Article pubs.acs.org/Langmuir
Modulation of Activity of Ultrashort Lipopeptides toward Negatively Charged Model Lipid Films Joanna M. Wenda, Joanna Juhaniewicz, Dagmara Tymecka, Dorota Konarzewska, and Sławomir Sęk* Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw, Ż wirki i Wigury 101, 02-089 Warsaw, Poland S Supporting Information *
ABSTRACT: Because of the increasing resistance of pathogens to commonly used antibiotics, there is an urgent need to find alternative antimicrobial compounds with different mechanisms of action. Among them, lipopeptides are recognized as promising candidates. In this work, the Langmuir technique and atomic force microscopy were employed to investigate the interactions of two novel lipopeptides with negatively charged phospholipid membranes, which served as a simplified model of inner membrane of Gram-negative bacteria. Lipid films contained phosphatidylethanolamine and phosphatidylglycerol extracts from E. coli bacteria. Lipopeptides were composed of palmitoyl chain covalently coupled to N-terminus of peptide with Trp-Lys-Leu-Lys amino acid sequence and the conformation of third residue was either D-Leu or L-Leu. It was found that chirality of leucine strongly affects interfacial behavior of these compounds, which was ascribed to the difference in effective size of the peptide portion of the molecules. Although the lipopeptides were the same in terms of amino acid sequence, charge, and identity of lipophilic chain, the experiments revealed that the barrier for their insertion into the lipid membrane is significantly different. Namely, it was lower for lipopeptide containing D-Leu residue. We have also found that insertion of the lipopeptides into the model membranes strongly alters lateral distribution of the membrane components and leads to its substantial fluidization. The dynamics of reorganization was noticeably faster in the presence of lipopeptide with smaller size of peptide moiety, i.e., containing D-Leu. It proves that effective size of the peptide headgroup is an important factor determining lipopeptide activity toward the lipid membranes. number of studies on living cells was reported.6−8 Unfortunately, the details of the mechanism of their action remain unclear, especially at the molecular level. Among various classes of such compounds, a promising group of peptide-based antibiotics includes synthetic ultrashort linear lipopeptides. They are amphiphilic molecules composed of 2−4 amino acids coupled to aliphatic carboxylic acids with different chain lengths and their net charge is usually positive. As demonstrated by the Shai group, the sequence of the amino acid residues and the length of the aliphatic chain are crucial factors in determining their range of antimicrobial activity.9,10 A major target of the ultrashort lipopeptides is the membrane of the pathogen. Such a conclusion was drawn based on microscopic observation of the bacterial and fungal cell damage. Increased permeability of their membranes was correlated with lipopeptides activity.9 Hypothetical mechanism assumes action similar to that observed for cationic antimicrobial peptides and involves interaction with membrane, insertion, and aggregation of the molecules.11 The latter step may lead to disruption of the membrane, for example, by pore formation or micellization of membrane components. On the other hand, the results of molecular dynamics simulations reported by the Grossfield
1. INTRODUCTION The increasing resistance of bacteria to available antibiotics has become a major clinical problem worldwide. This situation creates the need to develop new antibiotics with new modes of action. The potent group of compounds that meet these criteria include lipopeptides. In contrast to conventional antibiotics, which act on specific targets such as enzymes involved in synthesis of cell wall or DNA, many lipopeptides have the ability to penetrate and destroy the cell membrane, leading to its irreversible damage.1 It is postulated that lipopeptides can bind to the membrane as agglomerates which further dissociate and individual molecules insert themselves into the lipid bilayer, causing its destabilization.2 Importantly, in many cases, lipopeptide action seems to be less specific, compared to traditional drugs; therefore, microbial resistance may occur with lower probability than that observed with available antibiotics.2 For that reason, lipopeptides are considered as promising new class of antibiotics. In this context, the advantage of lipopeptides is also related to their uncomplicated structure. This feature simplifies the synthesis and possible chemical modifications of these compounds enabling optimization of their activity and selectivity.3,4 Lipopeptides are built of lipophilic chains attached to linear or cyclic peptides usually including 6 or 7 amino acids and its net charge could be either positive or negative.5 Bactericidal activity of lipopeptides is already recognized and an appreciable © XXXX American Chemical Society
Received: December 30, 2016 Revised: March 21, 2017 Published: May 1, 2017 A
DOI: 10.1021/acs.langmuir.6b04674 Langmuir XXXX, XXX, XXX−XXX
