Lipid Membrane Interactions of the Cationic Antimicrobial Peptide

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The lipid membrane interactions of the cationic antimicrobial peptide chimeras melimine and cys-melimine. Thomas Berry, Debarun Dutta, Renxun Chen, Andrea Leong, Huixin Wang, William Alexander Donald, Maryam Parviz, Bruce Cornell, Mark Willcox, Naresh Kumar, and Charles G. Cranfield Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01701 • Publication Date (Web): 17 Aug 2018 Downloaded from http://pubs.acs.org on August 18, 2018

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The lipid membrane interactions of the cationic antimicrobial peptide chimeras melimine and cys-melimine. Thomas Berry,a* Debarun Dutta,b,c Renxun Chen,c Andrea Leong,b Huixin Wang,c William A. Donald,c Maryam Parviz,e Bruce Cornell,d Mark Willcox,c Naresh Kumar c and Charles G. Cranfielda* a. School of Life Science, University of Technology Sydney, PO Box 123, Ultimo, NSW 2007, Australia. b. School of Optometry and Vision Science, The University of New South Wales, Sydney, NSW 2055, Australia. c. School of Chemistry, The University of New South Wales, Sydney, NSW 2055, Australia. d. SDx Tethered Membranes Pty Ltd, Unit 6, 30-32 Barcoo St, Roseville, NSW 2069, Australia. e. School of Mathematical and Physical Sciences, University of Technology Sydney, PO Box 123, Ultimo, NSW 2007, Australia.

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Abstract

Melimine and its derivatives are synthetic chimeric antimicrobial agents based on protamine and melittin. The binding of solubilised melimine and its derivative, with a cysteine on N-terminus, (cys-melimine) on tethered bilayer lipid membranes (tBLMs) was examined using AC electrical impedance spectroscopy. Addition of melimine and cys-melimine initially increased membrane conduction, which subsequently falls over time. Results were obtained for tBLMs comprising zwitterionic phosphatidylcholine, anionic phosphatidylglycerol or tBLMs made using purified lipids from Escherichia coli. The effect on conduction is more marked with the cysteine variant than the non-cysteine variant. The variation in membrane conduction most probably arises from individual melimines inducing increased ionic permeability which is then reduced as the melimines aggregate and phase separate within the membrane. The actions of these antimicrobials are modelled in terms of altering the critical packing parameter (CPP) of the membranes. Variations in the peptide length of cys-melimine were compared with a truncated version of the peptide, cys-mel4. Results suggest that the smaller molecule impacts the membrane by a mechanism that increases the average CPP, reducing membrane conduction. Alternatively, an uncharged alanine-replacement version of melimine still produced an increase in membrane conduction, further supporting the CPP model of geometry-induced toroidal pore alterations. All the data were then compared to their antimicrobial effectiveness for Gram positive and Gram negative strains of bacteria, and their fusogenic properties were examined using dynamic light scattering in 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine lipid spheroids. We conclude that a degree of correlation exists between the antimicrobial effectiveness of the peptides studies here with their modulation of membrane conductivity.


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INTRODUCTION Antimicrobial peptides (AMPs) are ancient weapons used as part of innate immune systems against bacteria, fungi and viruses.1 They consist of short chains of amino acids, between 10-50 residues long; are almost always positively charged (cationic); and are often amphipathic.2 There are many hypotheses as to how AMPs kill microbes,3-4 these include the creation of transmembrane pores;5 the activation of hydrolases that degrade the cell wall;6 the scrambling of lipids within the membranes of the microbe; disruption of membrane function;7 and disruption of intracellular processes after permeation of the cell membrane.8 Melimine is a 29 amino acid synthetic peptide with an amino acid sequence: TLISWIKNKRKQRPRVSRRRRRRGGRRRR It is a chimera of the natural AMPs melittin and protamine. This chimera was selected after it was shown that a combination of melittin and protamine could prevent the growth of Pseudomonas aeruginosa and Staphylococcus aureus more effectively than the original peptides alone.9 It is effective against both Gram-positive and Gram-negative bacteria,9-10 fungi and protozoa.11 It is not cytotoxic to mammalian cells at active concentrations, and bacteria do not appear to easily gain resistance as evidenced by repeated subculture experiments at subinhibitory concentrations.10 This peptide is resistant to heat sterilisation and results in over 99.9% reduction in adhesion of viable bacteria when it is tethered to substrates.12 We have previously shown that when melimine is covalently attached to a surface it is an effective antimicrobial and anti-adhesive agent in vitro10-11, 13-14 and in vivo/ex vivo.12, 15 An additional cysteine, added to the n-terminus of melimine (hereafter designated cys-melimine) has been employed to attach the peptide to substrates.13


