Structure-Activity Relationship of a U-Type Antimicrobial Microemulsion System

The structure-activity relationship of a U-type antimicrobial microemulsion system containing glycerol monolaurate and ethanol at a 1∶1 mass ratio as oil phase and Tween 20 as surfactant were investigated along a water dilution line at a ratio of 80∶20 mass% surfactant/oil phase, based on a pseudo-ternary phase diagram. The differential scanning calorimetry results showed that in the region of up to 33% water, all water molecules are confined to the hydrophilic core of the reverse micelles, leading to the formation of w/o microemulsion. As the water content increases, the water gains mobility, and transforms into bicontinuous in the region of 33–39% water, and finally the microemulsion become o/w in the region of above 39% water. The microstructure characterization was confirmed by the dynamic light scattering measurements and freeze-fracture transmission electron microscope observation. The antimicrobial activity assay using kinetics of killing analysis demonstrated that the microemulsions in w/o regions exhibited relatively high antimicrobial activity against Escherichia coli and Staphylococcus aureus due to the antimicrobial oil phase as the continuous phase, while the antimicrobial activity started to decrease when the microemulsions entered the bicontinuous region, and decreased rapidly as the water content increased in the o/w region, as a result of the dilution of antimicrobial oil droplets in the aqueous continuous phase.     

Bilayer localization of membrane-active peptides studied in biomimetic vesicles by visible and fluorescence spectroscopies.

Depth of bilayer penetration and effects on lipid mobility conferred by the membrane-active peptides magainin, melittin, and a hydrophobic helical sequence KKA(LA)7KK (denoted KAL), were investigated by colorimetric and time-resolved fluorescence techniques in biomimetic phospholipid/poly(diacetylene) vesicles. The experiments demonstrated that the extent of bilayer permeation and peptide localization within the membrane was dependent upon the bilayer composition, and that distinct dynamic modifications were induced by each peptide within the head-group environment of the phospholipids. Solvent relaxation, fluorescence correlation spectroscopy and fluorescence quenching analyses, employing probes at different locations within the bilayer, showed that magainin and melittin inserted close to the glycerol residues in bilayers incorporating negatively charged phospholipids, but predominant association at the lipid-water interface occurred in bilayers containing zwitterionic phospholipids. The fluorescence and colorimetric analyses also exposed the different permeation properties and distinct dynamic influence of the peptides: magainin exhibited the most pronounced interfacial attachment onto the vesicles, melittin penetrated more into the bilayers, while the KAL peptide inserted deepest into the hydrophobic core of the lipid assemblies. The solvent relaxation results suggest that decreasing the lipid fluidity might be an important initial factor contributing to the membrane activity of antimicrobial peptides.     

Formulation and Characterization of Phytophenol-Carrying Antimicrobial Microemulsions

Phytophenols were solubilized in nonionic surfactant micelles to form antimicrobially active and thermodynamically stable microemulsions. Formulation of phytophenols in microemulsions has previously been shown to improve their antimicrobial activity in model microbiological and food systems. Carvacrol and eugenol were incorporated in micellar solutions of two nonionic surfactants (Surfynol® 485W and Surfynol® 465) by mixing at room temperature. Particle size of formed microemulsions was determined by dynamic light scattering, and structural information about the mixed micellar system was obtained by nuclear magnetic resonance spectroscopy (NMR). Uptake of carvacrol and eugenol in surfactant micelles as determined by ultrasonic velocity measurements was very rapid, e.g., below the maximum additive concentration, the phytophenols were completely solubilized in the micelles in less than 30 min. Depending on the surfactant–phytophenol combination, the self-assembled surfactant–phytophenol aggregates had mean particle diameters between 3 and 17 nm. Elucidation of the structure of aggregates by 1H NMR studies indicated that micelles had a “bracket-like” structure with phytophenols being located inside the palisade layer of the micelle in direct contact with adjacent surfactant monomers. Encapsulation of phytophenols in surfactant micelles enables the incorporation of large amounts of hydrophobic antimicrobials in aqueous phases. Formulation of antimicrobial microemulsions may thus offer a means to deliver high concentrations of phytophenols to the bacterial surfaces of foodborne pathogens to affect kill.     

