Investigation of the interaction of antimicrobial peptides with lipids and lipid membranes 2

CHAPTER INVESTIGATION OF THE BINDING OF A NOVEL ANTIMICROBIAL PEPTIDE V4 TO MEMBRANE MIMICS 5.1 Introduction Although a large number of native antimicrobial peptides have been discovered by now, few of them have been extended to clinical research. One of the reasons is that many native antimicrobial peptides have a lack of selectivity with both antimicrobial activity and hemolytic activity, which limits further application. Therefore the design of antimicrobial peptides which allow modified antimicrobial activity and hemolytic activity to achieve better selectivity for bacterial cells, while not harming mammalian cells, have drawn a wide interest in the development of therapeutical application of antimicrobial peptides. V4 is designed with this purpose and it has been reviewed in Chapter 2. V4 displayed high antimicrobial activity, low cytotoxic activity and low hemolytic activity in vivo. Therefore it is interesting to investigate the binding affinity of V4 for different membranes in vitro to examine its nature of selectivity. In this chapter FCS is used to study the interaction of this novel artificial peptide V4 with different membrane components. The purpose of this study is to (i) obtain information about the oligomerization or aggregation state of V4, (ii) compare the binding of V4 to different lipid components of mammalian and microbial membranes. From the data we have gained insights into the properties of V4 and can obtain suggestions on how to improve on the design of artificial antimicrobial peptides. 78 5.2 Materials and methods Materials Rhodamine 6G chloride (Rho 6G), tetramethylrhodamine (TMR) and R18 are products from Molecular Probes. LPS from Escherichia coli strain 0111:B4, its fluorescent derivative FITC-LPS, Lipid A from Escherichia coli strain F583, Triton-X100 and PBS were purchased from Sigma-Aldrich. DMSO was purchased from Mallinckrodt Baker (Mallinckrodt Asia Pacific Pte. Ltd., Singapore). Phosphatidylcholine (PC), 1,2Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC), 1,2-Dipalmitoyl-sn-Glycero-3- Phosphoethanolamine (DPPE), 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DPPG), 1-Palmitoyl-2- Oleoyl-sn-Glycero-3-Phosphocholine (POPC), 1-Palmitoyl-2Oleoyl-sn-Glycero- 3-Phosphoethanolamine (POPE) and 1-Palmitoyl-2-Oleoyl-snGlycero-3- [Phospho-rac-(1-glycerol)] (POPG) were purchased from Avanti (Avanti Polar Lipids, Inc., Alabaster, AL). Peptides. The sequence of V4 is CVKVQVKVGSGVKVQVKVC with cyclization by a disulfide bond at the two terminal cysteines (C). Four lysine (K) residues provide high net positive charge and eight valine (V) residues make this peptide highly hydrophobic. V4-TMR is the V4 labeled with TMR at the N-terminus. Both peptides were synthesized by Genemed (Genemed Synthesis, Inc., South San Francisco, CA). According to the HPLC data provided by the company, the purity of V4-TMR is about 84% and the purity of V4 is above 97%. The stock solution of V4-TMR peptide was prepared as a mM solution in DMSO. The stock solution of V4 was prepared as mM in water. Both stock solutions were stored at –20 ºC in small aliquots until further use. 79 Small unilamellar vesicles (SUVs) preparation. All lipids were prepared as stock solutions in chloroform or a mixture of chloroform and ethanol (4:1). The solvent was evaporated under N2 gas and then the samples were placed into vacuum for at least one hour. PBS buffer was added to re-dissolve the lipids to give an aqueous suspension of phospholipids at a concentration of 0.5 mM. SUVs were prepared by freeze-thawing the lipid suspension times followed by extrusion through 0.05 µm polycarbonate membrane filters for 20 times using a mini-extruder syringe device (Avanti Polar Lipids). The extruded lipid solutions were diluted and mixed with 200 nM V4-TMR to study the interaction of peptide and lipid vesicles by FCS. Fluorescence Correlation Spectroscopy (FCS) The fluorescence yield Q is an important parameter in FCS. It determines the signal to noise ratio185 but is as well a characteristic value for a fluorophore in a certain environment. Therefore, determining the value of Q of a particle can yield information which can help identify a fluorophore and can give information about the local environment of the fluorophore. For a solution with a single fluorophore present and negligible background it is simply given by the number of average photon count rates, C1, divided by the average number of particles in the confocal volume, N1 as obtained from the ACF. Q1 = C1 N1 (5.1) In solutions with two different fluorophores and photon count rates C12, the autocorrelation amplitude which is inversely proportional to the apparent number of particles Napp is given by 80 ⎛ ⎞ ⎛Q ⎞ (1 − F2 ) + ⎜⎜ Q2 ⎟⎟ F2 F1 + ⎜⎜ ⎟⎟ F2 1 ⎝ Q1 ⎠ ⎝ Q1 ⎠ = × = × G (0) − G∞ = 2 N ⎛ N app N ⎛ ⎛ Q2 ⎞ ⎞ ⎛ Q2 ⎞ ⎞ ⎜ (1 − F2 ) + ⎜ ⎟ F2 ⎟ ⎜ F1 + ⎜ ⎟ F2 ⎟ ⎜Q ⎟ ⎟ ⎜Q ⎟ ⎟ ⎜ ⎜ ⎝ 1⎠ ⎠ ⎝ ⎠ ⎝ ⎝ ⎠ (5.2) C12 = N1Q1 + N