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Melimine in solution kills the Gram-negative bacterium (Pseudomonas aeruginosa) via a loss of cytoplasmic membrane integrity. However, for the Gram-positive bacteria (Staphylococcus aureus), cell death was not directly related to the loss of membrane integrity.16 The mechanisms by which many AMPs cause membrane disruption remain unresolved. To examine the mechanisms of melimine and cys-melimine membrane disruption, tethered bilayer lipid membranes (tBLMs) in conjunction with electrical impedance spectroscopy were employed. A recent model of peptide-membrane interactions describes the actions of antimicrobials in terms of altering the critical packing parameter (CPP) of the bilayer and thereby altering the size of membrane pores already present in the membrane.17-19 The CPP, first described by Israelachvili and colleagues, describes the geometry of lipid clusters as a function of the surface area (a0), the hydrophobic lipid chain length (l) and the overall volume of the individual lipids (v) such that the CPP = v/a0l.20 The CPP is a semi-predictive measure of the geometric arrangement surfactants will take. A CPP = 1 is predictive of a lipid bilayer structure, whilst a CPP of 1/3 is predictive of a micelle. Recently, we identified a decrease in the weighted CPP (CPPw) as a result of adding the cyclic antimicrobial peptides kalata B1 or kalata B2 to membranes containing phosphatidylethanolamine (PE) lipids.18 Decreases in the CPPw were suggested to arise from an increase in the effective hydrated lipid head-group region (a0) as a result of the peptide. This, in turn, leads to more lipids absorbing to the curved toroidal pore regions (where the CPP = 1/3). In order to accommodate the decrease in CPPw, an initial response is to share the CPPw between regions of planar lipid bilayer and regions of highly curved membrane associated with toroidal pores existing within the membrane, seen in Figure 1. A consequence of lowered CPPw is to increase the percentage of lipids possessing a low CPP within the existing bilayer resulting in an increase in the diameter of the existing toroidal pores


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within the membrane, increasing membrane conduction. Were the peptide to cause the CPPw to rise, then the reverse would be true, decreasing of toroidal pore diameters would cause a drop in membrane conduction. In the current investigations, we identify how minor structural alterations in melimine and its analogues (Table 1) can impact the lipid bilayer conduction using tethered bilayer lipid membranes (tBLMs) in conjunction with electrical impedance spectroscopy (Figure 2A and B). We interpret these impacts as modulating the CPPw. We correlate these membrane conduction changes with the peptides’ ability to kill Gram positive and Gram negative bacteria in vitro.

Figure 1. Cartoon depicting how peptide interactions can alter the size of intrinsic toroidal pores in lipid bilayers according the weighted critical packing parameter (CPPw) model. Peptide interactions can influence changes to the effective hydrated lipid head-group region of neighboring lipids, affecting lipid packing within the lipid bilayer.


