- Email: [email protected]
Antimicrobial Peptide NK-2 as an Emerging Therapeutic Agent: A Study with Phospholipid Membranes
Available online at www.sciencedirect.com
ScienceDirect Materials Today: Proceedings 18 (2019) 879–886
www.materialstoday.com/proceedings
ICN3I-2017
Antimicrobial Peptide NK-2 as an Emerging Therapeutic Agent: A Study with Phospholipid Membranes Sanat Karmakar*, Pabitra Maity, Animesh Halder Soft matter and Bio-Physics laboratory, Department of Physics, Jadavpur University, Raja . S. C. Mallick Road, Kolkata – 700032.
Abstract An antimicrobial peptide, NK-2, derived from a cationic core region of NK-lysin, is known to display antimicrobial activity towards negatively charged bacterial membranes. The aim of the present study is to get insights into the mechanism of antimicrobial activity. We have prepared unilamellar vesicles as model membranes composed of bacteria mimicking lipid composition. The interaction of NK-2 with phospholipid membranes has been investigated using a variety of experimental techniques, such as, isothermal titration calorimetry (ITC), zeta potential, dynamic light scattering (DLS). NK-2 exhibits strong binding affinity towards the negatively charged membranes, which indeed a characteristic of bacterial membrane. The weak binding affinity towards the neutral phospholipid suggests that NK-2 is expected to be inert towards normal eukaryotic cells. The ITC results show the overall endothermic response in the heat signal, suggesting the entropic contribution in the lipid –NK-2 interaction. One site binding model given by microcal origin, has been used to estimate the intrinsic binding constant and other thermodynamic parameters of binding kinetics of NK-2. The proliferation of size distribution of negatively charged vesicles containing NK-2 indicates the presence of large aggregates. Summarizing all experimental observations, we have suggested a possible mechanism of antimicrobial activity of NK-2.
© 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017). Keywords: Phospholipid membrane; Antimicrobial peptide; ITC ; Zeta potential; DLS
* Corresponding author. Tel.: +91-33-24572879 E-mail address: [email protected]
2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017).
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S. Karmakar et al. / Materials Today: Proceedings 18 (2019) 879–886
1. Introduction Antimicrobial peptides (AMP) are the evolutionary immune response towards many invading pathogens, such as viruses, bacteria, fungi etc [1, 2]. Their widespread distribution throughout the animals and plants and broad spectrum of targeted micro-organisms leads to successful development of multicellular organisms [3]. AMP target to bacterial membranes and create defects, such as pores without interacting with the specific receptors. There are several proposed mechanisms of antimicrobial action reported in literatures [4, 5]. Interestingly, different AMP uses a different mechanism to disrupt the bacterial membranes, which indeed depend on effective charge, hydrophobicity and length of the AMP. All pathways of antimicrobial action begins with the pore formation which eventually disintegrate the membrane [6, 7]. The driving force for the development of antimicrobial peptides as newer antiinfectives drugs is unpreventable emergence of bacterial resistance to conventional antibiotics. Therefore, the objective of the present study is to find out appropriate antimicrobial peptide which has potential for biomedical applications. NK-2 is one such potent peptide which has been studied systematically in order to obtain insights into the antimicrobial activity. Most AMP are cationic in nature and NK-2 is indeed derived from the cationic core regions of NK-lysin found in natural killer (NK) cell. The mechanisms of antimicrobial activity which cause disintegration of the lipid bilayer structure is difficult to infer in an in vivo experiment due to the high level of complexity of the cellular membrane [8]. Therefore, it is often useful to study model membrane composed of phospholipids in order to understand the underlying mechanism of antimicrobial activity [9]. As model membranes, large unilamellar vesicles (LUV) is an excellent model system to study the membrane-AMP interaction [10]. Unilamellar vesicles are the microscopic sac of single lipid bilayers. Besides model membranes, LUV are mostly used as vehicles of targeted drug delivery [11]. In order to study the antimicrobial activity, one needs to prepare LUV with bacterial membrane compositions. In the present article, we have systematically investigated the interaction of NK-2 with anionic membranes. A cationic peptide NK-2 possess +10 charges at physiological pH (7.4). The high positive charges within the NK2 promote strong binding to the negatively charged membranes. NK-2 exhibits an unordered structure in the form random coil configuration in aqueous solution and it adopts the α- helix in the presence of membranes [12]. NK-2 displays its antimicrobial activity against both gram-negative and –positive bacteria [13, 14], fungi, such as Candida albicans [13], the protozoan parasite Trypanosoma cruzi [15]. NK-2 exerts a potent activity against some cancer cell [16], but shows low hemolytic and cytotoxicity against human cells [13]. Besides the cell line studies, a variety of experimental studies with model membranes have been employed to elucidate antimicrobial activity of AMP [17]. It has been shown that NK-2 interacts specially with negatively charged lipids, such as lipopolysachharuse (LPS) which is a highly anionic component of the outer leaflet of gram-negative bacteria [6]. NK-2 binds to the LPS and induces a change in the endotoxin-lipid A aggregate structure, as revealed by small angle x-ray scattering. A recent study on the interaction of NK-2 with negatively charged membrane have shown the evidence of pores which is the prerequisite to the antimicrobial activity [10]. Differential scanning calorimetry and optical microscopy studies on the NKCS, an improved mutant of NK-2 have shown that it severely distorted, penetrated and perforated model lipid membranes that resembled bacterial membranes, but not those that were similar to human cell membranes [18]. Biophysical characterization of NK-2 at the air water interface has revealed adsorption and desorption kinetics which may be useful for understanding of its activity at different solution conditions [12]. The major problem that has impeded the development of drug therapeutics is the complexity of the biomembrane. Therefore, the present study focuses on the interaction of potent antimicrobial peptide NK-2 with negatively charged membranes which resembles with the composition of bacterial membranes. For comparison, interaction of NK-2 with neutral or zwetterionic lipid has also been investigated. A variety of experimental techniques, such as dynamic light scattering, zeta potential and isothermal titration calorimetry have been employed to study the NK-2 membrane interaction. The electrostatic behaviour of the charged membranes in the presence and absence of NK-2 has been envisaged using zeta potential. DLS is used to study the stability and to show the evidence of membrane-membrane interaction induced by NK-2. Finally the kinetics and thermodynamics of the interaction has been studied using isothermal titration calorimetry.