Q2 (5.3) To recover absolute concentrations we calibrated the system with a nM fluorophore solution and set all other values in relation to this calibration. The F2 (note that F2 = - F1) and Q2/Q1 can be calculated from Eqs. 5.1-3: ⎞⎛ C − C ⎞ −1 Q2 ⎛⎜ N1 ⎛ C12 ⎞ ⎜⎜ ⎟⎟ − 1⎟⎜⎜ 12 ⎟ = ⎜ ⎟ Q1 N app ⎝ C1 ⎠ C1 ⎟⎠ ⎝ ⎝ ⎠ 2 ⎛ N ⎛ C12 ⎞ ⎛ C1 ⎞ ⎞⎟ ⎟⎟ ⎟⎟ − ⎜⎜ F2 = ⎜ + ⎜⎜ ⎜ N app ⎝ C12 − C1 ⎠ ⎝ C12 − C1 ⎠ ⎟ ⎝ ⎠ (5.4) −1 (5.5) The values thus obtained can then be used to identify the second particle or make predictions of its environment. This method is used to calculate the fluorescence yield and concentration of lipid-bound V4-TMR in the presence of a constant fluorescent impurity with known fluorescence yield, assumed to be free TMR as discussed in results and discussion. In this chapter, because of the low fluorescence photon count rates obtained compared with the noise, all values have been corrected for background photon count rates185,210 and referred to the uncorrected values for the number of particles as Nmeas and to the corrected values as Nc. B is the photon count rates of background which in this work is referred to the photon count rates of PBS solution. The number of particles is corrected by the Eq. 5.6. 81 N c = N meas × F F+B (5.6) If one fluorescent species is present, Nc is N. If two fluorescent species are present, then Nmeas is used to describe the inverse of the amplitude, Napp is the background corrected value Nc (Eq. 5.2), N is the number of particles corrected for background and different fluorescence yields, and F2 describes the mole fraction of the second species. In other chapters, because of the high photon count rates detected, the noise is so small as to be neglected. FCS instrumentation. FCS experiments were performed using an Axiovert 200 inverted microscope. The samples were excited with the 530 nm line of laser beam from an Argon-Krypton laser (Melles Griot SP, Pte Ltd, Singapore). A dichroic filter (560DRLP) and an emitter (595AF60) were used to separate the excitation light from the emission fluorescence. The emitted fluorescence was detected by an APD (PerkinElmer Canada Inc., Canada) and then the signals were sent to a digital correlator. Interaction of V4 with R18. 200 nM unlabeled V4 was titrated by 10 nM R18 in PBS buffer. FCS experiments were performed at room temperature. Interaction of V4-TMR with LPS. The stock solution of V4-TMR in DMSO (2 mM) was diluted with PBS to 100 nM. LPS was dissolved in PBS to different concentrations (50, 100, 150, 200, 300, 400, 500, 600, 700, 1000, 2000 and 10000 nM). The mixture of 82 peptide and different concentrations of LPS were incubated for at least hours to reach equilibrium. FCS experiments were performed at room temperature. Interaction of V4 with FITC-LPS. 500 nM FITC-LPS and different concentrations of V4 (10 nM, 100 nM, µM) were mixed followed by at least four hours incubation. FCS measurements were performed using the 488 nm Argon-Krypton laser line for excitation at a power of 10 µW. The emission light was filtered by a dichroic filter (505DRLP) and an emitter (530DF30). Interaction of V4-TMR with lipid A and PC. Lipid A and PC were dissolved in PBS respectively and diluted to 10 µM. The mixture of V4-TMR and lipid A or PC was incubated for at least hours for FCS experiments. Interaction of V4-TMR with SUVs. The procedure is similar to that of interaction of V4- TMR with LPS. The SUV solutions with different lipid compositions were diluted to 50 µM (lipid concentration) and incubated with 200 nM V4-TMR for at least hours followed by FCS experiments. Fluorescence confocal imaging. Studies of V4-TMR attachment on glass coverslips (0.17 mm thick) was performed with a confocal microscope (FluoViewTM FV300,Olympus), equipped with a HeNe laser (543 nm) and a long pass emission filter at 560 nm. A PBS solution with either 100 nM TMR, µM V4-TMR or µM V4-TMR with 50 µM LPS were placed on the coverslilp and a stack of 60 confocal images of each solution were 83 acquired from 15 µm below coverslip to 45 µm above coverslip with a step size of µm. The average fluorescence intensity of each confocal image was calculated and the values for the surface and solution were reported. 5.3 Results 5.3.1 Calibration of the FCS setup The FCS setup was first calibrated with a nM solution of TMR in PBS. Measurements were performed in replicates and fitted with a one-particle model. The laser power was set to 100 µW before entering the microscope. The background photon count rates of PBS buffer were 0.9 kHz. The average number of particles measured was Nmeas = 0.388 ± 0.006 and the average diffusion times was τD = 56.9 ± 0.8 µs. After background correction the number of particle was N = 0.349 and fluorescence yield Q was calculated to be 51.6 kHz (Eq. 5.1). The diffusion time of micellar LPS was identified to be 1.77 ± 0.50 ms by using FITC-LPS (Table 5.1). The diffusion time of lipid SUVs was determined to be in the range of 1.3 to 3.5 ms by labeling with R18. 