Human gastric lipase. A kinetic study with dicaprin monolayers

The effects of several proteins on the hydrolysis at pH 3.0 of didecanoylglycerol monolayers by human gastric lipase were investigated. Among the six proteins tested (bovine serum albumin, myoglobin, a protein inhibiting lipase isolated from soya bean, melittin, beta-lactoglobulin and ovalbumin), only the first three proteins were found to inhibit lipase activity. The inhibition capacity of the proteins was not related to the decrease in interfacial tension or to their isoelectric points. However, inhibition of human gastric lipase by proteins may be correlated with the penetration power of the protein into the lipid interface. It is hypothesized that this lipase has a higher penetration power than that of pancreatic lipase, even though the former enzyme is more susceptible to interfacial denaturation.     

Effect of ionic strength on the organization and dynamics of membrane-bound melittin

Melittin, a cationic hemolytic peptide, is intrinsically fluorescent due to the presence of a single functionally important tryptophan residue. We have previously shown that the sole tryptophan of melittin is localized in a motionally restricted environment in the membrane interface. We have monitored the effect of ionic strength on the organization and dynamics of membrane-bound melittin utilizing fluorescence and circular dichroism (CD) spectroscopic approaches. Our results show that red edge excitation shift (REES) of melittin bound to membranes is sensitive to the change in ionic strength of the medium. This could be attributed to a change in the immediate environment around melittin tryptophan with increasing ionic strength due to differential solvation of ions. Interestingly, the rotational mobility of melittin does not appear to be affected with change in ionic strength. In addition, fluorescence parameters such as lifetime and acrylamide quenching of melittin indicate an increase in water penetration in the membrane interface upon increasing ionic strength. Our results suggest that the solvent dynamics and water penetration in the interfacial region of the membranes are significantly affected at physiologically relevant ionic strength. These results assume significance in the overall context of the influence of ionic strength in the organization and dynamics of membrane proteins and membrane-active peptides.     

The monolayer technique: a potent tool for studying the interfacial properties of antimicrobial and membrane-lytic peptides and their interactions with lipid membranes

Erudites of the antiquity already knew the calming effect of oil films on the sea waves. But one had to wait until 1774 to read the first scientific report on oil films from B. Franklin and again 1878 to learn the thermodynamic analysis on adsorption developed by J. Gibbs. Then, in 1891, Agnes Pockels described a technique to manipulate oil films by using barriers. Finally, in 1917, I. Langmuir introduced the experimental and theoretical modern concepts on insoluble monolayers. Since that time, and because it has been found to provide invaluable information at the molecular scale, the monolayer technique has been more and more extensively used, and, during the past decade, an explosive increase in the number of publications has occurred. Over the same period, considerable and ever-increasing interest in the antimicrobial peptides of various plants, bacteria, insects, amphibians and mammals has grown. Because many of these antimicrobial peptides act at the cell membrane level, the monolayer technique is entirely suitable for studying their physicochemical and biological properties. This review describes monolayer experiments performed with some of these antimicrobial peptides, especially gramicidin A, melittin, cardiotoxins and defensin A. After giving a few basic notions of surface chemistry, the surface-active properties of these peptides and their behavior when they are arranged in monomolecular films are reported and discussed in relation to their tridimensional structure and their amphipathic character. The penetration of these antimicrobial peptides into phospholipid monolayer model membranes, as well as their interactions with lipids in mixed films, are also emphasized.     

Organizations of melittin peptides after spontaneous penetration into cell membranes

The antimicrobial peptide, melittin, is a potential next-generation antibiotic because melittin can spontaneously form pores in bacterial cell membranes and cause cytoplasm leakage. However, the organizations of melittin peptides in cell membranes remain elusive, which impedes the understanding of the poration mechanism. In this work, we use coarse-grained and all-atom molecular dynamics (MD) simulations to investigate the organizations of melittin peptides during and after spontaneous penetration into DPPC/POPG lipid bilayers. We find that the peptides in lipid bilayers adopt either a transmembrane conformation or a U-shaped conformation, which are referred to as T- and U-peptides, respectively. Several U-peptides and/or T-peptides aggregate to form stable pores. We analyze a T-pore consisting of four T-peptides and a U-pore consisting of three U-peptides and one T-peptide. In both pores, peptides are organized in a manner such that polar residues face inward and hydrophobic residues face outward, which stabilizes the pores and produces water channels. Compared with the U-pore, the T-pore has lower energy, larger pore diameter, and higher permeability. However, the T-pore occurs less frequently than the U-pore in our simulations, probably because the formation of the T-pore is kinetically slower than the U-pore. The stability and permeability of both pores are confirmed by 300 ns all-atom MD simulations. The peptide organizations obtained in this work should deepen the understanding of the stability, poration mechanism, and permeability of melittin, and facilitate the optimization of melittin to enhance the antibacterial ability.     

Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides

Antimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response of Escherichia coli upon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σ^E envelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S₄ dermaseptin, also activate several E. coli envelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes to E. coli tolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show that E. coli senses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway to E. coli tolerance to antimicrobial peptides.     

Cooperative antimicrobial action of melittin on lipid membranes: A coarse-grained molecular dynamics study

We conducted a series of coarse-grained molecular dynamics (CG-MD) simulations to investigate the complicated actions of melittin, which is an antimicrobial peptide (AMP) derived from honey bee venom, on a lipid membrane. To accurately simulate the AMP action, we developed and used a protein CG model as an extension of the pSPICA force field (FF), which was designed to reproduce several thermodynamic quantities and structural properties. At a low peptide-to-lipid (P/L) ratio (1/102), no defect was detected. At P/L = 1/51, toroidal pore formation was observed due to collective insertion of multiple melittin peptides from the N-termini. The pore formation was initiated by a local increase in membrane curvature in the vicinity of the peptide aggregate. At a higher P/L ratio (1/26), two more modes were detected, seemingly not controlled by the P/L ratio but by a local arrangement of melittin peptides: 1. Pore formation accompanied by lipid extraction by melittin peptides:a detergent-like mechanism. 2. A rapidly formed large pore in a significantly curved membrane: bursting. Thus, we observed three pore formation modes (toroidal pore formation, lipid extraction, and bursting) depending on the peptide concentration and local arrangement. These observations were consistent with experimental observations and hypothesized melittin modes. Through this study, we found that the local arrangements and population of melittin peptides and the area expansion rate by membrane deformation were key to the initiation of and competition among the multiple pore formation mechanisms.     

Oxidized phospholipids as potential molecular targets for antimicrobial peptides

The effects of oxidatively modified phospholipids on the association with model biomembranes of four antimicrobial peptides (AMPs), temporin B and L, indolicidin, and LL-37(F27W) were studied by Langmuir balance and fluorescence spectroscopy. In keeping with previous reports the negatively charged phospholipid phosphatidylglycerol (PG) enhanced the intercalation of all four peptides into lipid monolayers and liposomal bilayers under low ionic strength conditions. Interestingly, similar effect was observed for 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde function at the end of its truncated sn-2 acyl chain. Instead, the structurally similar 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) containing a carboxylic moiety was less efficient in promoting the membrane association of these peptides. Physiological saline reduced the binding of the above peptides to membranes containing PG, whereas interactions with PoxnoPC were found to be insensitive to ionic strength. Notably, membrane intercalation of temporin L, the most surface active of the above peptides could be into PoxnoPC containing monolayers was strongly attenuated by methoxyamine, suggesting the importance of Schiff base formation between peptide amino groups and the lipid aldehyde function. PoxnoPC and similar aldehyde bearing oxidatively modified phospholipids could represent novel molecular targets for AMPs.     

Parameters modulating the maximum insertion pressure of proteins and peptides in lipid monolayers

The lipid monolayer model membrane is useful for studying the parameters responsible for protein and peptide membrane binding. Different approaches have been used to determine the extent of protein and peptide binding to lipid monolayers. This review focuses on the use of the “maximum insertion pressure” (MIP) to estimate the extent of protein and peptide penetration in lipid monolayers. The MIP data obtained with different proteins and peptides have been reviewed and discussed which allowed to draw conclusions on the parameters modulating the monolayer binding of proteins and peptides. In particular, secondary structure components such as amphipathic α-helices of proteins and peptides as well as electrostatic interactions play important roles in monolayer binding. The MIPs have been compared to the estimated lateral pressure of biomembranes which allowed to evaluate the possible association between proteins or peptides with natural membranes. For example, the MIP of a membrane-anchored protein with a glycosylphosphatidylinositol (GPI) was found to be far below the estimated lateral pressure of biomembranes. This allowed us to conclude that this protein is probably unable to penetrate the membrane and should thus be hanged at the membrane surface by use of its GPI lipid anchor. Moreover, the values of MIP obtained with myristoylated and non-myristoylated forms of calcineurin suggest that the myristoyl group does not contribute to monolayer binding. However, the acylation of a peptide resulted in a large increase of MIP. Finally, the physical state of lipid monolayers can have a strong effect on the values of MIP such that it is preferable to perform measurements with lipids showing a single physical state. Altogether the data show that the measurement of the maximum insertion pressure provides very useful information on the membrane binding properties of proteins and peptides although uncertainties must be provided to make sure the observed differences are significant.     