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2. Materials and Method 2.1. Materials Dioleoyl Phosphatidylcholine (DOPC), Dioleoyl phosphatidylethanolamine (DOPE), Dioleoyl Phosphatidylglycerol (DOPG) were obtained from Avanti Polar Lipids and peptide NK-2 from WITA GmbH, Berlin, Germany. They were used without further purification. 2.2. Preparation of LUV An appropriate amount of lipid in chloroform (10 mg/ml) was transferred to a 10 ml glass vial. A thin dried lipid film was prepared on the wall of the glass vial by gently passing nitrogen gas. The traces of the solvent were then removed by keeping the glass vial for a couple of hours in a vacuum desiccator. 2.5 ml of 1 mM HEPES (pH 7.4) was added to the dried lipid film so as to obtain the final desired concentration. Multilamellar vesicles (MLV) were produced by vortexing of hydrated lipid film for about 20-30 minutes. MLV suspensions are extruded successively through polycarbonate membranes having pore diameters of 400 , 200 and 100 nm using LiposoFast-Pneumatic from AVESTIN (Canada). This procedure results in a formation of LUV of diameter ∼ 100 nm, as measured by dynamic light scattering [19]. The polydispersity index obtained from the width of the distribution is much lower than 0.7 indicating that LUV are highly monodispersed. 2.3. Zeta potential and size distributions using DLS The size distributions and the zeta potential of LUV have been measured with a Zetasizer Nano ZS from Malvern Instruments. The instrument uses 2 mW He-Ne Laser of wavelength 633 nm to illuminate the sample. Zeta potential is determined from the electrophoretic mobility (µ) by laser Doppler velocimetry using Helmoltz-Smolushovski equation [20]. = 3 /2 ( ) (1) Where, η and ϵ are the coefficient of viscosity and the permittivity of the aqueous medium, respectively. Although, the Henry function, ( ) depends on the inverse Debye length (κ) and the radius (a) of the vesicle, the change in the size distribution of the vesicles does not alter the zeta potential significantly. This is due to the fact that zeta potential depends on the formation of electrostatic double layer. In this study, the zeta potential is estimated from the Smoluchowski approximation, in which the Henry function f(κa) takes its maximum value 1.5. In the present DLS setup, back scattered light at an angle of 173o is detected and sent to digital signal processing correlator. The rate of decay of intensity auto-correlation function G(τ) was measured by correlator which was fitted to a single exponential decay [ ( )~ ] to determine the diffusion constant. The Stokes-Einstein relation = /6 is being used to estimate the average hydrodynamic radius of the LUV. Here, D being the diffusion constant and is the thermal energy (KB, Boltzmann constant= 1.38 × 10-23 Joule/Kelvin ). The mean zeta potential and average size of LUV were obtained from three successive measurements. Each measurement includes 100 runs. Same cuvette is used for both zeta potential and DLS measurements. All experiments were performed at room temperature 25oC. 2.4. Isothermal Titration Calorimetry (ITC) Interaction of NK-2 with LUV produces either exothermic or endothermic reaction. Such change of heat is measured using a VP-ITC microcalorimeter produced by MicroCal Inc (Northampton, MA). In a typical ITC experiments, sample cell (1.442 ml) was filled various concentrations of NK-2 and injection syringe was loaded with LUV suspension. Both LUV and NK-2 solution were prepared with 1 mM HEPES buffer (pH 7.4) and degassed prior to the loading with ITC cell and syringe. Reference cell is filled with HEPES buffer only. A series of 28 injections, each 8 μl, were introduced into the sample cell at 300 second intervals. In a reference measurement,
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LUV was injected into only buffer to determine the heat of dilution. The amount of heat of reaction in each injection was obtained by integrating individual calorimetry traces. We have obtained net heat (due to lipid-NK-2 interaction) per injection by subtracting heat of dilution. Other heat of dilution (∼ - 0.07 μcal/injection), appearing from the dilution of NK-2 , was much smaller than that of former case. The binding constant (K), the change in molar enthalpy (ΔH) and Gibbs free energy (ΔG = ) can be estimated from the fitting binding model with the ITC isotherm and hence the entropic contributions (TΔS = ∆ ∆ ) can also be determined. The fitting routine given by microcal origin has been used to obtain all above thermodynamic parameters.
Fig. 1: Typical chemical structures of all phopholipids used in experiments. The structure of NK-2, an antimicrobial peptide, and its amino acid sequence is also shown.