5.3.2 Solubility of V4-TMR First the solubility was tested in PBS buffer. For V4-TMR solutions of nM the photon count rates were at background level and no correlations could be detected. We have subsequently chosen concentrations of 100 - 200 nM of V4-TMR for FCS measurements. At a concentration of 100 nM the average number of particles measured was Nmeas = 0.098 ± 0.004. There was one species in the solution with τD= 52.5 ± 1.8 µs. After background correction the number of particle was N = 0.064 and Q was determined to be 84 59.5 kHz. Because of the similar τD and Q of V4-TMR and of free TMR, it is likely that the measured particles correspond to free TMR and represent an impurity. Only on rare occasions some strong peaks could be observed in the photon count traces. A two-particle model had to be used in those cases where the second particle had a small fraction and a strongly variable τD of 684 ± 440 µs assumed the same fluorescence yield. These peaks might point towards peptide aggregation. Fig. 5.1 R18 incorporates into V4 aggregates. (A) ACF of 200 nM unlabeled V4 in the presence of 10 nM R18. The solid line is the fit to the data depicted in grey. Nmeas= 1.669; τD1 = 54.4 µs; τD2= 31.0 ms. (B) Photon count rates trace of V4 with R18. To test for peptide aggregation in PBS, unlabeled V4 solutions were titrated with the amphipathic dye R18. In these experiments, large aggregates were detected as shown by the large τDs and distinct peaks in the photon count traces (Fig. 5.1). The diffusion time distribution of aggregates was quite wide, ranging from several hundreds of microseconds to tens of milliseconds. These experiments indicate that V4-TMR is aggregated and the fluorescence is strongly quenched. Therefore we tried to dissolve V4TMR in different solvents. Due to the hydrophobic and positively charged characteristic of V4, we tried DMSO as well as the detergent Triton-X100 to overcome the 85 hydrophobicity, and alternatively pure de-ionized water to minimize ions that could shield the positive charges and facilitate aggregation (Fig. 5.2). Fig. 5.2 (A) ACFs of 200 nM V4-TMR in PBS, water and 0.05% Triton-X100. The measured particle numbers are as follows: NV4-TMRPBS = 0.182 ± 0.008, τD1 = 56.7 ± 0.5 µs, τD2 = 684 ± 440 µs; NV4-TMRwater = 0.635 ± 0.076, τD1 = 54 µs (fixed), τD2 = 447 ± 30 µs; NV4-TMRTriton-X100 = 5.167 ± 0.737, τD1 = 54 µs (fixed), τD2 = 317 ± 47 µs. (B) ACFs of 1nM rhodamine 6G and 100 nM V4-TMR in DMSO. In PBS, 200 nM V4-TMR yielded an Nmeas of 0.182 ± 0.008. In Triton-X100, at the same V4-TMR concentration Nmeas was 5.167 ± 0.737, which is an increase of a factor 28. A two-particle model was used to fit the data and besides a fast species with 53.7 ± 1.0 µs, which is assumed to be free TMR, a slow species was detected with τD = 317 ± 47 µs. In de-ionized water Nmeas was 0.635 ± 0.076 which is an increase of a factor 3.5 compared to PBS solutions. In this case, V4-TMR solutions also showed two τDs, one again similar to free TMR and the other 447 ± 30 µs. 86 In DMSO, strong quenching was observed and the measured number (compared to a calibration with nM Rho 6G in DMSO) rose by a factor of 2.1 (Fig. 5.2B). Although an increase in Nmeas can be observed in the different solvents, the value of Nmeas always remained below the expected value by almost a factor of 20 in the best case (TritonX100). In the rest of the work experiments have been performed in PBS solution since it is physiologically the most relevant condition. 5.3.3 Binding of V4-TMR to LPS The putative target molecule for antimicrobial peptides in the outer membrane of bacteria is LPS108. Therefore, at a concentration of 100 nM V4-TMR, increasing concentrations of LPS were added to test for binding activity. The dependence of the ACF on the concentration of LPS is shown in Fig. 5.3. Two components can be distinguished in solution, a fast diffusing species (fixed at τD1 = 52 µs) and an average slow diffusing species (τD2 = 1.36 ± 0.17 ms) with a diffusion time similar to that of LPS micelles (τD =1.77 ± 0.50 ms). With increasing concentrations of LPS, the amplitude of the ACF decreased continuously, indicating an increasing number of fluorescent particles in the confocal volume. At the same time the overall photon count rates increased synchronously with the apparent number Napp (Fig. 5.4A). 87 Fig. 6.9 Interaction of antimicrobial peptides with Rho-PE labeled DPPG LUVs. 125 Polymyxin B promoted aggregation at a low peptide/lipid ratio of 1:40 with an increase in amplitude of the ACF and a slight increase in diffusion time. At a peptide/lipid ratio of 1:20, the shape of the ACF changed markedly with the emergence of a much longer diffusion time, indicative of strong aggregation in solution. An increase of the peptide/lipid ratio to 1:8 resulted in the detection of fluorescent particle with much lower diffusion time in addition to the large aggregates. Little further change in the ACF could be observed with additional polymyxin B. The addition of V4 also promoted aggregation. With increasing V4 concentration, the amplitude of the ACF was initially constant at peptide/lipid ratios of 1:40 and 1:20, and then rose steeply at a peptide/lipid ratio of 1:8 followed by a decline at the ratio of 1:4. The normalized ACF showed that a small amount of V4 at a peptide/lipid ratio of 1:40 and 1:20 did generate little change in the diffusion time compared with that of Rho-PE labeled DPPG LUVs. An increased peptide/lipid ratio to 1:8 led to a strong increase in the diffusion time. When the peptide/lipid ratio increased to 1:4, V4 disrupted DPPG vesicles and the diffusion time decreased. In addition, occasional larger aggregates were detected in the solution. 