Spontaneous formation of interfacial lipid-protein monolayers during adsorption from vesicles.

Spread and adsorbed monolayers of lipid-protein mixtures have served as models for biomembranes and pulmonary surfactant, but their similarity was unclear. Epifluorescence microscopy of monolayers spontaneously adsorbed from vesicles of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylcholine plus surfactant protein C (SP-C) showed gas, liquid expanded, and liquid condensed (LC) domains. The shapes and distribution of LC domains in the adsorbed and solvent-spread monolayers were quite similar. Labeled SP-C adsorbed into the air-water interface in the company of the lipids. In both forms of monolayers, SP-C occupied the fluid phase and reduced the size and amount of the LC domains. The properties suggest that these adsorbed and spread monolayers are analogous to one another.     

Free energy analysis of membrane pore formation process in the presence of multiple melittin peptides

Understanding the molecular mechanism underlying pore formation in lipid membranes by antimicrobial peptides is of great importance in biological sciences as well as in drug design applications. Melittin has been widely studied as a pore forming peptide, though the molecular mechanism for pore formation is still illusive. We examined the free energy barrier for the creation of a pore in lipid membranes with and without multiple melittin peptides. It was found that six melittin peptides significantly stabilized a pore, though a small barrier (a few k_BT) for the formation still existed. With five melittin peptides or fewer, the pore formation barrier was much higher, though the established pore was in a local energy minimum. Although seven melittins effectively reduced the free energy barrier, a single melittin peptide left the pore after a long time MD simulation probably because of the overcrowded environment around the bilayer pore. Thus, it is highly selective for the number of melittin peptides to stabilize the membrane pore, as was also suggested by the line tension evaluations. The free energy cost required to insert a single melittin into the membrane is too high to explain the one-by-one insertion mechanism for pore formation, which also supports the collective melittin mechanism for pore formation.     

In situ study by polarization modulated Fourier transform infrared spectroscopy of the structure and orientation of lipids and amphipathic peptides at the air-water interface.

Free amphipathic peptides and peptides bound to dimyristoylphosphatidylcholine (DMPC) were studied directly at the air/water interface using polarization modulation infrared reflection absorption spectroscopy (PMIRRAS). Such differential reflectivity measurements proved to be a sensitive and efficient technique to investigate in situ the respective conformations and orientations of lipid and peptide molecules in pure and mixed films. Data obtained for melittin, a natural hemolytic peptide, are compared to those of L15K7, an ideally amphipathic synthetic peptide constituted by only apolar Leu and polar Lys residues. For pure peptidic films, the intensity, shape, and position of the amide I and II bands indicate that the L15K7 peptide adopts a totally alpha-helical structure, whereas the structure of melittin is mainly alpha-helical and presents some unordered domains. The L15K7 alpha-helix axis is oriented essentially parallel to the air-water interface plane; it differs for melittin. When injected into the subphase, L15K7 and melittin insert into preformed expanded DMPC monolayers and can be detected by PMIRRAS, even at low peptide content (> 50 DMPC molecules per peptide). In such conditions, peptides have the same secondary structure and orientation as in pure peptidic films.     

Interaction of cationic antimicrobial peptides with model membranes

A series of natural and synthetic cationic antimicrobial peptides from various structural classes, including alpha-helical, beta-sheet, extended, and cyclic, were examined for their ability to interact with model membranes, assessing penetration of phospholipid monolayers and induction of lipid flip-flop, membrane leakiness, and peptide translocation across the bilayer of large unilamellar liposomes, at a range of peptide/lipid ratios. All peptides were able to penetrate into monolayers made with negatively charged phospholipids, but only two interacted weakly with neutral lipids. Peptide-mediated lipid flip-flop generally occurred at peptide concentrations that were 3- to 5-fold lower than those causing leakage of calcein across the membrane, regardless of peptide structure. With the exception of two alpha-helical peptides V681(n) and V25(p,) the extent of peptide-induced calcein release from large unilamellar liposomes was generally low at peptide/lipid molar ratios below 1:50. Peptide translocation across bilayers was found to be higher for the beta-sheet peptide polyphemusin, intermediate for alpha-helical peptides, and low for extended peptides. Overall, whereas all studied cationic antimicrobial peptides interacted with membranes, they were quite heterogeneous in their impact on these membranes.     