3. Results and Discussions As model membranes, we have used negatively charged phospholipid DOPG and two zwetterionic phospholipids DOPC and DOPE to prepare LUV. DOPE and DOPG are abundant phospholipids found in bacterial cell surface, whereas, DOPC is mostly found in the eukarytotic plasma membrane. Therefore, DOPE and DOPG resemble the composition of bacterial membranes and DOPC has been used for comparison. These phospholipids in aqueous solvent also exhibit fluid phase above -18 0 C. Therefore, vesicles can easily be prepared at room temperature ( 250 C). Various concentrations of peptide NK-2 ranging from 10-200 µM were titrated with LUV. Size distributions and zeta potential of LUV have been measured before and after ITC experiments, i.e, in the absence and presence of NK-2. Typical ITC raw data representing the heat flow upon injecting the LUV into the NK-2 are shown in Fig 2. Titration of LUV into NK-2 produces endothermic heat when the heat of dilution has been subtracted. In the beginning of titration, there are plenty of NK-2 for binding with lipids. However, as the titration progresses, less and less NK-2 are available for binding, indicating by the gradual decrease in the heat signal. The titration curve attains its saturation value when all the NK-2 are exhausted and no peptide is remaining for binding to lipids. Besides binding of NK-2, contributions of initial heat also arise due to conformational change, i.e, transformation of random coil to α –helix, change of orientation of peptide when associates with the membrane. The endothermic heat could also arise from the liberation of water molecules from the hydration layer at the membrane-water interface, when NK-2 binds to the membrane. Normalized heat per injection, obtained from integrating each peak (see Fig. 2(a)), as a function of lipid/ NK-2 ratio is shown in Fig.3. One site binding model fits this isotherm very nicely and we have obtained thermodynamic parameters of the interaction. The binding constant (K) and molar binding enthalpy (ΔH) have been found to be (6.6 ± 1.1)× 104 M-1 and 269.8 ± 4.2 cal/mol , respectively. Other thermodynamic parameters, such as Gibbs free energy ( ΔG = - 8949 cal/mol ) and entropic contribution ( TΔS = 9245cal/mol) have been obtained from equations given in the experimental section.
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Fig. 2: ITC raw data showing the heat flow upon injecting 4 mM LUV into 50 µM of NK-2. Both LUV and peptide are prepared in 1 mM HEPES buffer at physiological pH (= 7.4). LUV are composed of DOPE-DOPG (4:1) (a) and DOPC (b).
Fig. 3. Isotherm obtained from the integrated heat per injection when 50 µM NK-2 were titrated with 4 mM LUV. Heat of dilution, obtained from injecting 4 mM LUV into buffer, was subtracted from the actual measurement. Here we assume, lipids from both monolayers interact with the NK-2. The solid line is obtained from the fit using one site model given by the microcal origin.
Although the one site model fits very well, it essentially provides the apparent binding constant (Kapp) which is expected to vary with NK-2 concentration. This model does not account for electrostatic contribution. Further, interaction of negatively charged LUV with cationic NK-2 is primarily driven by electrostatic attraction. Therefore, it's desirable to take into account for the electrostatic contribution in the model which eventually gives an intrinsic
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binding constant. In the present article, we have only mentioned the typical order of magnitude of the binding constant. Although, there have been one ITC study on the interaction of NK-2 with LPS, no further analysis were discussed [6]. Therefore, no previous reports on binding constant of NK-2 with negatively charged phospholipids have been found to compare our results. The binding constant obtained from this study is in agreement with that found in similar peptide Magainin II when binds to POPG (21). For comparison, we have performed ITC experiment where LUV composed of zwitterionic phospholipid DOPC in injected to NK-2 (Fig. 2 (b)). Interestingly, there is no significant heat flow observed in this case, suggesting a weak interaction with neutral lipids. As hydrophobic partitioning of NK-2 with negatively charged membranes are due to electrostatic attraction, it is important to look at the electrostatic behavior of the LUV in the presence of NK-2. We have measured the zeta potential of LUV at different composition of lipid/ NK-2 before and after ITC experiment. As shown in Fig. 4, zeta potential gradually decreases its negative value as more and more NK-2 adsorb onto the membrane. The charge neutralization occurs at NK-2/ lipids molar ratio 1/20. As the zeta potential is realized with respect to charge lipids, the charge compensation happens at PG/NK-2 ~ 4 which indeed the case where ITC thermogram shows saturation value. Further increasing NK-2 concentration leads to overcharge compensation and hence saturation of zeta potential value. On the contrary, there is no significant charge in zeta potential in case of DOPC, indicating weak binding and therefore, this result is consistent with ITC result. In summary, these results indicate that NK-2 has a strong binding affinity towards negatively charged membranes and very weak affinity to neutral lipid DOPC. This is an essential requirement for an AMP to use as therapeutics application for the replacement of conventional antibiotics. Zeta potential can be used to estimate the surface charge, surface potential and hence intrinsic binding constant using Gouy Chapman theory. Further research is going on in order to gain some insights into the electrostatic behaviour of the membranes.
Fig. 4 : Zeta potential of LUV composed of 4:1 DOPE-DOPG in the presence of various concentrations of NK-2. For comparison, zeta potential of DOPC is also depicted in the figure.
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The average size of the LUV measured before and after each ITC experiment for various NK-2/lipid ratio is shown in Fig. 5. DLS, in principle, show evidence of membrane-membrane interaction induced by NK-2. The width of the distribution profiles is a measure of polydispersity indicated by the error bar. This error is relevant in order to understand the effect of NK-2 on the membranes. However, errors, obtained from different measurements, are small and are not shown in the plot. It is clearly evident from Fig. 5 that the average size of anionic LUV increases with increasing NK-2 concentration. Interestingly, polydisperisity of the sample increases and the sample starts showing aggregation, as seen from the turbid LUV solution after ITC experiment. In case of DOPC LUV, average size does not change with increasing NK-2 concentration and no physical change in the appearance of the LUV solution after ITC experiment. This result is consistent with zeta potential and ITC, which essentially indicates that NK-2 primarily interacts with the negatively charged membranes and not with neutral phospholipid. For NK-2/Lipids ~ 0.10, sample becomes unsuitable for DLS measurement, as the error bar, suggesting the polydispersity is too high to fit with the single exponential in the correllogram. This behaviour confirms the membrane-membrane interaction
Fig. 5. Average size of LUV composed of a mixture of DOPE and DOPG at 4:1, and DOPC as measured from dynamic light scattering. Error bar represents the standard deviation of the distribution obtained from the width of the distribution.