126 Fig. 6.10 Comparison of Napp and diffusion time of antimicrobial peptides interacting with Rho-PE labeled DPPG LUVs (■ diffusion time;● Napp) The comparison of apparent particle number Napp and the diffusion time after addition of the studied antimicrobial peptides to Rho-PE labeled DPPG LUVs are shown in Fig. 6.10. Except magainin 2, which did not induce vesicle disruption in the range of examined peptide/lipid ratios, the other three antimicrobial peptides all promoted aggregation and induced vesicle disruption. The study of melittin showed that there was little change in both Napp and the diffusion time at a peptide/lipid ratio of 1:40. At a peptide/lipid ratio of 1:20, the diffusion time increased with a large variance. When the peptide/lipid ratio increased further, there was consistent appearance of aggregates in solution. At least two fluorescent species were detected in the solution. Only the smaller species was plotted (Fig. 6.10). With increasing peptide/lipid ratio, the diffusion time became smaller, 127 indicating that vesicles have been disrupted. Polymyxin B began to promote slight aggregation at a peptide/lipid ratio of 1:40. At a peptide/lipid ratio of 1:20 big aggregates were detected in solution. When the peptide/lipid ratio increased further, the diffusion time apparently decreased which we attribute to fragments of the disrupted vesicles. Similar to labeled POPG LUVs, and in addition to the smaller fragments seen, there were also aggregates with longer diffusion time than labeled DPPG LUVs (not shown). Similarly at a V4 peptide/lipid ratio from 1:4 to 1:2, only shorter diffusion times were shown indicating vesicle disruption. In this case there were also aggregates in solution as shown in the normalized ACFs. 6.3.5 Visualization of antimicrobial peptides interacting with Rho-PE labeled LUVs The diameter of LUVs is about 100 nm well below the optical resolution limit. However, aggregation leading to larger particles can be observed. Fig. 6.11 shows the confocal image of labeled LUVs with and without peptides. As expected, we cannot see any detail of LUVs in the solution. The LUV solution was homogeneous in the scanned range without any large aggregates. In the presence of magainin 2, at the highest studied peptide/lipid ratio of 1:2, there was no change in the confocal images indicating that there were no large aggregates induced by magainin 2. However the other three antimicrobial peptides induced aggregation to different extent. The aggregation caused by melittin, polymyxin B and V4 at peptide/lipid ratio of 1:4, 1:8 and 1:4, respectively (Fig. 6.11). 128 Fig. 6.11 Confocal images of Rho-PE labeled LUVs in the absence and presence of antimicrobial peptides. (Final concentration of lipids: 40 µM; final peptide/lipid ratio: magainin 2, 1:2; melittin, 1:4; polymyxin B, 1:8; V4, 1:4) 129 Fig. 6.12 Antimicrobial peptides interacting with rhodamine 6G entrapped POPG LUVs with different incubation times Fig. 6.13 Antimicrobial peptides interacting with rhodamine 6G entrapped DPPG LUVs with different incubation times 130 6.3.6 Effect of incubation time on the activity of antimicrobial peptides Antimicrobial peptides are known to be present shortly after bacterial infection and perform their function in a short time. In order to know the effect of incubation time on the ability of antimicrobial peptides interacting with membranes, an overnight incubation was performed on rhodamine 6G entrapped POPG and DPPG vesicles. Although the incubation time has been increased from one hour to overnight, there is little change in the ACF for all antimicrobial peptides (Fig. 6.12 amd 6.13). The similarity for both two incubation times indicated that incubation time has no effect on the interaction. A one hour incubation time is enough to induce maximal permeation. Compared with POPG, DPPG LUVs showed a slight decrease in fraction of rhodamine 6G entrapped vesicles for the longer incubation time. This is due to the different ability of lipids to entrap fluorophores inside. 