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MS); the molecular peak in both cases was found at m/z = 835.3 [M + Na]+. Lipid films at the air/liquid interface were obtained with L-αphosphatidylethanolamine (extract from E. coli), further referenced as PE, and L-α-phosphatidylglycerol sodium salt (extract from E. coli), further referenced as PG. Both were purchased from Avanti Polar Lipids, Inc. All other compounds were obtained from Sigma−Aldrich. In all experiments, ultrapure Milli-Q water was used with final resistivity of 18.2 MΩ cm. Lipid monolayers at the gas/liquid interface were prepared using a KSV NIMA L&LB trough (Biolin Scientific, Sweden) equipped with two hydrophilic barriers. A Wilhelmy plate made of filter paper was used to control the surface pressure. All lipid monolayers studied in this work were formed on 0.01 M phosphate buffer saline (PBS) aqueous solution at pH 7.4. In order to obtain the spreading solutions, PE and PG lipids were dissolved in a chloroform/ methanol mixture (3:1, v/v). Upon spreading, the films at the interface were left for 15−20 min to complete solvent evaporation. The surface pressure versus molecular area isotherms were recorded at a compression speed of 10 mm/min, and the temperature was kept constant at 22 ± 1 °C. Brewster angle microscopy (BAM) images were recorded using a Nanofilm EP3 setup equipped with an UltraBAM objective (Accurion) with a lateral resolution of 2 μm and a field-ofview of 800 μm × 430 μm. Images were taken simultaneously with compression of the monolayers at the air/liquid interface. Small unilamellar vesicles (SUVs) were prepared according to Barenholtz protocol.19 Stock solutions containing ∼5.0 mg/mL of PE and ∼5.0 mg/mL of PG in CHCl3/MeOH (3:1, v/v) were mixed in a test tube at a desired molar ratio of 8:2 (PE/PG). This particular ratio was chosen as being representative for the composition of inner membrane of Gram-negative bacteria.20 The solvent then was evaporated by vortexing the mixture under an argon stream. In order to remove the solvent residues completely, a test tube with dried lipid cake was placed in a vacuum desiccator for 2 h. Next, 1.5 mL of a 0.01 M PBS solution was added to the lipid cake and the mixture was sonicated at 37 °C for 1 h. The final suspension of SUVs was homogeneous and transparent. Dynamic light scattering (DLS) measurements indicated that the effective diameter of the vesicles was in the range of 30−50 nm. Supported lipid bilayers were obtained by spreading of SUVs onto freshly cleaved mica substrates. Formation of the bilayers was monitored by AFM imaging immediately upon injection of the vesicles into the liquid cell of the microscope. A steady state was achieved after ∼2 h, and then the cell was gently flushed with buffer in order to remove excessive lipid material. The thickness of the resulting lipid bilayers was estimated as an average height difference between the bare substrate and the area covered by the lipid assembly. AFM experiments were performed using Dimension Icon (Bruker) instrument. The images were acquired in 0.01 M PBS aqueous solution. For topography imaging, we have used Scan Asyst Fluid+ cantilevers (Bruker) with a nominal spring constant of 0.7 N/m and a nominal tip radius of 5 nm. The exact value of the spring constant for each cantilever was determined by the thermal tune method, and the curvature radius of the probes was evaluated using tip characterizer samples TGT1 (NT-MDT) and TC1 (BudgetSensors). The images were recorded in Peak Force QNM mode, which has the capability of mapping the topography and the nanomechanical properties of the sample simultaneously. During the imaging, the cantilever is modulated along the Z-axis at a certain frequency and at default amplitude. At each cycle, the force−distance curve is recorded for every pixel and the analysis of the retract curves allows determination of the adhesion force (Fadh) and the reduced modulus of elasticity (E*) according to the Derjaguin, Muller, Toporov (DMT) model:21
group indicate that the mechanism of lipopeptide action on lipid bilayer differs from classical poration or carpet models ascribed to antimicrobial peptides.12−14 It was found that lipopeptides disturb the structure of the lipid membrane through surface aggregation, which is followed by lipid demixing. Such an effect is a consequence of electrostatic interactions between the positively charged peptide portion of lipopeptide and negatively charged lipids and contributes to increased bilayer ordering upon lipopeptide insertion. In this case, the structure, as well as stability, of the lipid bilayer is distorted, leading to membrane deterioration. In the present work, the interactions of two novel lipopeptides with model lipid films were evaluated using the Langmuir technique and atomic force microscopy (AFM). These techniques proved to be useful tools to investigate interactions of biologically relevant compounds with lipids.15−18 The model membranes were composed of zwitterionic L-α-phosphatidylethanolamines and negatively charged L-αphosphatidylglycerols extracted from E. coli bacteria. Thus, their composition reflects, to some extent, the lipid content of the inner membrane of Gram-negative bacteria. Lipopeptides were built of palmitoyl chain coupled to tetrapeptide moiety consisting of the Trp-Lys-Leu-Lys amino acid sequence. However, the conformation of a third residue was either DLeu or L-Leu. This particular structure of the lipopeptides was designed to meet certain criteria, which are often considered to be important for membranolytic activity. These include (a) presence of hydrophobic portion, preferably with aromatic residue which is supposed to drive partitioning into the hydrophobic core of the lipid membrane; (b) polar amino acid residues, preferably possessing positively charged side groups to facilitate electrostatic interactions with negatively charged lipids; (c) introduction of D-amino acids to suppress in vivo enzymatic degradation of lipopeptide molecule; and (d) proper balance between polar and nonpolar portions. Surprisingly, we have found that the chirality of a single amino acid strongly affects the interfacial behavior of lipopeptides, as well as their activity against model lipid membranes, which seems to be related to a pronounced difference in size of the peptide moieties. The results of surface pressure measurements, combined with those obtained from AFM, enabled conclusion about susceptibility of the membranes to lipopeptides insertion and possible modes of action. We believe that the results of this study may contribute to a better understanding of the mechanisms of membranolytic activity of lipopeptides.