mediated by NK-2. It is important to mention that change in the size distribution of the vesicles does not influence the zeta potential significantly. Zeta potential is estimated from the Smoluchowski approximation, where the Henry function f(κa) takes its maximum value 1.5. In other word, zeta potential is determined from the electrostatic double layer formation which does not depend on the size of the particle [22]. Therefore, whatever change in zeta potential we observe is not due to change in size, but due to binding of peptide in the membrane. Based on all experimental observations, we proposed mechanisms of action of NK-2. It is known from the previous literature that pores form above a threshold concentration of AMP [5]. In the beginning of ITC experiment, there are plenty NK-2 which interact with the injected LUV and threshold concentration is already reached. Such interaction is also supported by the zeta potential result. NK-2 increases the membrane tension as in the case of other AMP and eventually form pores [23]. Once pores are formed NK-2 can translocate from outer leaflet to an inner monolayer and vice versa. At higher lipid/NK-2 concentration, it is possible that all NK-2 exhausted for binding and NK-2 can come out from the inner monolayer through transmembrne pores and binds to fresh vesicle. Therefore, NK-2 forms bridges between porated vesicles and fresh anionic vesicles. This situation can lead to an aggregation of vesicles which has been envisaged from the large average size and aggregation of LUV.
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4. Conclusion A systematic investigation on the interaction of an important antimicrobial peptide NK-2 with model membranes shows strong binding affinity towards negatively charged lipid, as revealed from ITC and zeta potential and DLS. Adsorption of NK-2 on membranes, as indicated by an increase in zeta potential (Fig. 1) is primarily driven by electrostatic interaction between negatively charge membranes and positively charged NK-2. This is indeed an essential requirement for an AMP to exhibit antimicrobial activity. However, the interaction is very weak in the case of neutral DOPC. Increase in average size as well as the polydispersity, as evidenced from DLS clearly suggest membrane-membrane interaction mediated by NK-2. Based on all our experimental observations, we proposed a mechanism of action of NK-2 with anionic membranes. We believe that aggregation of vesicles, as seen from turbid solution in the presence of NK-2 happenes through the formation of transmembrane pores. The very weak affinity of NK-2 towards the PC membrane (which is the major constituent of eukaryotic cell membranes) propels the development of peptide antibiotics. NK-2 can be used as therapeutics to kill malaria parasite Plasmodium falciparum. It also shows activity against Escherichia coli and preferentially kills cancer cells. Further, low toxicity towards human cells is a great advantage for the development of antibiotics. Therefore, the present study will definitely reinforce the therapeutics applications. Nevertheless, this study will provide insights into the NK-2 membrane interaction which are important for biomedical applications. Acknowledgements This work was funded by the Department of Biotechnology (DBT), Govt. of India (BT/PR8475/BRB/10/1248/2013). P. Maity and A. Halder thank to the Univeristy Grant Commission (UGC), Govt. of India for providing research fellowship. References [1] M. Zasloff, Nature 415 (2002) 389-395. [2] D. Ren, A. A. Bahar, Pharmaceuticals 6 (2003) 1543-1575. [3] B. Bechinger, S.-U. Gorr, J. Dent. Res. 96 (2017) 254 - 260. [4] K. A. Brogden, Nature 3 (2005) 238-250. [5] M-T. Lee, W-C. Hung, F-Yu. Chen, H.W. Huang, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 5087-5092. [6] J. Andrӓ, M.J.H. Koch, R. Bartels, K. Brandenburg, Animicrob. Agent and Chemtheraphy 48 (2004) 15931599. [7] L. K. Clifton, M. W. A. Skoda, A. P. Le Brun, F. Clieseilski, I. Kuzmenko, S. A. Holt, J. H. Lakey, Langmuir 31 (2015) 404-414. [8] Y. Shai, Biophys. J. 66 (2002) 236-248. [9] L. Zhang, A. Rozek, R. E. Hancock, J. Biol. Chem. 276 (2001) 35714-35722. [10] S. Karmakar, P. Maity, A. Halder, ACS Omega 2 (2018) 8859-8867. [11] T. M. Allen, P. R. Cullis, Advanced Drug Delivery Reviews, 65 (2013) 36-48. [12] C. Olak, A. Muenter, J. Andrӓ, G. Brezesinski, J. Pep. Sci. 14 (2008) 510-517. [13] J. Andrӓ, M. Leippe, Med. Microbiol Immuno. 188 (1999) 117-124. [14] J. Andrӓ, D. Monreal, G. M. de Tejada, C. Olak, G. BrezesinSKI, S. S. Gomez, T. Goldmann, R. Bartels, K. Brandenburg, I. Moriyon, J. Biol. Chem. 282 (2007) 14719-14728. [15] T. Jacob, H. Bruhn, I. Gaworski, B. Fleischer, M. Leippe, Antimicrobe. Agents Chemother. 47 (2003) 607613. [16] H. Schröder-Borm, R. Bakalova, J. Andrӓ, FEBS Lett. 579 (2005) 6128-6124. [17] O. G.Travkova, H. Moehwald, G. Brezesinski, Adv. Colloid Interface Sci. 247 (2017) 521-532. [18] C. Ciobanasu, A. Rzeszutek, U. Kubitscheck, R. Willumeit, Molecules 20 (2015) 6941-6958. [19] P. Maity, B. Saha, G. S. Kumar, S. Karmakar, Biochim. Biophys. Acta. 1858 (2016) 706-714. [20] R. Hunter, Zeta Potential in Colloids Science: Principles and Applications, R. H., Rowell, R. L Ottewill. (Eds.) Academic Press, New York, 1981. [21] M. R. Wenk, J. Seelig, Biochemistry 37 (1998) 3909-3916. [22] P. Maity, B. Saha. G. S. Kumar, S. Karmakar, 6 (2016) 83916-83925. [23] M-T. Lee, T-L. Sun, W-C. Hung, H. W. Huang, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 14243-14248.