6.3.7 Real time interaction of V4 with rhodamine 6G entrapped POPG LUVs LUVs composed of POPG were used to investigate the real time interaction of V4 with vesicles at a high peptide/lipid ratio. Fig. 6.14 depicts the process of the leakage of fluorophore entrapped LUVs (20 µM POPG lipid concentration) caused by V4 (10 µM). The leakage occurred in less than 10 minutes. At the beginning, the Rhodamine 6G entrapped LUVs diffused as large fluorescent particles, whose diffusion time was 6.46 ± 2.56 ms (Fig. 6.14A). After less than minutes incubation with V4 two diffusion times could be detected. The two fluorescent species in the solution have diffusion times of 47.2 ± 4.2 µs and 24.6 ± 8.7 ms (Fig. 6.14B). After minutes, there was only one fluorescent species detected in the solution with a diffusion time of 54.7 ± 4.8 µs (Fig. 131 6.14C). This value compared well with the diffusion time of Rhodamine 6G of 56.6 ± 1.3 µs, which indicated that the Rhodamine 6G entrapped inside the LUVs has been released into solution. Fig. 6.14 Leakage of fluorophore entrapped LUVs (20 µM POPG lipid) was caused by V4 (10 µM) in less than 10 minutes. (A) At the beginning LUVs with Rhodamine 6G entrapped diffused as large fluorescent particle with a diffusion time on the order of ms. (B) After less than minutes, leakage occurred with some Rhodamine 6G released into the solution from vesicles. The free Rhodamine 6G was detected as a particle with a diffusion time of 47.2 ± 4.2 µs. In addition, some large-size particles appeared in the solution with long diffusion time of more than 20 ms. (C) After minutes, only released Rhodamine 6G can be detected with the diffusion time of 54.7 ± 4.8 µs. 6.4 Discussion 6.4.1 Mechanisms of antimicrobial peptides Both POPG and DPPG are negatively charged phospholipids, which are used to mimic bacterial membranes, and antimicrobial peptides displayed similar action on both membrane mimics. Therefore we will focus on POPG to discuss the mechanism of 132 antimicrobial peptides inducing membrane permeation. The comparison of mechanism of different antimicrobial peptides is summarized in Table 6.1. Table 6.1 Comparison of mechanisms of antimicrobial peptides inducing membrane permeation Peptide Magainin Melittin Polymyxin B V4 Mechanism (Increasing peptide/lipid ratio →) Pore formation (no aggregation)→ membrane disruption Pore formation →membrane aggregation →membrane disruption+aggregation Membrane disruption + aggregation Membrane aggregation → membrane disruption +aggregation It is believed that maginin induces membrane permeation through the formation of a torroidal pore142,143. However there is also some evidence that magainin disrupts the membrane212. Our data suggest that magainin induces pore formation to cause membrane permeation. In this study at a peptide/lipid ratio of 1:8 and 1:4, magainin induced apparent leakage of entrapped POPG LUVs. However at these two ratios, no size change of the labeled POPG LUVs could be observed. This shows that magainin induces pore formation at these peptide/lipid ratios. When the peptide concentration increased further (1:2.67 and 1:2), magainin induced membrane destabilization, leading to membrane disruption indicated by a decrease in the size of the vesicles. Melittin is known to form pores on the membrane and lead to the death of the target cells213,214. The present study showed that melittin also induces pores to perform its function. At peptide/lipid ratios of 1:8 and 1:4, melittin induced partial or complete leakage of entrapped POPG LUVs. However, no particle smaller than POPG LUVs was detected in the experiment of labeled POPG LUVs in the presence of melittin, which suggests that POPG LUVs were not disrupted. Instead, pores were formed at these two 133 peptide/lipid ratios, leading to membrane permeation. When the peptide/lipid ratio increased to 1:2.67 and 1:2, smaller aggregates were found as shown by the decrease in diffusion time (Fig. 6.6). In contrast to magainin 2, melittin promoted aggregation during the process of inducing vesicle permeation. Melittin induced slight aggregation of POPG LUVs at peptide/lipid ratio of 1:8, and at peptide/lipid ratio of 1:4, the aggregates became bigger. Because melittin formed pores at these two ratios, the aggregates might be the cluster of the empty or intact POPG LUVs or fused POPG LUVs or all. When the peptide/lipid ratio increased to 1:2.67 and 1:2, except the fragments of broken LUVs in the solution, there were also some aggregates, which were not shown in Fig. 6.6 but were indicated in Fig. 6.5. These aggregates were probably the result of coalesced POPG LUVs fragments. Polymyxin B is an antimicrobial peptide similar to melittin with high toxicity. Current research focuses on the interaction between polymyxin B and the outer bacterial membrane in which LPS plays an important role215-217. Little is known about the fate of the inner membrane. This study demonstrates that polymyxin B disrupts the membrane at peptide/lipid ratio of 1:8. The great variance in the diffusion time (from 1.6 ms to s) indicated the presence of both fragments and aggregates of the vesicles in the solution. The fragments probably coalesced to form large aggregates. This was corroborated by increased ratio of peptide/lipid to 1:4, 1:2.67 and 1:2. Excluding large aggregates, a