2. EXPERIMENTAL SECTION Synthesis of lipopeptides CH3(CH2)14−C(O)-Trp-Lys-Leu-Lys (further referred to as C16−WKLK) and CH3(CH2)14−C(O)-Trp-Lys(D)Leu-Lys (further referenced as C16−WKLK) was performed on the solid phase using standard Fmoc protocols and Fmoc-Lys(Boc)-Wang resin (substitution level of 0.56 mmol/g) on 0.6 mmol scale. As the side chain protecting group of Lys and Trp residues, tertbutyloxycarbonyl (Boc) was used. The coupling steps were performed in DMF/DCM solution (1:1 v/v) using TBTU (3 equiv) as the coupling reagent and DIPEA (6 equiv) as the base. The same coupling procedure was used for coupling the fatty acids to the N-termini of the peptides. The final lipopeptides were cleaved from the resin using 10 mL of the TFA cocktail mixture (TFA-phenol-H2O-TIS: 88:5:5:2 v/v) per gram of the resin. Peptides were purified by RP-HPLC on a Supelco Discovery BIO Wide Pore C8 column (250 mm × 10 mm), using 0.1% TFA in water as solvent A and 0.1% TFA in acetonitrile as solvent B, at a flow rate of 3 mL/min. The identity of the lipopeptides was confirmed using electrospray ionization mass spectrometry (ESI-
Ftip =
4 E* Rd3 + Fadh 3
(1)
where Ftip is the force acting on the tip, d the distance between the tip and the sample, and R the tip radius. Furthermore, the sample modulus of elasticity (Es) can be estimated as B
DOI: 10.1021/acs.langmuir.6b04674 Langmuir XXXX, XXX, XXX−XXX
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Langmuir −1 ⎡1 − ν 2 1 − νs 2 ⎤ t ⎢ ⎥ + E* = Es ⎦ ⎣ Et
(2)
where νt and νs are Poisson’s ratios for the material of the tip and the sample respectively, and Et is the modulus of elasticity of the tip material. The accuracy of determination of modulus of elasticity was evaluated using the PDMS test sample (Bruker) with a nominal modulus of 3.5 MPa. AFM imaging with chemically modified tips was performed using gold-coated NPG probes (Bruker). First, the probes were gently rinsed with CH2Cl2/MeOH (1:1 v/v) and then placed in a Petri dish filled with solution of 16-mercaptohexadecanoic acid in the same solvent. Because of the strong affinity of sulfur to gold surface, carboxylic acid was chemisorbed on gold-coated AFM tips. After ∼1.5 h, the probes were rinsed again with CH2Cl2/MeOH (1:1 v/v) and left to dry. Next, they were placed for 30 min in an aqueous solution containing 0.4 mg of EDC and 0.6 mg of NHS in 3 mL of water, in order to activate carboxylic groups. The probes then were transferred to an aqueous solution of either WKLK or WKLK tetrapeptide and the coupling reaction was carried out for at least 6 h to complete attachment of peptide moiety to activated carboxylic groups of tip-bound 16mercaptohexadecanoic acid. After that, AFM probes were gently rinsed with water and dried. Finally, they were used to obtain maps of adhesion forces acting between the modified tip and the supported PE/PG lipid bilayers.
3. RESULTS AND DISCUSSION 3.1. Surface Pressure Measurements. Lipid films compressed at the air/water interface are considered as useful models to study interactions between lipids and variety of compounds, including proteins, peptides, and drugs.22 A similar approach was used in this work to investigate the influence of lipopeptides on PE/PG monolayers. First, we have evaluated the interfacial behavior of single-component lipopeptide films. The isotherms of C16−WKLK and C16−WKLK are shown in Figures 1A (red line) and 1B (red line), respectively. Both molecules are identical, in terms of the amino acid sequence, the effective charge, and the identity of lipophilic chain. The conformation of leucine residue is the only variation in their structure. Nevertheless, it seems to be sufficient to affect the interfacial behavior of these molecules noticeably. The mean molecular area obtained for C16−WKLK was ∼59 Å2, whereas, for C16−WKLK, its value was >73 Å2. Thus, we observe significant differences in molecular packing. The latter will be influenced mainly by the size of the peptide part of the molecule, because it is more bulky, compared with the crosssectional area of the palmitoyl chain. Apparently, different conformation of leucine residue influences the effective size of the lipopeptide headgroup, and this, in turn, affects the packing density at the air/buffer interface. Such a conclusion is reasonable, since numerous peptides show variation in aggregation behavior, depending on the chirality of the amino acid residues.23 Other parameters, such as the collapse pressure and the corresponding molecular area, are also different, when comparing lipopeptides with D- and L-Leu residues, indicating that the first one forms the more-stable film (see Table 1). Moreover, the maximum compression modulus, which allows conclusion about the state of the monolayer, also seems to be affected by the conformation of leucine residue. Although both systems are in liquid expanded state, particular values of the maximum compression modulus were different, i.e., 69 and 85 mN/m for C16−WKLK and C16−WKLK, respectively. Thus, the first one forms slightly more fluid films. The difference in interfacial behavior was also revealed by Brewster angle microscopy imaging (see the Supporting Information). We
Figure 1. Surface pressure versus area per molecule isotherms recorded for monolayers compressed on an aqueous 0.01 M PBS subphase. Panel (A) illustrates the isotherms obtained for pure C16− WKLK (red line), PE/PG (black line), and mixed monolayers composed of PE/PG and C16−WKLK at different lipopeptide concentrations. Panel (B) illustrates the isotherms obtained for pure C16−WKLK (red line), PE/PG (black line), and mixed monolayers composed of PE/PG and C16−WKLK with different lipopeptide concentrations.