ScienceDirect Materials Today: Proceedings 18 (2019) 879–886
www.materialstoday.com/proceedings
ICN3I-2017
Antimicrobial Peptide NK-2 as an Emerging Therapeutic Agent: A Study with Phospholipid Membranes Sanat Karmakar*, Pabitra Maity, Animesh Halder Soft matter and Bio-Physics laboratory, Department of Physics, Jadavpur University, Raja . S. C. Mallick Road, Kolkata – 700032.
Abstract An antimicrobial peptide, NK-2, derived from a cationic core region of NK-lysin, is known to display antimicrobial activity towards negatively charged bacterial membranes. The aim of the present study is to get insights into the mechanism of antimicrobial activity. We have prepared unilamellar vesicles as model membranes composed of bacteria mimicking lipid composition. The interaction of NK-2 with phospholipid membranes has been investigated using a variety of experimental techniques, such as, isothermal titration calorimetry (ITC), zeta potential, dynamic light scattering (DLS). NK-2 exhibits strong binding affinity towards the negatively charged membranes, which indeed a characteristic of bacterial membrane. The weak binding affinity towards the neutral phospholipid suggests that NK-2 is expected to be inert towards normal eukaryotic cells. The ITC results show the overall endothermic response in the heat signal, suggesting the entropic contribution in the lipid –NK-2 interaction. One site binding model given by microcal origin, has been used to estimate the intrinsic binding constant and other thermodynamic parameters of binding kinetics of NK-2. The proliferation of size distribution of negatively charged vesicles containing NK-2 indicates the presence of large aggregates. Summarizing all experimental observations, we have suggested a possible mechanism of antimicrobial activity of NK-2.
© 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017). Keywords: Phospholipid membrane; Antimicrobial peptide; ITC ; Zeta potential; DLS
* Corresponding author. Tel.: +91-33-24572879 E-mail address: [email protected]
2214-7853 © 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International Conference on Nanotechnology: Ideas, Innovations & Initiatives-2017 (ICN:3i2017).
880
S. Karmakar et al. / Materials Today: Proceedings 18 (2019) 879–886
1. Introduction Antimicrobial peptides (AMP) are the evolutionary immune response towards many invading pathogens, such as viruses, bacteria, fungi etc [1, 2]. Their widespread distribution throughout the animals and plants and broad spectrum of targeted micro-organisms leads to successful development of multicellular organisms [3]. AMP target to bacterial membranes and create defects, such as pores without interacting with the specific receptors. There are several proposed mechanisms of antimicrobial action reported in literatures [4, 5]. Interestingly, different AMP uses a different mechanism to disrupt the bacterial membranes, which indeed depend on effective charge, hydrophobicity and length of the AMP. All pathways of antimicrobial action begins with the pore formation which eventually disintegrate the membrane [6, 7]. The driving force for the development of antimicrobial peptides as newer antiinfectives drugs is unpreventable emergence of bacterial resistance to conventional antibiotics. Therefore, the objective of the present study is to find out appropriate antimicrobial peptide which has potential for biomedical applications. NK-2 is one such potent peptide which has been studied systematically in order to obtain insights into the antimicrobial activity. Most AMP are cationic in nature and NK-2 is indeed derived from the cationic core regions of NK-lysin found in natural killer (NK) cell. The mechanisms of antimicrobial activity which cause disintegration of the lipid bilayer structure is difficult to infer in an in vivo experiment due to the high level of complexity of the cellular membrane [8]. Therefore, it is often useful to study model membrane composed of phospholipids in order to understand the underlying mechanism of antimicrobial activity [9]. As model membranes, large unilamellar vesicles (LUV) is an excellent model system to study the membrane-AMP interaction [10]. Unilamellar vesicles are the microscopic sac of single lipid bilayers. Besides model membranes, LUV are mostly used as vehicles of targeted drug delivery [11]. In order to study the antimicrobial activity, one needs to prepare LUV with bacterial membrane compositions. In the present article, we have systematically investigated the interaction of NK-2 with anionic membranes. A cationic peptide NK-2 possess +10 charges at physiological pH (7.4). The high positive charges within the NK2 promote strong binding to the negatively charged membranes. NK-2 exhibits an unordered structure in the form random coil configuration in aqueous solution and it adopts the α- helix in the presence of membranes [12]. NK-2 displays its antimicrobial activity against both gram-negative and –positive bacteria [13, 14], fungi, such as Candida albicans [13], the protozoan parasite Trypanosoma cruzi [15]. NK-2 exerts a potent activity against some cancer cell [16], but shows low hemolytic and cytotoxicity against human cells [13]. Besides the cell line studies, a variety of experimental studies with model membranes have been employed to elucidate antimicrobial activity of AMP [17]. It has been shown that NK-2 interacts specially with negatively charged lipids, such as lipopolysachharuse (LPS) which is a highly anionic component of the outer leaflet of gram-negative bacteria [6]. NK-2 binds to the LPS and induces a change in the endotoxin-lipid A aggregate structure, as revealed by small angle x-ray scattering. A recent study on the interaction of NK-2 with negatively charged membrane have shown the evidence of pores which is the prerequisite to the antimicrobial activity [10]. Differential scanning calorimetry and optical microscopy studies on the NKCS, an improved mutant of NK-2 have shown that it severely distorted, penetrated and perforated model lipid membranes that resembled bacterial membranes, but not those that were similar to human cell membranes [18]. Biophysical characterization of NK-2 at the air water interface has revealed adsorption and desorption kinetics which may be useful for understanding of its activity at different solution conditions [12]. The major problem that has impeded the development of drug therapeutics is the complexity of the biomembrane. Therefore, the present study focuses on the interaction of potent antimicrobial peptide NK-2 with negatively charged membranes which resembles with the composition of bacterial membranes. For comparison, interaction of NK-2 with neutral or zwetterionic lipid has also been investigated. A variety of experimental techniques, such as dynamic light scattering, zeta potential and isothermal titration calorimetry have been employed to study the NK-2 membrane interaction. The electrostatic behaviour of the charged membranes in the presence and absence of NK-2 has been envisaged using zeta potential. DLS is used to study the stability and to show the evidence of membrane-membrane interaction induced by NK-2. Finally the kinetics and thermodynamics of the interaction has been studied using isothermal titration calorimetry.