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Figure 2. A. Schematic of a tethered bilayer lipid membrane (tBLM). Shorter benzyldisulfide tetra-ethyleneglycol hydroxy terminated “spacers” attached to a gold electrode are interspersed with phytanyl terminated “tethers”. The hydrophobic phytanyl tether groups anchor the lipid bilayer 2-2.5 nm from the gold tethering electrode. A counter gold electrode is then positioned ~100 µM distant from the tethered lipid bilayer membrane to complete the electrical circuit. B The equivalent circuit model used to fit the electrical impedance spectroscopy data. In this model Ge represents the conduction of the PBS electrolyte bathing solution, Gm, the membrane conductance, Cm, the membrane capacitance, Qs, the imperfect capacitance (constant phase element, CPE) of the gold tethering electrode

Table 1. Summary of the five peptide derivatives and their properties.


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EXPERIMENTAL Tethered bilayer lipid membranes (tBLMs) tBLMs (Figure 2) were fabricated using pre-prepared tethered benzyl-disulfide tetraethyleneglycol phytanyl coated gold slides (SDx Tethered Membranes Pty Ltd, Australia). To these tethers, 8µL of a 3mM solution of a mobile lipid phase mixture of 70 % zwitterionic C20 diphytanyl-glycero-phosphatidylcholine lipid (SDx tethered membranes, Pty Ltd, Australia), and 30% C20 diphytanyl-diglyceride ether (SDx tethered membranes Pty Ltd, Australia) dissolved in ethanol, was added, and after a 2 minute incubation, was repeatedly washed with phosphate buffered saline (PBS) solution (pH 7.4). The tBLMs were then studied by AC impedance spectroscopy, yielding a typical capacitance of 0.8 - 1.2 µF cm-2, and a conduction of 0.3 - 1.5 µS, for a 2.1 mm2 area electrode at ~20°C and pH 7.2. Conductances and capacitances were measured using a TethaPod™ (SDx Tethered Membranes Pty Ltd, Australia). Melimine and its derivatives were diluted in PBS to 10 µM for each impedance measurement. To assess how melamine and cys-melimine interact with lipids form a bacterial source, we created tBLMs using lipid extracts from E. coli (Avanti Polar Lipids, USA). Negatively

charged

lipid

bilayers

were

made

by incorporating

palmitoyl-oleoyl-

phosphatidylglycerol (POPG) (0-30%) (Avanti Polar Lipids, USA) into the mobile lipid phase mixture.

Peptide synthesis Peptides were synthesized by conventional solid-phase peptide synthesis protocols and were obtained from Auspep pty. Ltd. (Vic, Australia). Peptides with ~90% purity were used in experiments.


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Bacterial killing assays The minimal inhibitory concentration (MIC) was determined via a microplate dilution assay in 96-well flat-bottom polystyrene plates (Greiner Bio-One, Kremsmünster, Austria), chosen for ideal optical properties. The assay medium was Mueller-Hinton broth (MHB; Oxoid Ltd, Basingstoke, UK) containing 0.2% bovine serum albumin (Sigma Aldrich, St Louis, MO, USA) to minimise adhesion of peptides to the well plate surface, and 0.01% acetic acid to reduce precipitation of the cationic peptides.21 Serial twofold dilutions of the antimicrobial agent were made in a series including 1000 µg/ml (as per a widely-accepted MIC test series22). The test organisms were grown overnight in Trypticase soy broth (Oxoid), and then twice centrifuged and resuspended in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 10 mM Na2HPO4 in H2O, pH 7.4). This suspension was adjusted with PBS to an optical density at 660 nm (OD660) of 0.08 for P. aeruginosa and E. coli, or 0.12 for Staphylococcus spp., corresponding to approximately 1×108 CFU/ml as tested by retrospective agar plate counts. This suspension was diluted and added to the wells to give a final concentration of 1×105 CFU/ml (confirmed by retrospective plate count). Blank wells contained sterile medium, and positive growth controls containing no antimicrobial agent were inoculated with bacteria. Wells were prepared in triplicate and the experiment was repeated three times. The plates were incubated at 37°C for 24 h with shaking, and the MIC was recorded as the lowest concentration of peptide at which no microbial growth was observed. Results are reported as micromolar (µM) concentrations to allow for comparison between peptideS. The minimal bactericidal concentration (MBC) of peptides was determined within the same assay as the MIC. Aliquots (100 µl) from all wells which were visually growth-free were