have found that C16−WKLK forms uniform and featureless films within the surface pressure range of 0−35 mN/m, while C16−WKLK shows a tendency to form separated domains. Interestingly, the differences in interfacial behavior seem to be strongly diminished when lipopeptides are spread together with PE/PG lipids on buffer and then compressed to form monolayers. Figure 1 shows surface pressure versus molecular area isotherms recorded for two-component PE/PG monolayers and three-component PE/PG monolayers with lipopeptides added at different concentrations, ranging from 2% up to 15%. It is apparent that the shape as well as the position of the isotherms does not change significantly in the presence of lipopeptides. As demonstrated in Table 1, the shift toward larger molecular area upon increasing concentration of lipopeptides is rather moderate and does not exceed 8% of the initial area per molecule observed for a pure lipid film. The aforementioned shift is slightly more pronounced for C16− WKLK. Interestingly, the stability of the monolayers seems to be unaffected by the presence of lipopeptides, as can be concluded from the values of collapse pressure, which are very close to that determined for the PE/PG system. These observations may suggest that the lipopeptides are partially repelled from the interfacial film during monolayer compression at least at higher surface pressures, where some isotherms C
DOI: 10.1021/acs.langmuir.6b04674 Langmuir XXXX, XXX, XXX−XXX
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Langmuir Table 1. Characteristic Parameters of Monolayers Formed at the Air/Liquid Interface with 0.01 M PBS Subphase A0 [Å2] monolayer composition lipopeptide PE/PG PE/PG + lipopeptide PE/PG + lipopeptide PE/PG + lipopeptide PE/PG + lipopeptide
(2%) (5%) (10%) (15%)
C16-WKLK 59.2 76.1 72.2 75.2 80.4 81.5
± ± ± ± ± ±
1.7 1.7 0.2 0.4 2.5 1.3
C16-WKLK 73.5 76.1 74.9 77.9 79.3 78.9
± ± ± ± ± ±
0.8 1.7 0.6 0.6 1.2 0.6
C16-WKLK 35.0 51.5 50.5 52.4 54.0 54.1
± ± ± ± ± ±
1.7 0.8 0.8 0.5 1.3 0.5
C16-WKLK 49.7 51.5 51.7 53.0 51.1 51.9
Cs−1 [mN/m]
πcoll [mN/m]
Acoll [Å2] ± ± ± ± ± ±
0.8 0.8 0.4 0.2 0.8 0.6
C16-WKLK 38.2 43.8 44.8 45.0 43.7 43.0
± ± ± ± ± ±
1.0 0.3 0.1 1.9 0.1 0.2
C16-WKLK 35.3 43.8 46.4 43.5 42.4 42.7
± ± ± ± ± ±
0.8 0.3 1.1 0.3 0.3 0.5
C16-WKLK 69 138 121 119 102 96
± ± ± ± ± ±
6 4 2 1 3 1
C16-WKLK 85 138 122 107 101 86
± ± ± ± ± ±
4 4 4 2 3 1
parameters of monolayers formed on lipopeptide-containing subphase are shown in Table 2. It is apparent that the shift of the isotherms is dependent on lipopeptide concentration. Namely, the area per molecule becomes higher when the concentration of lipopeptide in subphase is increasing. However, this effect is much more pronounced for C16−WKLK, demonstrating its higher affinity to adsorb at the interface and incorporate into the lipid monolayer. Interestingly, the collapse pressure for all monolayers is almost unchanged, suggesting that the stability of the interfacial films is practically unaffected by the presence of lipopeptides. Nevertheless, substantial differences appear in the values of the maximum compression modulus. Their changes in the presence of C16−WKLK are rather small and the monolayer exists in the liquid condensed state, even at the highest lipopeptide concentration. In contrast, the influence of C16−WKLK is more dramatic and the maximum compression modulus decreases significantly already at the lowest lipopeptide concentration studied in this work. This reflects the strong fluidizing effect of C16−WKLK. Initially, the PE/PG monolayer is in the liquid condensed state, while in the presence of C16−WKLK, it becomes more fluid and exists in a liquid expanded state. Analysis of the maximum compression modulus, together with the changes in molecular area in the presence of lipopeptides dissolved in the subphase leads to the conclusion that PE/PG monolayers accommodate higher amounts of C16−WKLK molecules, compared to its analogue containing L-Leu residue. This suggests that significant differences may also be expected when spontaneous insertion of these lipopeptides into the lipid film is considered. In order to verify this assumption, the interactions of lipid monolayer with dissolved active compounds present in a subphase were investigated and the exemplary results are illustrated in Figure 3. The lipopeptide concentration was 3 μM in both cases. In this experiment, lipid monolayers were spread at the air/buffer interface and compressed to certain surface pressures. The barriers of the Langmuir trough then were stopped to maintain a constant area occupied by the lipid film. Furthermore, the given lipopeptide was injected into the subphase and the changes in the surface pressure were monitored as a function of time, until equilibrium adsorption pressure was achieved. This enabled calculation of the increase in maximum surface pressure, which is a difference between equilibrium pressure and initial pressure.24 Figure 3A presents exemplary data obtained for PE/PG monolayers compressed to a initial surface pressure of 10 mN/m upon injection of either C16−WKLK (gray line) or C16−WKLK (blue line) into the subphase. In both cases, we have observed an increase in surface pressure, which reflects the insertion of a soluble active compound present in the subphase into the hydrophobic region of the lipid film.25 Nevertheless, the kinetics of this process seems to be different. The injection of C16−WKLK results in a sharp
