependent. At 37 degrees C Mn2+ addition eliminates the entire 31P-NMR spectrum, demonstrating that all phospholipids interact with Mn2+. At 4 degrees C a 43% reduction in signal intensity is observed, indicating that at this temperature 43% of the phospholipids are located in the outer monolayer of a lipid bilayer. 5. In liver slices incubated in oxygenated Krebs-Ringer buffer at 4 degrees C there is a rapid decrease in ATP level such that within 20 min almost all ATP is degraded. In these ATP-depleted liver slices at 4 degrees C, virtually all phospholipids in all membranes have 31P-NMR spectra indicating bilayer structure. At 37 degrees C approx. 14% of the phospholipids undergo isotropic motion. 6. Preliminary experiments on perfused rat liver show stable ATP levels for 4 h at 37 degrees C. The spectra, furthermore, suggest similar behavior for the membrane phospholipids to that observed in the liver slices. 7. The possible sources of teh observed isotropic motion of the membrane phospholipids are discussed.

The action of pimaricin, etruscomycin and amphotericin B on liposomes with varying sterol content.

1. The effect of pimaricin, etruscomycin and amphotericin B on the K+ release from liposomes is strongly dependent on their sterol concentration. Pimaricin and etruscomycin induce K+ release from egg lecithin liposomes with cholesterol contents of more than 25 and 10 mol%, respectively, at polyene concentrations of 100 and 10 microgram/ml, respectively. Amphotericin B shows a maximal effect at a cholesterol content of 20 mol% at a concentration of 0.4 microgram/ml. 2. For liposomes containing ergosterol the sensitivity is shifted to a lower sterol content. All three polyenes show activity at 10 mol% ergosterol. The sensitivity for amphothericin B is increased approx. 15 times by the incorporation of ergosterol compared to cholesterol. The increase in sensitivity is much less for pimaricin and etruscomycin. The K+ release is maximal at an ergosterol concentration of 30 mol%. 3. Pimaricin, etruscomycin and amphotericin B can induce K+ release from erythrocytes without the release of haemoglobin at concentrations of 20, 2 and 1 microgram/ml, respectively. For these polyenes a selective permeability change is also demonstrated for liposomes since K+ is released but no [14C]dextran. Filipin shows a nonselective release of solutes from erythrocytes and liposomes. 4. At cholesterol concentrations higher than 20 mol% and ergosterol concentrations higher than 10 mol%, etruscomycin, pimaricin and amphotericin B show little dependence of the bilayer thickness and are able to release K+ from didocosenoyl phosphatidylcholine liposomes after addition of the polyene to one side of the membrane. A possible mechanism is discussed.

Effect of the phase transition on the transbilayer movement of dimyristoyl phosphatidylcholine in unilamellar vesicles.

Dimyristoyl phosphatidylcholine rapidly exchanges between vesicles at 37 degrees C without vesicle fusion. The rate of the transbilayer movement of dimyristoyl phosphatidylcholine in sonicated vesicles has been measured employing 13C NMR using N-13CH3-labeled lipids which are introduced into the outer monolayer of non-labeled vesicles by a phosphatidylcholine exchange protein. The rate of transbilayer movement of dimyristoyl phosphatidylcholine shows a distinct maximum (half-time 4 h) in the temperature range at which the hydrocarbon phase transition occurs. The activation energy of the flip-flop rate above the phase transition is 23.7 +/- 2.0 kcal/mol.