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transferred onto Mueller-Hinton agar (Oxoid). The plates were incubated at 37°C for 24 h and the resulting colonies were counted. The concentration that resulted in a 99.9% from the inoculum was recorded as the MBC.23

Mass spectroscopy Mass spectrometry experiments were performed using a linear trap quadrupole MS coupled to a 7 Tesla Fourier transform ion cyclotron resonance MS (LTQ-FTICR; Thermo Scientific) that is equipped with a custom external nanoelectrospray ionization (nESI) source. nESI emitters were prepared by pulling borosilicate capillaries to an inner orifice diameter of less than 1 µm. The electrospray capillaries were sputter-coated with a thin layer of Au and Pd for 20 s. A voltage of 1 kV was applied to the metal coating of the ESI emitter to initiate and maintain electrospray. nESI solutions were prepared containing 10 µM of cys-melimine and cys-mel4 in 49.5/49.5/1 methanol/water/acetic acid.

Dynamic light scattering Dynamic light scattering was run using a Zetasizer Nano S (Malvern Instruments Ltd., Southborough, UK) equipped with a 632.8 nm He–Ne laser (4 mW). The detection angle was 173° using a non-invasive backscatter technique, and the measurements were run at 25 ± 0.1 °C. The measurements were taken 15 minutes after the addition of the peptides. Lipid spheroids were composed of 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (PC 18:1) (Avanti Polar Lipids, USA) and were used in a 48.5:1 ratio with the peptides. 15 scans with 20 s interval were accumulated for each reading, and the measurements were repeated 3 times on each sample. Malvern Instruments software (DTS) was utilized to analyse the averaged size (nm) and intensity (%). The standard deviation (Std Dev) were calculated based on the acquired values of each


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measurement as these errors were significantly higher than the errors of readings. The optical properties of the examined molecules were not considered critical as their size is lower than the wavelength of the employed laser.24 A measurement of the zeta potential brought by the arginine rich peptides showed that each of the PC 18:1 lipid-peptide structures carried a positive charge demonstrating the associating of the peptides with the PC 18:1 lipid spheroids.

RESULTS AND DISCUSSION Electrical impedance spectroscopy Figure 3A shows that the presence of a cysteine group at the N-terminal side of the peptide induces an increase in membrane conduction. The membrane conduction initially increases, but then over a period of ~20 min returns to an approximate baseline value. The increase in conduction is more marked in tBLMs that are composed of E. coli derived lipids (Figure 4C with expanded ordinate scale). E. coli lipids are reported to be significantly more negative than the zwitterionic diphytanyl lipids used to obtain Figure 4A and B.25 The effect of negatively charged lipids was explored using tBLMs containing increasing amounts of POPG lipids seen in Figure 4D. Negatively charged lipids in tBLMs have previously been employed in a study of the Xenopus antimicrobial peptide PGLa.26 It is apparent that cys-melimine induces a greater response in the presence of these negatively charged lipids, suggesting the cationic peptide may be attracted to the negatively charged membrane surface. A truncated version of the melimine peptide, termed mel4 was also studied.27-28 As can be seen in Figures 3A and B, this truncated version causes a smaller response to the original melimine, while cys-mel4 causes a decrease in membrane conduction. The concentration of Mel4 was further increased to 30 µM try and illicit a change in the conduction at a concentration that matched its MIC value in S. aureus 31 (Table


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2). The observed response showed no significant change upon increasing the concentration of the peptide (see supporting information). This suggests that the peptide molecular shape, in addition to its charge, affects ion transport across lipid bilayers. To further explore the effect of charged groups within the peptide, another variant of the melimine without the cysteine residue, and with some of the arginines replaced by alanines, was used to observe its modulation of the conduction of zwitterionic tBLMs. This peptide was designated melAlanine and has the amino acid sequence: TLISWIKNKRKQRPRVSAAAAAAGGRRRR. As shown in Figure 3B, this peptide induced a marked increase in the membrane ionic conduction despite having a reduced net cationic charge.