are very close in shape or even overlap. Nevertheless, the presence of C16−WKLK and C16−WKLK affects the fluidity of lipid films. The value of the maximum compression modulus for the pure PE/PG monolayer was 138 mN/m, which is within the range of 100−250 mN/m, corresponding to a liquid condensed state. This parameter is noticeably lowered when the content of lipopeptides in lipid films was increased, and, at the highest concentration, the monolayers were in a liquid expanded state. In order to evaluate further the affinity of lipopeptides to the PE/PG monolayer spread at the air/buffer interface, we have performed experiments where the surface pressure changes of the lipid film were monitored during compression on a 0.01 M PBS subphase containing either C16−WKLK or C16−WKLK. As shown in Figure 2, the presence of lipopeptides in the subphase causes a shift of the isotherms toward higher values of molecular area. This proves that lipopeptides are incorporated into the interfacial film during its formation. Characteristic
Figure 2. Surface pressure versus area per molecule isotherms recorded for monolayers of PE/PG compressed on pure 0.01 M PBS subphase and 0.01 M PBS subphase containing different concentrations of (A) C16−WKLK and (B) C16−WKLK. D
DOI: 10.1021/acs.langmuir.6b04674 Langmuir XXXX, XXX, XXX−XXX
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Table 2. Characteristic Parameters of PE/PG (8:2) Monolayers Formed at the Air/Liquid Interface with 0.01 M PBS Subphase Containing Different Concentrations of Lipopeptide A0 [Å2] lipopeptide concentration 0 1 × 10−7 M 5 × 10−7 M 1 × 10−6 M
C16-WKLK 69.4 87.9 128.4 226.5
± ± ± ±
1.4 0.8 2.6 3.3
C16-WKLK 69.4 71.6 78.9 90.5
± ± ± ±
1.4 0.2 1.6 0.3
C16-WKLK 42.5 52.3 72.6 112.5
± ± ± ±
C16-WKLK
0.6 0.6 0.8 0.7
42.5 42.1 44.8 49.5
Cs−1 [mN/m]
πcoll [mN/m]
Acoll [Å2] ± ± ± ±
0.6 0.5 0.8 0.4
C16-WKLK 43.7 44.3 43.4 44.7
± ± ± ±
0.8 0.7 0.3 0.7
C16-WKLK 43.7 44.3 43.8 44.5
± ± ± ±
0.8 0.6 0.6 0.9
C16-WKLK 123 64 62 59
± ± ± ±
7 4 3 4
C16-WKLK 123 118 114 103
± ± ± ±
7 3 4 2
contribution. Based on it, we conclude that the magnitude of surface pressure response will be dictated mainly by the number of lipopeptide molecules accommodated in the lipid film, which, in turn, will reflect their affinity to lipids forming the monolayer. The larger increase in the pressure observed for C16−WKLK suggests that the ability of these molecules to penetrate the lipid film is much higher, compared with C16− WKLK. The same trend was noted for other values of initial surface pressure, as demonstrated in Figure 3B. By extrapolating the regression of the plots presented in Figure 3B to the xaxis, it is possible to determine a maximum insertion pressure (MIP).26 This parameter reflects the maximum surface pressure of lipid monolayer up to which insertion of the lipopeptide is feasible. The values of MIP found for C16−WKLK and C16− WKLK were 55 and 32 mN/m, respectively. These numbers show clearly that activity of lipopeptide with D-Leu residue against PE/PG film is enhanced, compared with its analogue that contains L-Leu. Thus, the surface pressure measurements demonstrate that C16−WKLK, as well as C16−WKLK, can penetrate PE/PG membranes but the barrier for insertion is apparently lower for the first one. We assume that such an effect is related to the smaller size of the polar headgroup in C16−WKLK, which facilitates the initial penetration of peptide moiety into the hydrophilic region of the lipid film, and, as a consequence, further reorientation of lipopeptide molecules becomes easier. Interestingly, if we consider that the estimated value of lateral pressure in natural membranes is in the range of 30−35 mN/m,27,28 MIP values obtained for C16−WKLK and C16−WKLK suggest that both can penetrate cell membranes; however, the first one could be much more efficient in bilayer destabilization. 3.2. Atomic Force Microscopy Studies. Atomic force microscopy (AFM) was employed to investigate interactions of lipopeptides with PE/PG (8:2) bilayers supported on freshly cleaved mica. Lipid films were obtained by spontaneous spreading of SUVs. The AFM images of resulting bilayers show separated domains (Figures 4A and 4D), corresponding to topographically lower and higher regions. These can be ascribed to the presence of a liquid disordered phase (Lα) and a gel phase (Lβ), respectively. PE and PG E. coli extracts used in this work are mixtures of lipids with different saturation ratios of fatty acid chains. Therefore, phase separation is a rather expected phenomenon. Detailed analysis of the images obtained for the series of several independent samples revealed that the mean thickness of the Lα phase and Lβ phase is 4.1 ± 0.3 nm and 5.5 ± 0.2 nm, respectively, which is in very good agreement with our previous observations.29 Interestingly, exposure of the PE/PG bilayer to C16-WKLK leads to substantial variation in the structure of the lipid film. We observed that topographically higher Lβ domains substantially reduce their area and shape after ∼20 min upon injection of the lipopeptide into the buffer solution (Figure 4B) and finally they disappear after 90 min. The resulting film was uniform and
