Molecular mechanism of membrane permeabilization by the peptide antibiotic surfactin

Surfactin, an acidic lipopeptide produced by various strains of Bacillus subtilis, behaves as a very powerful biosurfactant and possesses several other interesting biological activities. This work deals with the molecular mechanism of membrane permeabilization by incorporation of surfactin. The surfactin-induced vesicle contents leakage was monitored by following release of carboxyfluorescein entrapped into unilamellar vesicles made of palmitoyloleoylphosphatidylcholine (POPC). The effect of the addition of cholesterol, dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylethanolamine (POPE) was also checked. It was observed that surfactin was able to induce content leakage at concentrations far below the onset surfactin/lipid ratio for membrane solubilization to occur, which in our system was around 0.92. Electron microscopy showed that vesicles were present after addition of surfactin at a ratio below this value, whereas no vesicles could be observed at ratios above it. Cholesterol and POPE attenuated the membrane-perturbing effect of surfactin, whereas the effect of DPPC was to promote surfactin-induced leakage, indicating that bilayer sensitivity to surfactin increases with the lipid tendency to form lamellar phases, which is in agreement with our previous observation that surfactin destabilizes the inverted-hexagonal structure. Fourier-transform infrared spectroscopy (FTIR) was used to specifically follow the effect of surfactin on different parts of the phospholipid bilayer. The effect on the C=O stretching mode of vibration of POPC indicated a strong dehydration induced by surfactin. On the other hand, the C-H stretching bands showed that the lipopeptide interacts with the phospholipid acyl chains, resulting in considerable membrane fluidization. The reported effects could be useful to explain surfactin-induced 'pore' formation underlying the antibiotic and other important biological actions of this bacterial lipopeptide.     

Leakage and lysis of lipid membranes induced by the lipopeptide surfactin

Surfactin is a lipopeptide produced by Bacillus subtilis which possesses antimicrobial activity. We have studied the leakage and lysis of POPC vesicles induced by surfactin using calcein fluorescence de-quenching, isothermal titration calorimetry and ³¹P solid state NMR. Membrane leakage starts at a surfactin-to-lipid ratio in the membrane, R _b ≈ 0.05, and an aqueous surfactin concentration of C _S^w ≈ 2 μM. The transient, graded nature of leakage and the apparent coupling with surfactin translocation to the inner leaflet of the vesicles, suggests that this low-concentration effect is due to a bilayer-couple mechanism. Different permeabilization behaviour is found at R _b ≈ 0.15 and attributed to surfactin-rich clusters, which can induce leaks and stabilize them by covering their hydrophobic edges. Membrane lysis or solubilization to micellar structures starts at R _b^sat = 0.22 and C _S^w = 9 μM and is completed at R _m^sol = 0.43 and C _S^w = 11 μM. The membrane–water partition coefficient of surfactin is obtained as K = 2 × 10⁴ M⁻¹. These data resolve inconsistencies in the literature and shed light on the variety of effects often referred to as detergent-like effects of antibiotic peptides on membranes. The results are compared with published parameters characterizing the hemolytic and antibacterial activity. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Ionic channels induced by surfactin in planar lipid bilayer membranes.

Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion-conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process.     

Detergent-like effects of alamethicin on lymphocyte plasma membranes.

Alamethicin enhanced adenylate cyclase and Na⁺-K⁺-ATPase activities in microsomes and purified plasma membranes from pig lymphocytes. As this stimulation was also found in inside-out vesicles obtained from these membranes and as we showed that lymphocyte membrane vesicles are not impermeable to 5′AMP, ATP and concanavalin A, it appears clearly that alamethicin effects are not related to its channel-forming properties, but rather to its detergent-like character. Indeed sodium dodecylsulfate and Lubrol PX mimicked alamethicin effects. Moreover alamethicin treatment of plasma membranes induced protein and phospholipid solubilization.     

