Elmar J. Prenner

Differential scanning calorimetry and X-ray diffraction studies of the specificity of the interaction of antimicrobial peptides with membrane-mimetic systems.

Interest in biophysical studies on the interaction of antimicrobial peptides and lipids has strongly increased because of the rapid emergence of antibiotic-resistant bacterial strains. An understanding of the molecular mechanism(s) of membrane perturbation by these peptides will allow a design of novel peptide antibiotics as an alternative to conventional antibiotics. Differential scanning calorimetry and X-ray diffraction studies have yielded a wealth of quantitative information on the effects of antimicrobial peptides on membrane structure as well as on peptide location. These studies clearly demonstrated that antimicrobial peptides show preferential interaction with specific phospholipid classes. Furthermore, they revealed that in addition to charge-charge interactions, membrane curvature strain and hydrophobic mismatch between peptides and lipids are important parameters in determining the mechanism of membrane perturbation. Hence, depending on the molecular properties of both lipid and peptide, creation of bilayer defects such as phase separation or membrane thinning, pore formation, promotion of nonlamellar lipid structures or bilayer disruption by the carpet model or detergent-like action, may occur. Moreover, these studies suggest that these different processes may represent gradual steps of membrane perturbation. A better understanding of the mutual dependence of these parameters will help to elucidate the molecular mechanism of membrane damage by antimicrobial peptides and their target membrane specificity, keys for the rationale design of novel types of peptide antibiotics.

The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes

Gramicidin S (GS) is a cyclic decapeptide of primary structure [cyclo-(Val-Orn-Leu-D-Phe-Pro)(2)] secreted by Bacillus brevis. It is a powerful antimicrobial agent with potent cidal action on a wide variety of Gram-negative and Gram-positive bacteria as well as on several pathogenic fungi. Unfortunately, however, GS is rather non-specific in its actions and also exhibits a high hemolytic activity, limiting its use as an antibiotic to topical applications. In a wide variety of environments, the GS molecule exists as a very stable amphiphilic antiparallel beta-sheet structure with a polar and a non-polar surface. Moreover, the large number of structure-activity studies of GS analogs which have been carried out indicate that this sidedness structure is required for its antimicrobial action. In this review, we summarize both published and unpublished biophysical studies of the interactions of GS with lipid bilayer model and with biological membranes. In general, these studies show that GS partitions strongly into liquid-crystalline lipid bilayers in both model and biological membranes, and seems to be located primarily in the glycerol backbone region below the polar headgroups and above the hydrocarbon chains. The presence of GS appears to perturb lipid packing in liquid-crystalline bilayers and GS can induce the formation of inverted cubic phases at lower temperatures in lipids capable of forming such phases at higher temperature in the absence of peptide. The presence of GS at lower concentrations also increases the permeability of model and biological membranes and at higher concentrations causes membrane destabilization. There is good evidence from studies of the interaction of GS with bacterial cells that the destruction of the integrity of the lipid bilayer of the inner membrane is the primary mode of the antimicrobial action of this peptide. The considerable lipid specificity of GS for binding to and destabilization of lipid bilayer model membranes indicates that the design of GS analogs with an improved antimicrobial potency and a markedly decreased toxicity for eukaryotic cell plasma membranes should be possible.

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.

X-ray studies on the interaction of the antimicrobial peptide gramicidin S with microbial lipid extracts: evidence for cubic phase formation

We have investigated the effect of the interaction of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of model lipid bilayer membranes generated from the total membrane lipids of Acholeplasma laidlawii B and Escherichia coli. The A. laidlawii B membrane lipids consist primarily of neutral glycolipids and anionic phospholipids, while the E. coli inner membrane lipids consist exclusively of zwitterionic and anionic phospholipids. We show that the addition of GS at a lipid-to-peptide molar ratio of 25 strongly promotes the formation of bicontinuous inverted cubic phases in both of these lipid model membranes, predominantly of space group Pn3m. In addition, the presence of GS causes a thinning of the liquid-crystalline bilayer and a reduction in the lattice spacing of the inverted cubic phase which can form in the GS-free membrane lipid extracts at sufficiently high temperatures. This latter finding implies that GS potentiates the formation of an inverted cubic phase by increasing the negative curvature stress in the host lipid bilayer. This effect may be an important aspect of the permeabilization and eventual disruption of the lipid bilayer phase of biological membranes, which appears to be the mechanism by which GS kills bacterial cells and lysis erythrocytes.

Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes.

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.

Interaction of the antimicrobial peptide gramicidin S with dimyristoyl^phosphatidylcholine bilayer membranes: a densitometry and sound velocimetry study

We determined changes in the volume and adiabatic compressibility of large multi- and unilamellar vesicles composed of dimyristoylphosphatidylcholine containing various concentrations of the antimicrobial peptide gramicidin S (GS) by applying densitometry and sound velocimetry. Gramicidin S incorporation was found to progressively decrease the phase transition temperature of DMPC vesicles as well as to decrease the degree of cooperativity of the main phase transition and to increase the volume compressibility of the vesicles. GS probably enhanced thermal fluctuations at the region of main phase transition and provide more freedom of rotational movement for the phospholipid hydrocarbon chains. The ability of GS to increase the membrane compressibility and to decrease the phase transition temperature is evidence for regions of distorted membrane structure around incorporated gramicidin S molecules. At relatively high GS concentration (10 mol%), more significant changes of specific volume and compressibility appear. This might suggest changes in the integrity of the lipid bilayer upon interaction with high concentrations of GS.

Structure-activity relationships of the antimicrobial peptide gramicidin S and its analogs: aqueous solubility, self-association, conformation, antimicrobial activity and interaction with model lipid membranes.

GS10 [cyclo-(VKLdYPVKLdYP)] is a synthetic analog of the naturally occurring antimicrobial peptide gramicidin (GS) in which the two positively charged ornithine (Orn) residues are replaced by two positively charged lysine (Lys) residues and the two less polar aromatic phenylalanine (Phe) residues are replaced by the more polar tyrosine (Tyr) residues. In this study, we examine the effects of these seemingly conservative modifications to the parent GS molecule on the physical properties of the peptide, and on its interactions with lipid bilayer model and biological membranes, by a variety of biophysical techniques. We show that although GS10 retains the largely β-sheet conformation characteristic of GS, it is less structured in both water and membrane-mimetic solvents. GS10 is also more water soluble and less hydrophobic than GS, as predicted, and also exhibits a reduced tendency for self-association in aqueous solution. Surprisingly, GS10 associates more strongly with zwitterionic and anionic phospholipid bilayer model membranes than does GS, despite its greater water solubility, and the presence of anionic phospholipids and cholesterol (Chol) modestly reduces the association of both GS10 and GS to these model membranes. The strong partitioning of both peptides into lipid bilayers is driven by a large favorable entropy change opposed by a much smaller unfavorable enthalpy change. However, GS10 is also less potent than GS at inducing inverted cubic phases in phospholipid bilayer model membranes and at inhibiting the growth of the cell wall-less bacterium Acholeplasma laidlawii B. These results are discussed in terms of the comparative antibiotic and hemolytic activities of these peptides.

Interaction of gramicidin S and its biologically active analogs with phospholipid bilayers

Gramicidin S (GS) is a cyclic decameric peptide, which is biologically active as a potent antibiotic against a wide range of bacteria and fungi [1]. GS is also very hemolytic against human erythrocytes. Interaction of GS with lipid bilayers of cell membranes is believed to play a major role in its biological activity. In order to develop insight into the mechanism of interaction of GS and GS-like biologically active peptides with lipid bilayers, we have utilized a series of GS analogs with distinct structural and functional features in a comparative study.