Figure 3. (A) Changes in surface pressure in time recorded for PE/PG monolayers compressed to 10 mN/m on an aqueous 0.01 M PBS upon injection of lipopeptide into the subphase; the total concentration of lipopeptide was 3 μM. (B) Maximum increase of surface pressure, as a function of initial surface pressure of PE/PG monolayers upon injection of 3 μM of lipopeptide into the 0.01 M PBS subphase.
increase in the pressure, reflecting fast incorporation of lipopeptide into the interfacial film, whereas for C16−WKLK, the time dependence is rather shallow. Therefore, it can be concluded that C16−WKLK have higher barrier for penetration of lipid film. Interestingly, the maximum increase in surface pressure observed in the presence of C16−WKLK is almost 30 mN/m, which is significantly higher, compared to the value of ∼10 mN/m obtained for C16−WKLK. The magnitude of increase in surface pressure resulting from the insertion of lipopeptide molecules into the lipid monolayer is dependent on several factors, but two of them are crucially important. First one is the amount of lipopeptide incorporated into the lipid monolayer, while the second one is related to the mechanical properties of the lipid film. Since the composition of lipid monolayers is fixed and all experiments were performed under the same conditions, the mechanical properties of the lipid monolayer can be considered to be a factor with constant E
DOI: 10.1021/acs.langmuir.6b04674 Langmuir XXXX, XXX, XXX−XXX
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between positively charged amines in lysine side chains and negatively charged PG lipids are crucially important. However, in our case, both active compounds have exactly the same charge, which means that electrostatic forces provide a constant contribution to initial lipopeptide binding. Therefore, it is reasonable to assume that the insertion of lipopeptide is preceded by the immersion of peptide moieties between the polar heads of lipids. The latter concomitantly changes their spatial arrangement to facilitate the reorientation and insertion of lipopeptide molecules. In such cases, smaller peptide moieties will be preferred, because of the lower energetic input required to rearrange lipid polar heads. Moreover, the size of the peptide moiety will also affect the rate of lateral diffusion of the molecules in the fully inserted state. Faster movement of lipopeptide molecules with smaller headgroups can facilitate changes in molecular arrangement of membrane assembly. This scenario shares some features with the model proposed by the Grossfield group, based on the molecular dynamics simulations.14 These authors have demonstrated that lipopeptide C16−KGGK spontaneously binds to the surface of membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and causes demixing of the components by attracting anionic lipids and increasing bilayer ordering. Our results seem to confirm this model in some aspects, since lateral reorganization of the lipid molecules within the membrane is evident from AFM images where the reduction of Lβ domains was observed. However, thinning of the membrane, as well as partial perforation observed in the presence of C16−WKLK, suggests a rather fluidizing effect of lipopeptides. In order to verify this assumption, we have determined the moduli of elasticity of lipid films before and after exposure to lipopeptides. Figure 5A shows the spread of the modulus of elasticity obtained for an intact PE/PG bilayer supported on mica. The distribution is bimodal, with mean values of 19.1 ± 3.5 MPa and 35.4 ± 7.5 MPa reflecting different molecular packing within liquid-like Lα and gel-like Lβ domains, respectively. Interestingly, the modulus of elasticity determined for the PE/ PG bilayer after exposure to C16−WKLK (at steady state) is significantly lower and its mean value was found to be 9.4 ± 3.1 MPa (see Figure 5B). This clearly shows that the molecular packing density was altered in the presence of lipopeptide and the bilayer became more fluid. A similar trend was observed in the presence of C16−WKLK; however, in this case, the decrease in the modulus of elasticity was less dramatic. The distribution is still bimodal, with mean values of 12.4 ± 2.4 MPa and 17.1 ± 1.3 MPa (see Figure 5B), which may reflect nonuniform packing density of the molecules within the film. Nevertheless, these results also prove a substantial change in elastic properties related to increased disorder within the membrane and confirm observations from surface pressure measurements where fluidization of the lipid films was noted in the presence of lipopeptides. As was demonstrated previously, the structure of the intact PE/PG membrane studied in this work is characterized by the coexistence of separated Lα and Lβ domains. Such a specific distribution of lipids may imply some significant differences in lipopeptide binding and insertion. In order to verify this hypothesis, we have performed AFM-based experiments enabling assessment of the affinity of lipopeptides to particular domains. Figure 6 demonstrates the results obtained from simultaneous topography imaging and adhesion force mapping of PE/PG bilayers with chemically modified tips. Such