decrease in the diffusion time of the labeled POPG LUVs was detected, possibly representing vesicle fragments. Correspondingly, at these ratios polymyxin B induced complete leakage from the entrapped POPG LUVs. Taken together, the results suggest 134 that polymyxin B adopts the carpet mechanism to cause membrane permeation without a pore formation process. V4 peptide did not induce membrane permeation until a peptide/lipid ratio of 1:2. However, the membrane did change at peptide/lipid ratios lower than 1:2. At peptide/lipid ratios of 1:8 and 1:4, V4 induced a small increase in the diffusion time of the labeled POPG LUVs. At a peptide/lipid ratio of 1:2.67, the increase was more apparent and the diffusion time reached about twice the value of POPG LUVs. Correspondingly the particle number in the confocal volume decreased when the peptide/lipid ratio was increased from 1:8 to 1:2.67. These results indicate that V4 induces POPG LUVs aggregation but without leakage. When the peptide/lipid ratio increased to 1:2, the diffusion time of the labeled POPG LUVs decreased strongly with a concomitant increase in Napp. Therefore V4 induces membrane permeation at this ratio. It should be noted that except for the fragments of the vesicles at a peptide/lipid ratio of 1:2, there were also large aggregates which might be the result of coalesced POPG LUV fragments after V4 induced vesicle disruption. Similar to polymyxin B, no pore formation was detected. However, different from polymyxin B, membrane aggregation is promoted by V4 before membrane disruption. The results of confocal imaging were consistent with those of FCS. Magainin did not induce any aggregation in contrast to the other three antimicrobial peptides (Fig. 6.11). It should be noted that the concentration of large aggregates is relatively low and in FCS 135 experiments, the large aggregates are seen only occasionally, thus explaining why in the majority of FCS measurements a homogeneous solution is observed. 6.4.2 Comparison of different antimicrobial peptides Although all the studied antimicrobial peptides finally disrupt the membrane, adopting the carpet mechanism or a modified carpet mechanism to perform their function, there were apparently some differences between them. Firstly, before the vesicles were disrupted, magainin and melittin formed pores in the vesicle membranes, which were detected at a certain intermediate peptide/lipid ratio. However, polymyxin B and V4 disrupted the membrane directly. No pore formation was detected. This difference might result from the structure of the peptides. It is known that magainin and melittin form αhelices upon interaction with lipid membranes. This structure makes it possible that hydrophobic and hydrophilic amino acids of the peptide spatially separate to form two faces with different functions. The hydrophobic face of this secondary structure interacted hydrophobically with the alkyl chains of the phospholipids. Correspondingly, the hydrophilic face not only interacted with the head group of the phospholipids through electrostatic interaction but also pointed inwards to form pores. When the peptide/lipid ratio was further increased, the peptides penetrate into the inner layer of the vesicle, destabilize the membrane and finally cause membrane disruption at high concentrations. Different from the α-helical antimicrobial peptides, polymyxin B and V4 are cyclic peptides which have a relatively rigid structure. It is difficult for these cyclic peptides to change the conformation and form a similar structure to α-helical peptides. Therefore the polymyxin B and V4 possibly act in a detergent-like manner to disrupt membranes. 136 The differential propensity to aggregate differentiates the antimicrobial peptides under investigation. Within the studied range of peptide/lipid ratio, mellitin, polymixin and V4 showed apparent aggregation, which makes them different from magainin 2. Whether aggregates formed or not is possibly related to hydrophobic interactions. Study of the amino acid distribution of magainin in the α-helical conformation showed that it is amphipathic with positive charges concentrated in the half circle in the helical wheel projection and hydrophobic amino acids distributed in the other half circle5; some polar amino acids separated them into several small hydrophobic portions which weakened the hydrophobicity. Therefore when magainin absorbed on the surface of lipid vesicles or vesicle fragments, the hydrophobic interaction was not strong enough to bring the vesicles close together for aggregation. Different from magainin 2, melittin was observed to induce leakage of POPG LUVs at a peptide/lipid ratio of 1:8. When the peptide/lipid ratio increased, melittin induced aggregation of the fragments of the broken vesicles. The aggregation is probably caused by the strong hydrophobic force between the adsorbed melittin and phospholipids. The αhelical conformation shows that the polar amino acids of melittin are concentrated in a region smaller