13 C NMR measurements of unsonicated phosphatidylcholine bilayers of different fatty acid and sterol composition.

(1) 13C NMR linewidths were measured for various 13 C resonances in unsonicated dispersions of synthetic and natural phosphatidylcholines both in the absence and presence of cholesterol at temperatures where the acyl chains are in the liquid-crystalline state. (2) In the absence of cholesterol the linewidths of the various resolved chain resonances were decreased with increasing unsaturation and decreasing chain length. The motion of the delta9-cis olefinic carbon atoms in dioleoylphosphatidylcholine was more restricted than the motion of the delts9-cis olefinic carbon atoms in 1-stearoyl-2-oleoyl-phosphatidylcholine despite the higher overall fluidity of dioleoylphosphatidylcholine. The polar head-group motion was not dependent upon the fatty acid composition. (3) Incorporation of cholesterol broadens all observed chain resonances of all phosphatidylcholines, thus demonstrating a reduction in chain motion by cholesterol. For both the saturated and unsaturated phosphatidylcholines the reduction of the chain motion is decreased with increasing chain length. (4) The chemical shift of the carbonyl resonances of sonicated dipalmitoylphosphatidylcholine vesicle labelled with 13C in both chains in the 1-position was slightly decreased by the incorporation of 50 mol% of cholesterol. In contrast, 50 mol% of epicholesterol, the 3alpha0OH isomer, produced a large downfield shift and a splitting of the carbonyl resonance.

Optimal posttranslational translocation of the precursor of PhoE protein across Escherichia coli membrane vesicles requires both ATP and the protonmotive force.

In order to reach their final destination, periplasmic and outer membrane proteins have to pass the cytoplasmic membrane of Escherichia coli cells. To study the transport of PhoE protein, we developed an in vitro transcription-translation and translocation system. In this in vitro system, the protein is synthesized as a larger precursor, which can be processed by purified leader peptidase. The precursor can be translocated into inverted inner membrane vesicles as judged by the protection against externally added protease. Only part of the translocated protein is in the processed mature form. Translocation can occur posttranslationally and requires both ATP and the protonmotive force for an optimal process. Upon incubation of vesicles with mature PhoE protein or precursor PhoE in the absence of ATP, the proteins are bound to the vesicles, but they are not translocated, since they are still sensitive to externally added protease.

Preferential lipid association and mode of penetration of apocytochrome c in mixed model membranes as monitored by tryptophanyl fluorescence quenching using brominated phospholipids.

The fluorescence of the single tryptophan residue at position 59 in apocytochrome c, the biosynthetic precursor of the inner mitochondrial membrane protein cytochrome c, was studied in small unilamellar vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with or without specifically Br-labelled acyl chains at the sn-2 position. The protein has a very high affinity for PS-containing vesicles (dissociation constant Kd less than 1 microM). From the relative quenching efficiency by the brominated phospholipids, it could be concluded that the protein specifically associates with the PS component in mixed vesicles and that maximal quenching occurred with phospholipids in which the bromine was present at the 6,7-position of the 2-acyl chain suggesting that (part of) the bound protein penetrates 7-8 A deep into the hydrophobic core of the bilayer.

Influence of heme and importance of the N-terminal part of the protein and physical state of model membranes for the apocytochrome c-lipid interaction.

The interaction between cytochrome c and its heme-free precursor apocytochrome c and chemically prepared fragments of these basic proteins with phosphatidylserine containing model membrane systems was studied by differential scanning calorimetry and carboxyfluorescein release experiments. Addition of apocytochrome c and fragments derived from the N-terminus cause a pronounced and linear decrease of the enthalpy (delta H) of the gel to liquid-crystalline phase transition of dielaidoylphosphatidylserine. In contrast, fragments derived from the C-terminus cause a smaller reduction in delta H; a similar trend was observed for the ability of the fragments to cause an increased carboxyfluorescein release from unilamellar vesicles. In addition, the covalent attachment of the heme at cysteine residues 14 and 17 greatly reduced the ability of both the intact protein and the N-terminal fragments to decrease delta H. Using a protein translocation assay based on large unilamellar vesicles containing enclosed trypsin it was found that at gel state temperatures the ability of apocytochrome c to partially translocate the bilayer (reach the opposite membrane/water interface) was greatly reduced. The implications of these findings for the import mechanism of apocytochrome c in mitochondria are shortly indicated.