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Biochimica et Biophysica Acta 1858 (2016) 2699–2708

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Selective amino acid substitution reduces cytotoxicity of the antimicrobial peptide mastoparan Luz N. Irazazabal a, William F. Porto b, Suzana M. Ribeiro b,e, Sandra Casale c, Vincent Humblot c, Ali Ladram d, Octavio L. Franco a,b,e,⁎ a

Molecular Pathology Post-graduate Program, University of Brasília, Brasília, Distrito Federal, Brazil Centro de Análises Proteômicas e Bioquímicas, Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, Brazil c Sorbonne Universités, Université Pierre et Marie Curie (UPMC), CNRS, UMR 7197, Laboratoire de Réactivité de Surface (LRS), Paris, France d Sorbonne Universités, Université Pierre et Marie Curie (UPMC), CNRS, Institut de Biologie Paris-Seine (IBPS), Biogenèse des Signaux Peptidiques (BIOSIPE), Paris, France e S-Inova Biotech, Pós-Graduação em Biotecnologia, Universidade Católica Dom Bosco, Campo Grande, MS, Brazil b

a r t i c l e

i n f o

Article history: Received 16 April 2016 Received in revised form 28 June 2016 Accepted 12 July 2016 Available online 14 July 2016 Keywords: Antimicrobial peptides Mastoparan Rational design Hydrophobic moment Membrane permeabilization/depolarization

a b s t r a c t The emergence of antibiotic-resistant clinical isolates and the decreased rate of development of new antibiotics are a constant threat to human health. In this context, the therapeutic value of mastoparan (MP), a toxin from wasp venom, has been extensively studied. However, since MP shows significant cytotoxic activities, further optimization is needed. Here we evaluated the antimicrobial and cytolytic activities of an MP analog created by Ala-substitution in positions 5 and 8, named [I5, R8] mastoparan ([I5, R8] MP). We found that [I5, R8] MP displayed a broad-spectrum antimicrobial activity against bacteria and fungi (MIC in the range 3–25 μM), without being hemolytic or cytotoxic toward HEK-293 cells. In addition, [I5, R8] MP-amide was highly potent (MIC = 3 μM) against antibiotic-resistant bacteria. The interaction with microbial membranes was investigated revealing that [I5, R8] MP is able to form an active amphipathic α-helix conformation and to disturb membranes causing lysis and cell death. Based on our findings, we hypothesize that [I5, R8] MP follows a mechanism of action similar to that proposed for MP, where the pore-forming activity leads to cell death. Our results indicate that hydrophobic moment modified by amino acid substitution may enhance MP selectivity. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The need for innovative antibiotics is a pressing concern for public health, due to the alarming worldwide growth in resistance of pathogens to conventional drugs [1]. Taking this scenario into account, over the last two decades, antimicrobial peptides (AMPs) have been proposed as a new option for the development of novel drugs [2–4]. Ubiquitous in nature, AMPs are usually small (up to 100 amino acids), charged, amphipathic molecules found as components of an organism's innate immune system [5]. AMPs can be produced in a wide variety of structures but typically include positively charged amino acids. To date, in the APD antimicrobial peptide database [6], from 2621 AMPs deposited, about 14% of them are known to adopt an α-helical structure (accessed in February 2016). Despite their sequence heterogeneity, in general, they share physicochemical properties such as cationicity and amphipathicity, which appear to be the driving forces in the interaction with the negatively charged microbial cytoplasmic membrane as a

Abbreviations: MP, mastoparan; AMP, antimicrobial peptide; LC50, lytic concentration 50; IC50, inhibitory concentration 50; MIC, minimal inhibitory concentration. ⁎ Corresponding author at: Molecular Pathology Post-graduate Program, University of Brasília, Brasília, Distrito Federal, Brazil. E-mail address: [email protected] (O.L. Franco).

http://dx.doi.org/10.1016/j.bbamem.2016.07.001 0005-2736/© 2016 Elsevier B.V. All rights reserved.

primary target in most cases [7]. In fact, it is known that the degree of helicity and amphipathicity seems to be important for antimicrobial efficiency [8,9]. Regardless of their specific target, AMPs often exhibit multiple modes of action and operate via non-specific binding to microbial membranes, emerging as potential new therapeutic compounds with the ability to rapidly kill a broad-spectrum of microorganisms. AMPs affect the integrity of membranes. Some peptides translocate across the membrane and disrupt key cellular processes whereas others can even use multiple mechanisms where both lytic action and intracellular targets are involved [10,11]. Unfortunately, there are restrictions on the clinical use of AMPs due to the toxicity against eukaryotic cells and also to the growing evidence that pathogens acquire resistance to AMPs. It has been demonstrated that bacteria can neutralize AMPs as a result of diverse molecular mechanisms, including membrane net charge alteration, extracellular proteolytic degradation, removal of AMPs from the cell via active transport, and biofilm development, among others [12,13]. Thus, the development of new strategies to overcome the obstacles in the therapeutic use of AMPs is required. In this regard, the rational design of AMP emerges as an important tool for efficiently produced drug peptides [14,15]. Despite decades of work, a consensual understanding of the exact molecular mechanism of action of AMPs is still lacking [16]. However,