Figure 4. Time-lapse sequence of AFM images illustrating the changes in morphology of PE/PG bilayer supported on mica in the presence of 3 μM C16−WKLK (panels (A), (B), and (C)) and 3 μM C16−WKLK (panels (D), (E), and (F)). Each sequence was taken at the same spot on the sample and arrows indicate domains that substantially changed their size during the first interval. Vertical scale: Δz = 6 nm.
featureless, with an average thickness of 4.2 ± 0.5 nm. Longer exposure caused additional transformation of the bilayer morphology and, after 180 min, the film had visible irregular perforation but its thickness remained unchanged (Figure 4C). Since continuous imaging up to 360 min did not reveal any further structural changes, we assume that the morphology demonstrated in Figure 4C represents a steady state. Interestingly, the perforation observed in Figure 4C may suggest that, at a certain stage, the amount of lipopeptide molecules that accumulated within the membrane is high enough to solubilize the lipids and disrupt the membrane. The latter may occur through the formation of mixed lipopeptide− lipid micelles. Slightly different structural transitions of the PE/ PG bilayer were observed after its exposure to C16−WKLK. In this case, Lβ domains remained almost intact up to 60 min. Only a few domains slightly changed their size or shape, as can be deduced from a comparison of AFM images that are presented in Figures 4D and 4E. More-pronounced changes were observed after 180 min when the topographically higher domains disappeared and the bilayer became featureless. Its average thickness at this stage was determined to be 4.1 ± 0.4 nm. Further imaging revealed that, after ∼300 min, steady state was achieved. However, the final morphology of the film was different, compared to the PE/PG bilayer exposed to C16− WKLK. In the presence of C16−WKLK, the membrane retained its structural integrity, since we did not detect any lipopeptideinduced perforation, even after overnight exposure. Nevertheless, membrane thinning was clearly visible, as demonstrated in Figure 4F, where darker regions are ∼0.7 nm lower than the surrounding domains. Aforementioned results indicate that both lipopeptides affect molecular organization within PE/PG films by changing the lateral distribution of the components. This is reflected by vanishing Lβ domains and decreasing the average membrane thickness. Apparently, the action of C16−WKLK is more effective, compared with C16−WKLK, since reorganization of the membrane structure occurs within a shorter time scale. The difference in the size of the lipopeptide polar headgroups may explain such behavior. When the lipopeptide molecule approaches the bilayer, its peptide portion interacts with the polar heads of lipids. At this stage, electrostatic interactions F
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experiment enables direct correlation of surface morphology with forces acting between the probe and the sample. Since the tip is chemically modified with 16-mercaptohexadecanoic acid to which peptide moieties are coupled, the main contribution to the measured forces will come from the interaction between external hydrophilic region of the bilayer and peptide pendants attached to the AFM probe. Figures 6A and 6B present topography and adhesion maps recorded for PE/PG bilayer patches immobilized on mica. The images were obtained using the tip modified with the WKLK peptide moiety. Morphology of the lipid film is typical with easily distinguishable regions covered by Lα and Lβ domains (Figure 6A). Darkest regions represent bare mica. The corresponding map of adhesion forces (Figure 6B) shows that the tip−sample interactions are nonuniform over the scanned area, since the region covered by Lα domain is noticeably brighter, compared with Lβ domain and bare mica. It means that higher forces act between the tip and Lα domain. Exactly the same conclusion can be drawn from analysis of the images obtained with WKLK-modified tip (Figures 6C and 6D). The differences in adhesion forces between Lα and Lβ domains may be attributed to the different strength of interactions between peptide moieties and polar heads of lipids, which determine the measured pull-off forces. This would indicate that WKLK and WKLK peptides preferentially interact with more liquid-like domains. As the tip approaches the surface of the membrane, peptide moieties are in contact with the hydrophilic region of the bilayer and some fraction may interdigitate with polar heads of lipids. The extent of such interdigitation will be dependent on the strength of electrostatic interactions, the size of the peptide moieties, and the packing density of lipid polar heads. Since the latter is lower for the more-liquid-like Lα domain, the interaction is stronger and the resulting adhesion forces are noticeably higher, compared with those measured over the stiffer Lβ domain. Thus, the region of more fluid domain can be considered as the preferential entry site for lipopeptide insertion.