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2. Materials and Method 2.1. Materials Dioleoyl Phosphatidylcholine (DOPC), Dioleoyl phosphatidylethanolamine (DOPE), Dioleoyl Phosphatidylglycerol (DOPG) were obtained from Avanti Polar Lipids and peptide NK-2 from WITA GmbH, Berlin, Germany. They were used without further purification. 2.2. Preparation of LUV An appropriate amount of lipid in chloroform (10 mg/ml) was transferred to a 10 ml glass vial. A thin dried lipid film was prepared on the wall of the glass vial by gently passing nitrogen gas. The traces of the solvent were then removed by keeping the glass vial for a couple of hours in a vacuum desiccator. 2.5 ml of 1 mM HEPES (pH 7.4) was added to the dried lipid film so as to obtain the final desired concentration. Multilamellar vesicles (MLV) were produced by vortexing of hydrated lipid film for about 20-30 minutes. MLV suspensions are extruded successively through polycarbonate membranes having pore diameters of 400 , 200 and 100 nm using LiposoFast-Pneumatic from AVESTIN (Canada). This procedure results in a formation of LUV of diameter ∼ 100 nm, as measured by dynamic light scattering [19]. The polydispersity index obtained from the width of the distribution is much lower than 0.7 indicating that LUV are highly monodispersed. 2.3. Zeta potential and size distributions using DLS The size distributions and the zeta potential of LUV have been measured with a Zetasizer Nano ZS from Malvern Instruments. The instrument uses 2 mW He-Ne Laser of wavelength 633 nm to illuminate the sample. Zeta potential is determined from the electrophoretic mobility (µ) by laser Doppler velocimetry using Helmoltz-Smolushovski equation [20]. = 3 /2 ( ) (1) Where, η and ϵ are the coefficient of viscosity and the permittivity of the aqueous medium, respectively. Although, the Henry function, ( ) depends on the inverse Debye length (κ) and the radius (a) of the vesicle, the change in the size distribution of the vesicles does not alter the zeta potential significantly. This is due to the fact that zeta potential depends on the formation of electrostatic double layer. In this study, the zeta potential is estimated from the Smoluchowski approximation, in which the Henry function f(κa) takes its maximum value 1.5. In the present DLS setup, back scattered light at an angle of 173o is detected and sent to digital signal processing correlator. The rate of decay of intensity auto-correlation function G(τ) was measured by correlator which was fitted to a single exponential decay [ ( )~ ] to determine the diffusion constant. The Stokes-Einstein relation = /6 is being used to estimate the average hydrodynamic radius of the LUV. Here, D being the diffusion constant and is the thermal energy (KB, Boltzmann constant= 1.38 × 10-23 Joule/Kelvin ). The mean zeta potential and average size of LUV were obtained from three successive measurements. Each measurement includes 100 runs. Same cuvette is used for both zeta potential and DLS measurements. All experiments were performed at room temperature 25oC. 2.4. Isothermal Titration Calorimetry (ITC) Interaction of NK-2 with LUV produces either exothermic or endothermic reaction. Such change of heat is measured using a VP-ITC microcalorimeter produced by MicroCal Inc (Northampton, MA). In a typical ITC experiments, sample cell (1.442 ml) was filled various concentrations of NK-2 and injection syringe was loaded with LUV suspension. Both LUV and NK-2 solution were prepared with 1 mM HEPES buffer (pH 7.4) and degassed prior to the loading with ITC cell and syringe. Reference cell is filled with HEPES buffer only. A series of 28 injections, each 8 μl, were introduced into the sample cell at 300 second intervals. In a reference measurement,
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LUV was injected into only buffer to determine the heat of dilution. The amount of heat of reaction in each injection was obtained by integrating individual calorimetry traces. We have obtained net heat (due to lipid-NK-2 interaction) per injection by subtracting heat of dilution. Other heat of dilution (∼ - 0.07 μcal/injection), appearing from the dilution of NK-2 , was much smaller than that of former case. The binding constant (K), the change in molar enthalpy (ΔH) and Gibbs free energy (ΔG = ) can be estimated from the fitting binding model with the ITC isotherm and hence the entropic contributions (TΔS = ∆ ∆ ) can also be determined. The fitting routine given by microcal origin has been used to obtain all above thermodynamic parameters.
Fig. 1: Typical chemical structures of all phopholipids used in experiments. The structure of NK-2, an antimicrobial peptide, and its amino acid sequence is also shown.