Figure 3. A. A comparison of the conduction responses of melimine and cysteine terminated cys-melimine in diphytanyl PC tBLMs. B, Conduction changes induced by an uncharged equivalent peptide, melAlanine where the some of the arginines are replaced by alanine residues.


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Figure 4. A, tBLM conductance changes with time as a result of 10 µM melimine and 10 µM of truncated mel4. The truncated version leads to an almost negligible change in membrane conduction (n=3 for both peptides). B, tBLM conduction changes as a result of adding 10 µM cys-melimine and 10 µM of truncated cys-mel4. The truncated version leads to a decrease in membrane conduction (n=3 for both peptides). Note that the scales on the ordinate axes in A and B are equivalent. C, melimine and cys-melimine induced conduction changes in a tBLM created using E. coli derived lipids. D, cys-melimine causes increased conduction responses in various tBLMs containing increasing proportions of negatively charged POPG lipids.


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Mass spectrometry It was reasoned that cysteine variants of melimine could be dimerising in solution. To test this, high-resolution nanoelectrospray ionization mass spectrometry was used to identify whether disulfide-bridged homodimers of cys-mel4 and cys-melimine were formed in solution prior to addition to the tBLM. Figure 5A essentially shows only a monomeric form of truncated form of cys-melimine, cys-mel4 (> 99 % of peptide ion signal detected). For the full peptide, any crosslinking is also negligible (Figure 5B).

Figure 5. nESI mass spectra of (A) cys-Mel4 and (B) cys-Melimine. The peaks labelled with “†” and “‡” are assigned to [M+2Na+nH+mH3PO4](2+n)+ (n = 1 to 5) for m = 1 and 2, respectively. The peaks labelled with “*” are assigned to a minor impurity of cysmelimine, in which the N-terminal residues Cys-1 to Ile-4 are truncated (with 0 to 2 H3PO4 adducts).


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Inhibitory concentrations of peptides against bacteria To determine if there is any correlation in the peptides’ ability to increase membrane conduction with their ability to kill bacteria, various bactericidal assays were undertaken. Table 2 shows the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) for all the peptides used on four strains of staphylococci (Gram positive), and one strain of Pseudomonas aeruginosa and E. coli (Gram negative). Melimine without the cysteine moiety was highly effective against all strains with the exception of P. aeruginosa where its effectiveness was only moderate. Mel4 without the cysteine moiety had the highest potency against S. aureus 31 and S. epidermidis 013 but was variable on the other strains tested, both Gram negative and Gram positive. For both melimine and mel4, adding the cysteine amino acid to the N-terminus resulted in increased antimicrobial activity for some strains tested, most notably with cys-melimine for S. epidermidis 013 and P. aeruginosa 6294, and with cys-mel4 for S. aureus 152, S. epidermidis 013 and E. coli ATCC0157. Removing six arginines and replacing them with alanine (producing the peptide melAlanine) not only reduced the overall positive charge of the peptide, but also resulted in complete lack of antimicrobial activity. This reduced inhibitory effect seen may be due to melAlanine being unable pass through to the bacterial inner membrane. We recently developed a model in which AMPs are proposed to alter the CPPw of membranes.17-18 According to this model, the effect of AMP addition is to alter the CPPw which, in turn, can result in a variation in the diameter of pre-existing toroidal pores within the membrane. Examples of increased conduction are seen following the addition of AMPs to tBLMs include human cathelicidin antimicrobial peptide, LL-37,29 and the cyclotides, kalata B1