than a half circle. The hydrophobic amino acids are also concentrated in a region to form a hydrophobic N-terminus which has higher hydrophobicity than magainin 25. Therefore the strong hydrophobic interaction might bring the POPG LUVs or fragments of disrupted vesicles close together to form aggregates. It should be noted that melittin might induce vesicle fusion during the aggregation. However, using FCS, it is impossible to distinguish whether these vesicles or vesicle fragments fused or just form 137 aggregates. Ohki and colleagues studied melittin-induced PC and PS vesicle leakage and fusion and membrane micellization. They found that at high peptide/lipid ratio, melittin induced both vesicle fusion and micellization for both lipids129. This result is consistent with our findings. Similar to melittin, hydrophobic interactions might also play an important role for polymyxin B induced aggregation. Although polymyxin B is a small antimicrobial peptide, the N-terminal fatty acid 6-methylheptanoic/octanoic and D-Phe5-Leu6 also formed the hydrophobic part of polymyxin B218. Possibly the hydrophobic interaction between the hydrophobic part of polymyxin B and vesicle fragments connected a large number of vesicle fragments to form aggregates. V4 is a highly hydrophobic antimicrobial peptide. It even aggregated in the buffer itself because of the high hydrophobic interaction between the peptide molecules219. In the presence of POPG LUVs, V4 began to aggregate the POPG LUVs at peptide/lipid ratio of 1:8. When peptide/lipid ratio increased to 1:2, it induced membrane disruption, and further aggregated fragments of disrupted vesicle to form larger aggregates. Comparison of the diffusion time of the labeled POPG LUVs after addition of different antimicrobial peptides showed that the particle size after magainin induced vesicle disruption was much larger than for the other studied antimicrobial peptides. At peptide/lipid ratio of 1:2, the diffusion time of the broken vesicle fragments caused by magainin was 1.13 ms. However, the diffusion times of the vesicle fragments caused by 138 other antimicrobial peptides were about 100 µs or less, excluding the aggregates in all cases. This difference indicated that magainin formed complexes of lipid and peptide molecules whereas the other antimicrobial peptides broke the vesicles into smaller complexes, possibly phospholipid monomers or oligomers. It should be noted that the comparison of the particle number of the labeled POPG LUVs showed that the particle number of fragments of the broken vesicles caused by magainin was much higher than that caused by the other antimicrobial peptides. This can be attributed to the aggregation caused by melittin, polymyxin B and V4. Most broken vesicle fragments aggregate leading to a relatively low number of small complexes detected in the solution. Therefore the aggregation caused by melittin, polymyxin B and V4 resulted in the smaller Napp than magainin did as well as a big variance. Nrho which is the particle number of rhodamine 6G, including the pre-existing dyes and the released dyes from the entrapped vesicles, gives an indication of the extent of leakage. Combined with Nvesicle and F2, the status of the solution at different peptide/lipid ratio is achieved. Magainin 2, melittin and polymyxin B began to induce an obvious increase in Nrho at peptide/lipid ratio of 1:8 and Nvesicle and F2 dramatically decreased at the same peptide/lipid ratio, indicating that magainin 2, melittin and polymyxin B have comparable ability to induce membrane permeation. V4 induced membrane permeation at peptide/lipid ratio of 1:2, which was higher than other peptides did. Nevertheless, V4 is a special case. Considering that the large peptide aggregates are not active and only about 0.77% of the peptide is active, the active peptide/lipid ratio is low to 1/260219. This value is much lower than the other studied antimicrobial peptides. This result is consistent with 139 the previous observation that V4 kills bacteria at much lower concentration compared with polymyxin B in vivo161. Therefore, V4 is potentially an effective antimicrobial agent, although its strong propensity to aggregate hinders its solubility and application. The diameter of LUVs is around 100 nm and the area of each lipid molecule is 0.4 nm2, therefore the ratio of peptide to LUVs is estimated to be 606:1103. Compared to V4-TMR binding to different lipid SUVs, the peptide/SUVs is about 1.2:1 which is much lower and more peptide is needed to make the vesicles permeable. Therefore binding is the first step of V4 interacting with membrane and when more V4 binds to membrane, V4 begins to perform its function, inducing membrane permeation. The study of the incubation time indicates that antimicrobial peptides perform their function in a short time. Longer incubation time cannot lead to membrane permeation. It is peptide/lipid ratio that determines whether membrane is permeated or not. The real time experiment of V4 inducing membrane permeation shows that V4 acts on the membrane very rapidly with 10 minutes, confirming the rapid ability of antimicrobial peptides to target membranes. 