Tylopeptin B peptide antibiotic in lipid membranes at low concentrations: Self-assembling,mutual repulsion and localization

The medium-length peptide Tylopeptin B possesses activity against Gram-positive bacteria. It binds to bacterial membranes altering their mechanical properties and increasing their permeability. This action is commonly related with peptide self-assembling, resulting in the formation of membrane channels. Here, pulsed double electron-electron resonance (DEER) data for spin-labeled Tylopeptin B in palmitoyl-oleoyl-glycero-phosphocholine (POPC) model membrane reveal that peptide self-assembling starts at concentration as low as 0.1 mol%; above 0.2 mol% it attains a saturation-like dependence with a mean number of peptides in the cluster <n> = 3.3. Using the electron spin echo envelope modulation (ESEEM) technique, Tylopeptin B molecules are found to possess a planar orientation in the membrane. In the peptide concentration range between 0.1 and 0.2 mol%, DEER data show that the peptide clusters have tendency of mutual repulsion, with a circle of inaccessibility of radius around 20 nm. It may be proposed that within this radius the peptides destabilize the membrane, providing so the peptide antimicrobial activity. Exploiting spin-labeled stearic acids as a model for free fatty acids (FFA), we found that at concentrations of 0.1–0.2 mol% the peptide promotes formation of lipid-mediated FFA clusters; further increase in peptide concentration results in dissipation of these clusters.     

Structural rearrangement of model membranes by the peptide antibiotic NK-2

We have developed a novel alpha-helical peptide antibiotic termed NK-2. It efficiently kills bacteria, but not human cells, by membrane destruction. This selectivity could be attributed to the different membrane lipid compositions of the target cells. To understand the mechanisms of selectivity and membrane destruction, we investigated the influence of NK-2 on the supramolecular aggregate structure, the phase transition behavior, the acyl chain fluidity, and the surface charges of phospholipids representative for the bacterial and the human cell cytoplasmic membranes. The cationic NK-2 binds to anionic phosphatidylglycerol liposomes, causing a thinning of the membrane and an increase in the phase transition temperature. However, this interaction is not solely of electrostatic but also of hydrophobic nature, indicated by an overcompensation of the Zeta potential. Whereas NK-2 has no effect on phosphatidylcholine liposomes, it enhances the fluidity of phosphatidylethanolamine acyl chains and lowers the phase transition enthalpy of the gel to liquid cristalline transition. The most dramatic effect, however, was observed for the lamellar/inverted hexagonal transition of phosphatidylethanolamine which was reduced by more than 10 degrees C. Thus, NK-2 promotes a negative membrane curvature which can lead to the collapse of the phosphatidylethanolamine-rich bacterial cytoplasmic membrane.     

Selectivity of cation transport across lipid membranes by the antibiotic salinomycin

The ionophoric antibiotic salinomycin is in the phase of preclinical tests against several types of malignant tumors including breast cancer. Notwithstanding, the data on its ion selectivity, although being critical for its therapeutic activity, are rather scarce. In the present work, we studied the ability of salinomycin to exert cation/H⁺-exchange across artificial bilayer lipid membranes (BLM) by measuring electrical potential on planar BLM in the presence of a protonophore and fluorescence responses of the pH-sensitive dye pyranine entrapped in liposomes. The following order of ion selectivity was obtained by these two methods: K⁺ > Na⁺ > Rb⁺ > Cs⁺ > Li⁺. Measurements of the monovalent cation-induced quenching of fluorescence of thallium ions in methanol showed that salinomycin effectively binds potassium and calcium but poorly binds sodium and lithium ions. At high concentrations, salinomycin transports Ca²⁺ through membranes of liposomes and mitochondria, as measured by using the calcium-sensitive dye Fluo-5 N. The data obtained can be used in the mechanistic studies of the anti-tumor activity of salinomycin and its selective cytotoxicity towards cancer stem cells.     