3. Results and Discussions As model membranes, we have used negatively charged phospholipid DOPG and two zwetterionic phospholipids DOPC and DOPE to prepare LUV. DOPE and DOPG are abundant phospholipids found in bacterial cell surface, whereas, DOPC is mostly found in the eukarytotic plasma membrane. Therefore, DOPE and DOPG resemble the composition of bacterial membranes and DOPC has been used for comparison. These phospholipids in aqueous solvent also exhibit fluid phase above -18 0 C. Therefore, vesicles can easily be prepared at room temperature ( 250 C). Various concentrations of peptide NK-2 ranging from 10-200 µM were titrated with LUV. Size distributions and zeta potential of LUV have been measured before and after ITC experiments, i.e, in the absence and presence of NK-2. Typical ITC raw data representing the heat flow upon injecting the LUV into the NK-2 are shown in Fig 2. Titration of LUV into NK-2 produces endothermic heat when the heat of dilution has been subtracted. In the beginning of titration, there are plenty of NK-2 for binding with lipids. However, as the titration progresses, less and less NK-2 are available for binding, indicating by the gradual decrease in the heat signal. The titration curve attains its saturation value when all the NK-2 are exhausted and no peptide is remaining for binding to lipids. Besides binding of NK-2, contributions of initial heat also arise due to conformational change, i.e, transformation of random coil to α –helix, change of orientation of peptide when associates with the membrane. The endothermic heat could also arise from the liberation of water molecules from the hydration layer at the membrane-water interface, when NK-2 binds to the membrane. Normalized heat per injection, obtained from integrating each peak (see Fig. 2(a)), as a function of lipid/ NK-2 ratio is shown in Fig.3. One site binding model fits this isotherm very nicely and we have obtained thermodynamic parameters of the interaction. The binding constant (K) and molar binding enthalpy (ΔH) have been found to be (6.6 ± 1.1)× 104 M-1 and 269.8 ± 4.2 cal/mol , respectively. Other thermodynamic parameters, such as Gibbs free energy ( ΔG = - 8949 cal/mol ) and entropic contribution ( TΔS = 9245cal/mol) have been obtained from equations given in the experimental section.
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Fig. 2: ITC raw data showing the heat flow upon injecting 4 mM LUV into 50 µM of NK-2. Both LUV and peptide are prepared in 1 mM HEPES buffer at physiological pH (= 7.4). LUV are composed of DOPE-DOPG (4:1) (a) and DOPC (b).
Fig. 3. Isotherm obtained from the integrated heat per injection when 50 µM NK-2 were titrated with 4 mM LUV. Heat of dilution, obtained from injecting 4 mM LUV into buffer, was subtracted from the actual measurement. Here we assume, lipids from both monolayers interact with the NK-2. The solid line is obtained from the fit using one site model given by the microcal origin.
Although the one site model fits very well, it essentially provides the apparent binding constant (Kapp) which is expected to vary with NK-2 concentration. This model does not account for electrostatic contribution. Further, interaction of negatively charged LUV with cationic NK-2 is primarily driven by electrostatic attraction. Therefore, it's desirable to take into account for the electrostatic contribution in the model which eventually gives an intrinsic
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binding constant. In the present article, we have only mentioned the typical order of magnitude of the binding constant. Although, there have been one ITC study on the interaction of NK-2 with LPS, no further analysis were discussed [6]. Therefore, no previous reports on binding constant of NK-2 with negatively charged phospholipids have been found to compare our results. The binding constant obtained from this study is in agreement with that found in similar peptide Magainin II when binds to POPG (21). For comparison, we have performed ITC experiment where LUV composed of zwitterionic phospholipid DOPC in injected to NK-2 (Fig. 2 (b)). Interestingly, there is no significant heat flow observed in this case, suggesting a weak interaction with neutral lipids. As hydrophobic partitioning of NK-2 with negatively charged membranes are due to electrostatic attraction, it is important to look at the electrostatic behavior of the LUV in the presence of NK-2. We have measured the zeta potential of LUV at different composition of lipid/ NK-2 before and after ITC experiment. As shown in Fig. 4, zeta potential gradually decreases its negative value as more and more NK-2 adsorb onto the membrane. The charge neutralization occurs at NK-2/ lipids molar ratio 1/20. As the zeta potential is realized with respect to charge lipids, the charge compensation happens at PG/NK-2 ~ 4 which indeed the case where ITC thermogram shows saturation value. Further increasing NK-2 concentration leads to overcharge compensation and hence saturation of zeta potential value. On the contrary, there is no significant charge in zeta potential in case of DOPC, indicating weak binding and therefore, this result is consistent with ITC result. In summary, these results indicate that NK-2 has a strong binding affinity towards negatively charged membranes and very weak affinity to neutral lipid DOPC. This is an essential requirement for an AMP to use as therapeutics application for the replacement of conventional antibiotics. Zeta potential can be used to estimate the surface charge, surface potential and hence intrinsic binding constant using Gouy Chapman theory. Further research is going on in order to gain some insights into the electrostatic behaviour of the membranes.
Fig. 4 : Zeta potential of LUV composed of 4:1 DOPE-DOPG in the presence of various concentrations of NK-2. For comparison, zeta potential of DOPC is also depicted in the figure.
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The average size of the LUV measured before and after each ITC experiment for various NK-2/lipid ratio is shown in Fig. 5. DLS, in principle, show evidence of membrane-membrane interaction induced by NK-2. The width of the distribution profiles is a measure of polydispersity indicated by the error bar. This error is relevant in order to understand the effect of NK-2 on the membranes. However, errors, obtained from different measurements, are small and are not shown in the plot. It is clearly evident from Fig. 5 that the average size of anionic LUV increases with increasing NK-2 concentration. Interestingly, polydisperisity of the sample increases and the sample starts showing aggregation, as seen from the turbid LUV solution after ITC experiment. In case of DOPC LUV, average size does not change with increasing NK-2 concentration and no physical change in the appearance of the LUV solution after ITC experiment. This result is consistent with zeta potential and ITC, which essentially indicates that NK-2 primarily interacts with the negatively charged membranes and not with neutral phospholipid. For NK-2/Lipids ~ 0.10, sample becomes unsuitable for DLS measurement, as the error bar, suggesting the polydispersity is too high to fit with the single exponential in the correllogram. This behaviour confirms the membrane-membrane interaction
Fig. 5. Average size of LUV composed of a mixture of DOPE and DOPG at 4:1, and DOPC as measured from dynamic light scattering. Error bar represents the standard deviation of the distribution obtained from the width of the distribution.