140 [...]... compared to TMR of Q2/Q1 of 3 .21 90 Fig 5.5 Comparison of ACFs of V4-TMR and the complexes of V4-TMR with LPS, lipid A and PC The concentration of V4-TMR was 100 nM; the concentrations of LPS, lipid A and PC were 10 µM Fits to the data are given in solid lines Table 5.3 Comparison of interaction of V4-TMR peptide with LPS, lipid A and PC C (kHz)a V4-TMR Napp τD1 [µs] τD2 [ms] F2 [%] Q2/Q1b N2 3.8 ± 0.3... data fitting, with the first diffusion time τD1 fixed to 52 µs The values for the second particle, τD2, F2 and N2 are shown in Fig 5.6 The diffusion time of the larger particle τD2 was in all cases between 0.6 and 1.7 ms, similar to the expected diffusion time of lipid SUVs However, F2 and N2 differed markedly depending on the lipid used In the group of lipids with unsaturated lipid tails, the highest... µM The concentrations of antimicrobial peptides were 1, 2, 5, 10, 15 and 20 µM with corresponding peptide /lipid ratio of 1:40, 1 :20 , 1:8, 1:4, 1 :2. 67 and 1 :2 The laser power was 100 µW A dichroic filter ( 525 DRLP) and an emitter (545AF35) were used in experiments Interaction of antimicrobial peptides with Rho-PE labeled LUVs The same protocol was applied on the interaction of antimicrobial peptides with. .. Q2/Q1 ≈ 2 Both, V4-TMR: LPS, V4-TMR: lipid A had a τD of 1 to 2 ms 91 The molar fraction of complexes in solution, F2, were 80.8 % and 43.5 % for V4-TMR: LPS and V4-TMR: lipid A, respectively 5.3.7 Binding of V4-TMR to SUVs of pure lipids The interaction of V4-TMR (20 0 nM) with POPG, POPC, POPE, DPPG, DPPC and DPPE was compared by studying mixtures of V4-TMR with SUVs in PBS The concentration of lipids. .. unsaturated lipids increases the fluidity of the lipid bilayer, leading to less dense packing of the unsaturated lipid molecules This provides V4-TMR better access to the unsaturated lipid molecules and increases the chance of insertion into the bilayer In addition, the interaction between V4-TMR and the lipid tail groups is facilitated for the unsaturated lipids due to the larger flexibility of the tail... that of pure POPG SUVs The value of F2 and N2 were both very close to those of pure POPG SUVs, although the mole fraction of POPG in the mixture lipid was only 0.33 However POPC/POPE SUVs had smaller values of F2 and N2 compared to POPG SUVs The diffusion time of V4TMR: mixed lipids SUVs complex was similar to that of pure lipids SUVs, between 0.6 and 1 ms Fig 5.7 Binding of V4-TMR to SUVs of mixed lipid. .. 9.50 0.40 ± 0. 02 100 nM TMR 34 32 ± 6 82 423 ± 5 5 µM V4-TMR 306 ± 22 0 23 ± 2 5 µM V4-TMR: 50 µM LPS 1511 ± 455 727 ± 3 5.3.6 Comparison of V4-TMR binding to LPS, lipid A and PC Upon addition of V4-TMR to LPS, lipid A, and PC, the ACF changed significantly with the different binding processes Fig 5.5 shows the ACF of 100 nM of V4-TMR mixed with 10 µM of LPS, lipid A or PC A concentration of 10 µM was chosen... flexibility of the tail groups211 Both effects, the packing of lipid molecule in the bilayer, and the flexibility of the tail groups could contribute to the higher binding affinity of V4-TMR for unsaturated lipids 100 CHAPTER 6 INVESTIGATION OF THE MECHANISMS OF ANTIMICROBIAL PEPTIDES INTERACTING WITH MEMBRANES 6.1 Introduction The functional mechanism of antimicrobial peptides has become an important... V4-TMR with lipid A was lower than that of LPS despite the fact that lipid A is considered to be the bioactive part of LPS 99 5.4.4 Saturation of lipids affects the binding with V4-TMR The affinity of V4-TMR for lipids SUVs with the same head group was always larger for unsaturated lipids (POPG, POPE, POPC) than for saturated lipids (DPPG, DPPE, DPPC) as shown in Fig 5.6B The double bond of the unsaturated... peptide /lipid ratio of 1 :2, permeation of POPG vesicles occurred Below peptide /lipid ratio of 1 :2, there was no obvious leakage Nvesicle and F2 showed that there was no change up to peptide /lipid ratio of 1:4 At peptide /lipid ratio of 1 :2. 67 due to the apparent aggregation of the POPG vesicles, Nvesicle and F2 decreased to some extent When peptide /lipid ratio increased to 1 :2, vesicles disappeared and F2 . Comparison of ACFs of V4-TMR and the complexes of V4-TMR with LPS, lipid A and PC. The concentration of V4-TMR was 100 nM; the concentrations of LPS, lipid A and PC were 10 µM. Fits to the data. solutions of 20 0 nM V4-TMR and 50 µM of these lipid mixtures, the POPE/POPG SUVs showed a similar ACF with that of pure POPG SUVs. The value of F 2 and N 2 were both very close to those of pure. proportional to the apparent number of particles N app is given by 81 () () 2 2 1 2 2 2 2 1 2 2 2 2 1 2 1 2 2 1 2 1 1 1 111 )0( ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ +− ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ +− ×= ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ + ×==− ∞ F Q Q F F Q Q F N F Q Q F F Q Q F NN GG app