Structural rearrangement of model membranes by the peptide antibiotic NK-2

We have developed a novel α-helical peptide antibiotic termed NK-2. It efficiently kills bacteria, but not human cells, by membrane destruction. This selectivity could be attributed to the different membrane lipid compositions of the target cells. To understand the mechanisms of selectivity and membrane destruction, we investigated the influence of NK-2 on the supramolecular aggregate structure, the phase transition behavior, the acyl chain fluidity, and the surface charges of phospholipids representative for the bacterial and the human cell cytoplasmic membranes. The cationic NK-2 binds to anionic phosphatidylglycerol liposomes, causing a thinning of the membrane and an increase in the phase transition temperature. However, this interaction is not solely of electrostatic but also of hydrophobic nature, indicated by an overcompensation of the Zeta potential. Whereas NK-2 has no effect on phosphatidylcholine liposomes, it enhances the fluidity of phosphatidylethanolamine acyl chains and lowers the phase transition enthalpy of the gel to liquid cristalline transition. The most dramatic effect, however, was observed for the lamellar/inverted hexagonal transition of phosphatidylethanolamine which was reduced by more than 10 °C. Thus, NK-2 promotes a negative membrane curvature which can lead to the collapse of the phosphatidylethanolamine-rich bacterial cytoplasmic membrane.     

Effect of peptide PV on the ionic permeability of lipid bilayer membranes

This paper reports the effects of peptide PV (primary structure: cyclo-(D-val-L-pro-L-val-D-pro)_δ) on the electrical properties of sheep red cell lipid bilayers. The membrane conductance (G_m) induced by PV in either Na⁺ or K⁺ medium is proportional to the concentration of PV in the aqueous phase. The PV concentration required to produce a comparable increase in G_m in K⁺ medium is about 10⁴ times greater than for its analogue, valinomycin (val). Although the selectivity sequence for PV and val is similar, K⁺ ≳ Rb⁺ > Cs⁺ > NH₄ ⁺ > TI⁺ > Na⁺ > Li⁺; the ratio of GGm in K⁺ to that in Na⁺ is about 10 for PV compared to > 10³ for val. When equal concentrations of PV are added to both sides of a bilayer, the membrane current approaches a maximum value independent of voltage when the membrane potential exceeds 100 mV. When PV is added to only one side of a bilayer separating identical salt solutions of either Na⁺ or K⁺ salts, rectification occurs such that the positive current flows more easily away rather than toward the side containing the carrier. Under these conditions, a large, stable, zero-current potential (VVm) is also observed, with the side containing PV being negative. The magnitude of this VVm is about 90 mV and relatively independent of PV concentration when the latter is larger than 2 Times; 10^–5 M. From a model which assumes that V_m equals the equilibrium potential for the PV-cation complexes (MS ⁺) and that the reaction between PV and cations is at equilibrium on the two membrane surfaces, we compute the permeability of the membrane to free PV to be about 10^–5 cm s^–1, which is about 10^–7 times the permeability of similar membranes to free val. This interpretation is supported by the fact that the observed values of V_m are in agreement with the calculated equilibrium potential for MS⁺ over a wide range of ratios of concentrations of total PV in the two bathing solutions, if the unstirred layers are taken into account in computing the MS⁺ concentrations at the membrane surfaces.     

Charge-dependent translocation of the Trojan peptide penetratin across lipid membranes

We studied the interaction of the cell-penetrating peptide penetratin with mixed dioleoylphosphatidylcholine/dioleoylphoshatidylglycerol (DOPC/DOPG) unilamellar vesicles as a function of the molar fraction of anionic lipid, X(PG), by means of isothermal titration calorimetry. The work was aimed at getting a better understanding of factors that affect the peptide binding to lipid membranes and its permeation through the bilayer. The binding was well described by a surface partitioning equilibrium using an effective charge of the peptide of z(P) approximately 5.1 +/- 0.5. The peptide first binds to the outer surface of the vesicles, the effective binding capacity of which increases with X(PG). At X(PG) approximately 0.5 and a molar ratio of bound peptide-to-lipid of approximately 1/20 the membranes become permeable and penetratin binds also to the inner monolayer after internalization. The results were rationalized in terms of an "electroporation-like" mechanism, according to which the asymmetrical distribution of the peptide between the outer and inner surfaces of the charged bilayer causes a transmembrane electrical field, which alters the lateral and the curvature stress acting within the membrane. At a threshold value these effects induce internalization of penetratin presumably via inversely curved transient structures.     