Figure 5. (A) AFM-derived distribution of the modulus of elasticity determined for intact PE/PG bilayer supported on mica. Inset shows the map of modulus of elasticity obtained using Peak Force QNM mode with clearly distinguished Lα and Lβ domains. (B) AFM-derived distribution of modulus of elasticity determined for a PE/PG bilayer supported on mica upon exposure to 3 μM C16−WKLK (gray) and 3 μM C16−WKLK (navy blue). Insets show respective maps of modulus of elasticity obtained using Peak Force QNM mode.
4. CONCLUSIONS We have found that C16−WKLK and C16−WKLK both form liquid monolayers at the air/buffer interface. As it can be concluded from surface pressure measurements, the chirality of leucine residue affects molecular packing density within the films. This is interpreted in terms of different sizes of the peptide portion of molecules, which are smaller when D-Leu occurs in the amino acid sequence. When incorporated into the PE/PG lipid monolayer, lipopeptides show similar behavior and both increase the fluidity of the lipid films. Although the molecules of C16−WKLK and C16−WKLK are the same, in terms of the amino acid sequence, charge, and identity of lipophilic chain, penetration experiments revealed that the barrier for insertion is significantly lower for lipopeptide containing D-Leu residue. The same conclusion can be drawn from the analysis of AFM data obtained for supported PE/PG bilayers exposed to lipopeptides. It is evident from our experiments that, upon insertion of the lipopeptide into the membrane, lateral distribution of the components is strongly altered and the membrane becomes more fluid. However, the dynamics of reorganization is faster in the presence of C16− WKLK. It proves that the effective size of the peptide moiety is an important factor modulating lipopeptide activity toward the lipid membrane. Based on these results, we hypothesize that full insertion of the lipopeptide is most likely preceded by the immersion of peptide moieties within the hydrophilic region of
Figure 6. (A and C) AFM topography and (B and D) simultaneously recorded adhesion maps obtained for the patches of PE/PG bilayer supported on mica. Images shown in panels (A) and (C) were taken using the tip modified with WKLK peptide, while the images shown in panels (B) and (D) were obtained using the tip modified with WKLK peptide. All images were recorded in an aqueous 0.01 M PBS. G
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the bilayer occurring upon initial electrostatic interaction, which drives the molecules toward the outer plane of the membrane. The immersion depth, as well as the dynamics of this process, will be dependent on the size of the peptide portion of lipopeptide and the lateral spacing between lipid polar heads in the external plane of the membrane. Thus, the process will be facilitated when small peptide moieties enter liquid-like domains of the membrane. This scenario is supported by the results of above-mentioned penetration experiments, as well as adhesion mapping, which demonstrates that the interactions of peptide moieties are stronger within the regions of less densely packed Lα domains. Therefore, the latter can be considered as preferential entry sites for lipopeptide insertion. As it was already mentioned, insertion of lipopeptides into PE/PG membrane results in substantial increase in its fluidity, which is reflected by a significant decrease in the modulus of elasticity well below the value initially observed for liquid disordered domains. This may reflect the fact that perturbation of the bilayer structure results in accumulation of noticeable amounts of water molecules, which are dragged into the membrane, together with lipopeptides. Such interpretation is consistent with the assumptions of the interfacial activity model proposed by Wimley to explain the mechanism of action of antimicrobial peptides.30,31 The model assumes that peptides partition into the bilayer and locally disturb organization of lipid moieties without the formation of any specific aggregates, such as channels or pores. As a result, polar solutes pass across the membrane, along with peptide molecules, because of large perturbations in the bilayer structure. Such mechanism can be applicable to lipopeptides as well, since neither the formation of pores nor carpeting was observed in our experiments.
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ASSOCIATED CONTENT
S Supporting Information *
Supporting Information file includes The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b04674. Compression modulus plots, as a function of surface pressure obtained for monolayers composed of C16− WKLK, C16−WKLK, and PE/PG with different content of lipopeptides (Figure 1S); BAM images recorded during compression of monolayers composed of C16− WKLK, C16−WKLK and PE/PG with different content of lipopeptides (Figure 2S) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Sławomir Sęk: 0000-0002-7741-6448 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by Polish National Science Centre (Project No. 2013/10/E/ST4/00343). The study was carried out at the Biological and Chemical Research Centre, University of Warsaw, established within the project cofinanced by European Union from the European Regional Development Fund under the Operational Program Innovative Economy, 2007−2013. H
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