Effects of palmitoylation on dynamics and phospholipid-bilayer-perturbing properties of the N-terminal segment of pulmonary surfactant protein SP-C as shown by 2H-NMR

It has been proposed that palmitoylation of the N-terminal segment of surfactant protein SP-C is important for maintaining association of pulmonary surfactant complexes with interfacial films compressed to high pressures at the end of expiration. In this study, we examined surfactant membrane models containing palmitoylated and nonpalmitoylated synthetic peptides, based on the N-terminal SP-C sequence, in dipalmitoylphosphatidylcholine (DPPC)/egg phosphatidylglycerol (7:3, w/w) by ²H-NMR. Perturbations of lipid properties by the peptide versions were compared in samples containing chain- and headgroup-deuterated lipid (DPPC-d₆₂ and DPPC-d₄ respectively). Also, deuterated peptide palmitate chains were compared with those of DPPC in otherwise identical lipid-protein mixtures. Palmitoylated peptide increased average DPPC-d₆₂ chain orientational order slightly, particularly for temperatures spanning gel and liquid crystalline coexistence, implying penetration of palmitoylated peptide into ordered membrane. In contrast, the nonpalmitoylated peptide had a small disordering effect in this temperature range. Both peptide versions perturbed DPPC-d₄ headgroup orientation similarly, suggesting little effect of palmitoylation on the largely electrostatic peptide-headgroup interaction. Deuterated acyl chains attached to the SP-C N-terminal segment displayed a qualitatively different distribution of chain order, and lower average order, than DPPC-d₆₂ in the same membranes. This likely reflects local perturbation of lipid headgroup spacing by the peptide portion interacting with the bilayer near the peptide palmitate chains. This study suggests that SP-C-attached acyl chains could be important for coupling of lipid and protein motions in surfactant bilayers and monolayers, especially in the context of ordered phospholipid structures such as those potentially formed during exhalation, when stabilization of the respiratory surface by surfactant is the most crucial.     

Interaction of glycosphingolipids with melittin and myelin basis protein in monolayers.

The penetration of melittin and myelin basic protein into glycosphingolipid monolayers depends on the lipid polar head group, the protein concentration available and the initial surface pressure. The lipid-protein interaction leads to modification of the surface properties of both the glycosphingolipid and the proteins.     

Rational design of cationic antimicrobial peptides by the tandem of leucine-rich repeat

Antimicrobial peptides represent ancient host defense effector molecules present in organisms across the evolutionary spectrum. Lots of antimicrobial peptides were synthesized based on well-known structural motif widely existed in a variety of lives. Leucine-rich repeats (LRRs) are sequence motifs present in over 60,000 proteins identified from viruses, bacteria, and eukaryotes. To elucidate if LRR motif possesses antimicrobial potency, two peptides containing one or two LRRs were designed. The biological activity and membrane–peptide interactions of the peptides were analyzed. The results showed that the tandem of two LRRs exhibited similar antibacterial activity and significantly weaker hemolytic activity against hRBCs than the well-known membrane active peptide melittin. The peptide with one LRR was defective at antimicrobial and hemolytic activity. The peptide containing two LRRs formed α-helical structure, respectively, in the presence of membrane-mimicking environment. LRR-2 retained strong resistance to cations, heat, and some proteolytic enzymes. The blue shifts of the peptides in two lipid systems correlated positively with their biological activities. Other membrane-peptide experiments further provide the evidence that the peptide with two LRRs kills bacteria via membrane-involving mechanism. The present study increases our new understanding of well-known LRR motif in antimicrobial potency and presents a potential strategy to develop novel antibacterial agents.     

KL₄ peptide induces reversible collapse structures on multiple length scales in model lung surfactant

We investigated the effects of KL₄, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL₄ improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).     

KL4 Peptide Induces Reversible Collapse Structures on Multiple Length Scales in Model Lung Surfactant

We investigated the effects of KL₄, a 21-residue amphipathic peptide approximating the overall ratio of positively charged to hydrophobic amino acids in surfactant protein B (SP-B), on the structure and collapse of dipalmitoylphosphatidylcholine and palmitoyl-oleoyl-phosphatidylglycerol monolayers. As reported in prior work on model lung surfactant phospholipid films containing SP-B and SP-B peptides, our experiments show that KL₄ improves surfactant film reversibility during repetitive interfacial cycling in association with the formation of reversible collapse structures on multiple length scales. Emphasis is on exploring a general mechanistic connection between peptide-induced nano- and microscale reversible collapse structures (silos and folds).