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and kalata B2.18 These peptides possess low MICs against Gram negative and Gram positive bacteria.30-31 The modulation of membrane conduction by cys-melimine can be correlated with its MIC on all tested Gram positive and Gram negative bacteria (Table 2) and the insertion of the peptide also correlates with the number of hydrophobic residues present, see supporting information. However, other amphiphilic antimicrobial agents that have a low MIC show only moderate changes to membrane conductivity.17, 29 Alternatively, melAlanine had little effect on the growth of bacteria, whilst still causing a substantial change in membrane conduction. This may indicate that membrane interactions are not the only bactericidal mechanisms of melimine and mel4. Cys-mel4 possesses high antimicrobial activity against Gram positive and Gram negative bacteria whilst decreasing the membrane conduction. This would suggest the addition of cysmel4 would increase the CPPw for the membrane. The results presented here would suggest that decreasing the permeability of bilayers (by increasing the CPPw) can lead to an alteration in the membrane geometry. Whether this is responsible for inducing a significant loss in bacterial cell viability is still to be determined. One possibility is that the peptides, by altering membrane geometry, modulate the properties of other membrane associated proteins in bacteria which subsequently affects their viability. Such changes in membrane geometry have been demonstrated to affect the functioning of bacterial ion channels such as gramicidin-A.19, 32

Table 2. The minimum inhibitory concentrations and the minimum bactericidal concentrations for the peptides against various Gram positive and Gram negative strains of


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Figure 6. Size comparison of the DLS results. The introduction of Melimine, cys-melimine and MelAlanine into the existing lipid spheroids, in a 48.5:1 lipid:peptide ratio, resulted in similar fusogenic properties. The introduction of the truncated peptides mel4 and cys-mel4 results in smaller lipid spheroids.

Dynamic light scattering (DLS) To test fusogenic properties of the peptide family, 1-oleoyl-2-hydroxy-sn-glycero-3phosphocholine (PC 18:1) lipid spheroids33 were prepared and the change in apparent diameter five minutes following the addition of the peptide was measured. A comparison of the apparent lipid spheroid diameter is given in Figure 6. The addition of the greater molecular weight peptides; melimine, cys-melimine and melAlanine, induce a greater change in the PC 18:1 lipid-


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peptide structures. It is evident that an approximate correlation exists between the changes induced by the peptides from the resultant size of PC 18:1 lipid-peptide structures over 10 minutes and the molecular weight of the peptides. A similar correlation with the molecular weight of the peptides exists with , the change in conduction in the diphytanyl PC tBLMs with the addition of the peptides and their MIC values. This may arise from the differing geometries of the peptide family and, in particular, the disposition of the polar charged amino acid residues to the non-polar surfaces of each molecule. That is highlighted through the loss of the correlation upon replacing the arginine residues with alanines.

CONCLUSIONS The actions of the chimeric peptide melimine and its analogues were investigated against tBLMs of varying lipid composition, and these results were compared to their antimicrobial actions in vivo. The conduction changes induced by these peptides on lipid bilayers are described in terms of their ability to alter the weighted critical packing parameter (CPPw) of the membrane. A subset of the analogues showed a strong correlation between the modulation of the membrane ionic permeability and the in vivo MIC values. The measurement of changes in conductivity of tBLMs provides a convenient and rapid method of identifying the likely physiological effects of drug analogues. Changes in membrane permeability may increase the metabolic burden experienced by bacteria in the presence of these chimeric peptides.


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ASSOCIATED CONTENT Supporting information (word document)

AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected] Funding Sources This study was supported by an ARC Discovery Project (DP160101664). .ACKNOWLEDGMENT The Authors acknowledge Dr Paul Duckworth for valuable discussions concerning this work. We declare that Bruce Cornell is a shareholder of Surgical Diagnostics Pty Ltd.