mediated by NK-2. It is important to mention that change in the size distribution of the vesicles does not influence the zeta potential significantly. Zeta potential is estimated from the Smoluchowski approximation, where the Henry function f(κa) takes its maximum value 1.5. In other word, zeta potential is determined from the electrostatic double layer formation which does not depend on the size of the particle [22]. Therefore, whatever change in zeta potential we observe is not due to change in size, but due to binding of peptide in the membrane. Based on all experimental observations, we proposed mechanisms of action of NK-2. It is known from the previous literature that pores form above a threshold concentration of AMP [5]. In the beginning of ITC experiment, there are plenty NK-2 which interact with the injected LUV and threshold concentration is already reached. Such interaction is also supported by the zeta potential result. NK-2 increases the membrane tension as in the case of other AMP and eventually form pores [23]. Once pores are formed NK-2 can translocate from outer leaflet to an inner monolayer and vice versa. At higher lipid/NK-2 concentration, it is possible that all NK-2 exhausted for binding and NK-2 can come out from the inner monolayer through transmembrne pores and binds to fresh vesicle. Therefore, NK-2 forms bridges between porated vesicles and fresh anionic vesicles. This situation can lead to an aggregation of vesicles which has been envisaged from the large average size and aggregation of LUV.
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4. Conclusion A systematic investigation on the interaction of an important antimicrobial peptide NK-2 with model membranes shows strong binding affinity towards negatively charged lipid, as revealed from ITC and zeta potential and DLS. Adsorption of NK-2 on membranes, as indicated by an increase in zeta potential (Fig. 1) is primarily driven by electrostatic interaction between negatively charge membranes and positively charged NK-2. This is indeed an essential requirement for an AMP to exhibit antimicrobial activity. However, the interaction is very weak in the case of neutral DOPC. Increase in average size as well as the polydispersity, as evidenced from DLS clearly suggest membrane-membrane interaction mediated by NK-2. Based on all our experimental observations, we proposed a mechanism of action of NK-2 with anionic membranes. We believe that aggregation of vesicles, as seen from turbid solution in the presence of NK-2 happenes through the formation of transmembrane pores. The very weak affinity of NK-2 towards the PC membrane (which is the major constituent of eukaryotic cell membranes) propels the development of peptide antibiotics. NK-2 can be used as therapeutics to kill malaria parasite Plasmodium falciparum. It also shows activity against Escherichia coli and preferentially kills cancer cells. Further, low toxicity towards human cells is a great advantage for the development of antibiotics. Therefore, the present study will definitely reinforce the therapeutics applications. Nevertheless, this study will provide insights into the NK-2 membrane interaction which are important for biomedical applications. Acknowledgements This work was funded by the Department of Biotechnology (DBT), Govt. of India (BT/PR8475/BRB/10/1248/2013). P. Maity and A. Halder thank to the Univeristy Grant Commission (UGC), Govt. of India for providing research fellowship. References [1] M. Zasloff, Nature 415 (2002) 389-395. [2] D. Ren, A. A. Bahar, Pharmaceuticals 6 (2003) 1543-1575. [3] B. Bechinger, S.-U. Gorr, J. Dent. Res. 96 (2017) 254 - 260. [4] K. A. Brogden, Nature 3 (2005) 238-250. [5] M-T. Lee, W-C. Hung, F-Yu. Chen, H.W. Huang, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 5087-5092. [6] J. Andrӓ, M.J.H. Koch, R. Bartels, K. Brandenburg, Animicrob. Agent and Chemtheraphy 48 (2004) 15931599. [7] L. K. Clifton, M. W. A. Skoda, A. P. Le Brun, F. Clieseilski, I. Kuzmenko, S. A. Holt, J. H. Lakey, Langmuir 31 (2015) 404-414. [8] Y. Shai, Biophys. J. 66 (2002) 236-248. [9] L. Zhang, A. Rozek, R. E. Hancock, J. Biol. Chem. 276 (2001) 35714-35722. [10] S. Karmakar, P. Maity, A. Halder, ACS Omega 2 (2018) 8859-8867. [11] T. M. Allen, P. R. Cullis, Advanced Drug Delivery Reviews, 65 (2013) 36-48. [12] C. Olak, A. Muenter, J. Andrӓ, G. Brezesinski, J. Pep. Sci. 14 (2008) 510-517. [13] J. Andrӓ, M. Leippe, Med. Microbiol Immuno. 188 (1999) 117-124. [14] J. Andrӓ, D. Monreal, G. M. de Tejada, C. Olak, G. BrezesinSKI, S. S. Gomez, T. Goldmann, R. Bartels, K. Brandenburg, I. Moriyon, J. Biol. Chem. 282 (2007) 14719-14728. [15] T. Jacob, H. Bruhn, I. Gaworski, B. Fleischer, M. Leippe, Antimicrobe. Agents Chemother. 47 (2003) 607613. [16] H. Schröder-Borm, R. Bakalova, J. Andrӓ, FEBS Lett. 579 (2005) 6128-6124. [17] O. G.Travkova, H. Moehwald, G. Brezesinski, Adv. Colloid Interface Sci. 247 (2017) 521-532. [18] C. Ciobanasu, A. Rzeszutek, U. Kubitscheck, R. Willumeit, Molecules 20 (2015) 6941-6958. [19] P. Maity, B. Saha, G. S. Kumar, S. Karmakar, Biochim. Biophys. Acta. 1858 (2016) 706-714. [20] R. Hunter, Zeta Potential in Colloids Science: Principles and Applications, R. H., Rowell, R. L Ottewill. (Eds.) Academic Press, New York, 1981. [21] M. R. Wenk, J. Seelig, Biochemistry 37 (1998) 3909-3916. [22] P. Maity, B. Saha. G. S. Kumar, S. Karmakar, 6 (2016) 83916-83925. [23] M-T. Lee, T-L. Sun, W-C. Hung, H. W. Huang, Proc. Natl. Acad. Sci. U.S.A. 110 (2013) 14243-14248.