Detergent-like action of tetraphenylborate on phospholipid labelling in guinea pig cortex subfractions

—The addition of 2 × 10^?4m sodium tetraphenylborate to particulate preparations of guinea pig cerebral cortex increases labelling of phosphatidic acid from γ-[³²P]-ATP two- to four-fold. The effect ‘was observed in all subcellular fractions studied (nuclear, synaptosomal, mitochondrial and microsomal) and is not blocked by the addition of atropine. Changes in phospholipid labelling similar to those induced by tetraphenylborate can be demonstrated with sodium dodecyl sulphate or sodium desoxycholate. It is suggested that tetraphenylborate stimulates lipid labelling by a detergent-like activation of diglyceride kinase.     

Opioid peptide interactions with lipid bilayer membranes.

The interaction of the delta-opioid receptor selective peptides, cyclic [D-Pen2, D-Pen5]-enkephalin [DPDPE] and its acyclic analog, DPDPE(SH)2, with neutral phospholipid bilayer membranes was examined by permeability and calorimetry measurements. The permeabilities were accomplished by entrapping either peptide inside of unilamellar liposomes (composed of a mixture of a molar ratio 65:25:10 phosphatidylcholine/phosphatidylethanolamine/cholesterol) then monitoring the peptide efflux through the bilayer. The initial permeability of DPDPE (first 12 h) averaged over four experiments was (0.91 +/- 0.47).10(-12) cm s-1. In contrast the average permeability of the acylic DPDPE(SH)2 was (4.26 +/- 0.23).10(-12) cm s-1. The effect of these peptides on the phase transition, Tm, of 1,2-dipalmitoylphosphatidylcholine (DPPC) bilayers was examined by high sensitivity differential scanning calorimetry. The Tm, the calorimetric enthalpy, and the van 't Hoff enthalpy of DPPC were not significantly altered by the presence of DPDPE, whereas the calorimetric data for DPPC with DPDPE(SH)2 showed a small, yet significant, increase (0.2 degrees C) in the Tm with a 30% decrease in the cooperative unit. Both the permeability and calorimetry data reveal a stronger peptide-membrane interaction in the case of the more flexible acyclic peptide.     

The effect of peptide/lipid hydrophobic mismatch on the phase behavior of model membranes mimicking the lipid composition in Escherichia coli membranes

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by (31)P NMR spectroscopy. The peptides (WALPn peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at approximately 60 degrees C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by approximately 15 degrees C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H(II)) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.     

The action of gramicidin S on the ionic permeability of bilayer lipid membranes

The effect of cyclic decapeptide of gramicidin S on electrical conductivity of bilayer lipid membranes has been studied. The integral conductivity of bilayer has been shown to increase with the growth of antibiotic concentration. The integral conductivity increase occurs as series of conductivity discrete leaps, differing in amplitude from fluctuations of conductivity caused by linear gramicidins. In the series of selectivity of bilayer membranes for cations of alkaline metals the rubidium ion is before the cesium ion. This is the only difference between this series and the series of relative ionic mobility series of cations of alkaline metals in water solutions.     

Interaction of an artificial antimicrobial peptide with lipid membranes

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.     

Production of a lipopeptide antibiotic surfactin with recombinantBacillus subtilis

Summary Production of a lipopeptide antibiotic surfactin was carried out using a recombinantBacillus subtilis. Surfactin yield of the recombinant strain was about one and half times as much as that ofBacillus subtilis RB 14, the strain in which the surfactin gene was originated. This system is especially noteworthy because a recombinant strain surpassed the original strain in the production of a bacterial antibiotic as a secondary metabolite of the bacterium.     

Effects of surfactin on membrane models displaying lipid phase separation

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.     

Effect of changing the size of lipid headgroup on peptide insertion into membranes.

Adsorption of amphiphilic peptides to the headgroup region of a lipid bilayer is a common mode of protein-membrane interactions. Previous studies have shown that adsorption causes membrane thinning. The degree of the thinning depends on the degree of the lateral expansion caused by the peptide adsorption. If this simple molecular mechanism is correct, the degree of lateral expansion and consequently the membrane thinning should depend on the size of the headgroup relative to the cross section of the hydrocarbon chains. Previously we have established the connection between the alamethicin insertion transition and the membrane thinning effect. In this paper we use oriented circular dichroism to study the effect of varying the size of the headgroup, while maintaining a constant cross section of the lipid chains, on the insertion transition. A simple quantitative prediction agrees very well with the experiment.