REFERENCES 1. Zasloff, M., Antimicrobial peptides of multicellular organisms. Nature 2002, 415 (6870), 389-395. 2. Hamamoto, K.; Yutaka, K.; Ye, Z.; Takashi, S.; Koichi, K., Antimicrobial activity and stability to proteolysis of small linear cationic peptides with D-amino acid substitutions. Mircrobiol. Immunol. 2002, 46 (11), 741 - 749. 3. Brogden, K. A., Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology 2005, 3 (3), 238-250. 4. Sani, M.-A.; Separovic, F., How Membrane-Active Peptides Get into Lipid Membranes. Accounts of Chemical Research 2016, 49 (6), 1130-1138. 5. Yang, L.; Weiss, T. M.; Lehrer, R. I.; Huang, H. W., Crystallisation of antimicrobial pores in membranes: magainin and protegrin. Biophysical Journal 2000, 79, 2002-2009.


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22. Rodríguez-Tudela, J. L.; Barchiesi, F.; Bille, J.; Chryssanthou, E.; Cuenca-Estrella, M.; Denning, D.; Donnelly, J. P.; Dupont, B.; Fegeler, W.; Moore, C.; Richardson, M.; Verweij, P. E., Method for the determination of minimum inhibitory concentration (MIC) by broth dilution of fermentative yeasts. Clinical Microbiology and Infection 9 (8), i-viii. 23. French, G., Bactericidal agents in the treatment of MRSA infections—the potential role of daptomycin. Journal of Antimicrobial Chemotherapy 2006, 58 (6), 1107-1117. 24. Kaszuba, M.; McKnight, D.; Connah, M. T.; McNeil-Watson, F. K.; Nobbmann, U., Measuring sub nanometre sizes using dynamic light scattering. Journal of Nanoparticle Research 2008, 10 (5), 823-829. 25. Matsuzaki, K., Why and how are peptide–lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes. Biochimica et Biophysica Acta (BBA)-Biomembranes 1999, 1462 (1), 1-10. 26. Cranfield, C. G.; Cornell, B. a.; Grage, S. L.; Duckworth, P.; Carne, S.; Ulrich, A. S.; Martinac, B., Transient potential gradients and impedance measures of tethered bilayer lipid membranes: Pore-forming peptide insertion and the effect of electroporation. Biophysical Journal 2014, 106, 182-189. 27. Dutta, D.; Zhao, T.; Cheah, K. B.; Holmlund, L.; Willcox, M. D. P., Activity of a melimine derived peptide Mel4 against Stenotrophomonas, Delftia, Elizabethkingia, Burkholderia and biocompatibility as a contact lens coating. Contact Lens and Anterior Eye 2017, 40 (3), 175-183. 28. Rasul, R. Novel Antimicrobial Biomaterials. [PhD Thesis] Sydney, Australia: University of New South Wales, 2010. 29. Nizalapur, S.; Ho, K. K. K.; Kimyon, O.; Yee, E.; Berry, T.; Manefield, M.; Cranfield, C. G.; Willcox, M.; Black, D. S.; Kumar, N., Synthesis and biological evaluation of N-naphthoylphenylglyoxamide-based small molecular antimicrobial peptide mimics as novel antimicrobial agents and biofilm inhibitors. Organic & Biomolecular Chemistry 2016, 14 (14), 3623-3637. 30. Dutta, D.; Kumar, N.; DP Willcox, M., Antimicrobial activity of four cationic peptides immobilised to poly-hydroxyethylmethacrylate. Biofouling 2016, 32 (4), 429-438. 31. Craik, D. J., Host-defense activities of cyclotides. Toxins 2012, 4 (2), 139-156. 32. Lundbaek, J. A.; Andersen, O. S., Lysophospholipids modulate channel function by altering the mechanical properties of lipid bilayers. The Journal of General Physiology 1994, 104 (4), 645-673. 33. Carnie, S.; Israelachvili, J. N.; Pailthorpe, B. A., Lipid packing and transbilayer asymmetries of mixed lipid vesicles. Biochimica et Biophysica Acta (BBA) - Biomembranes 1979, 554 (2), 340-357.


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TABLE OF CONTENTS GRAPHIC


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Lipid Membrane Interactions of the Cationic Antimicrobial Peptide

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