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since the biological activities of AMPs start at their interaction with cellular membranes, various models proposed to explain their mechanism agree that the physicochemical properties of both the peptide and the target membrane are important to AMP function [17, 18]. Furthermore, there is evidence that upon interaction with a lipid surface, peptides undergo a significant conformational transition as a requirement for antimicrobial activity to occur [19,20]. Indeed, a particularly successful class of AMPs adopts an amphipathic α-helical conformation upon interaction with lipid bilayers in membranes, while they lack a secondary structure in aqueous environments. Based on existing natural AMPs, α-helical AMPs rationally designed by minimal amino acid substitutions proved to be productive in the development of synthetic analogs with optimized antimicrobial activity and reduced cytotoxicity [21,22]. Previous structure/activity relationship studies on amphipathic helical peptides have proposed that the AMPs' activity and specificity is modulated by physicochemical properties, especially the hydrophobicity, hydrophobic moment, angle of charged residues [8] and net charge [23]. The hydrophobic moment is a measure of amphipathicity and is defined as the vectorial sum of individual amino acid hydrophobicity, normalized to an ideal α-helix [24]. In this work, a new synthetic peptide derived from mastoparan L (MP) and named [I5, R8] mastoparan ([I5, R8] MP) was developed, aiming to decrease mammalian cytotoxicity, taking into account such physicochemical properties. MP is one of the most effective α-helical AMPs of great medical interest. Previously isolated from wasp venom, MP is a 14-residue peptide toxin [25], known to mediate mast cell degranulation and histamine release. This peptide is capable of forming amphipathic α-helical structures, penetrating the cell membrane and interacting with cytoplasmic proteins [26]. Also, experiments with model membranes demonstrate a permeabilization and formation of ion channels [27,28]. Here, the design, antimicrobial activities and possible mechanism of action of [I5, R8] MP are described. 2. Materials and methods 2.1. Peptide synthesis and mass spectrometry analysis [I5, R8] MP and [I5, R8] MP-amide were synthesized via Fmoc chemistry using a Liberty Blue™ automated microwave peptide synthesizer (CEM Corporation), a PAL-NovaSyn TG resin (Merck Millipore), and a systematic double-coupling protocol. Fmoc-protected amino acids were purchased from Iris Biotech GMBH, and solvents from Carlo Erba. Clivage and deprotection of the peptidyl resin were achieved by incubation of the resin with an acidic cocktail (95% TFA, 2.5% triisopropylsilane, 2.5% water) for 3 h at room temperature. After removal of the resin by filtration, the crude peptide was precipitated with cold diethyl ether (3000 × g, 15 min, 4 °C), washed with the same solvent, and dried under a stream of nitrogen. The crude peptide was subjected to purification by reversed-phase HPLC on a semi-preparative Nucleosil C-18 column (5 μm, 250 × 10 mm, Interchim SA). The homogeneity and identity of synthetic peptides were assessed by MALDI-TOF MS/MS analysis on UltraFlex III (Bruker Daltonics). The peptides' monoisotopic mass was obtained in reflector mode with external calibration, using the Peptide Calibration Standard for Mass Spectrometry calibration mixture (up to 4000 Da mass range, Bruker Daltonics). 2.2. Determination of minimum inhibitory concentration (MIC) The microorganisms used to determine the MIC of [I5, R8] MP included the Gram-positive bacteria Staphylococcus aureus (ATCC 25923), a clinical isolate of methicillin-resistant S. aureus (7133623), and Enterococcus faecalis (ATCC 29212), the Gram-negative bacteria Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Acinetobacter baumannii (ATCC 19606), Klebsiella pneumoniae (ATCC 13883), and a clinical isolate of carbapenem-resistant K. pneumoniae

(3,259,271). These strains were cultured in Lysogeny Broth (LB). The two Gram-positive species, Streptococcus pyogenes (ATCC 19615) and Listeria ivanovii (Li 4pVS2), were cultured in Brain Heart Infusion (BHI), whereas the fungi Candida albicans ATCC 90028 and C. parapsilosis ATCC 22019 were cultured in Yeast Dextrose medium (YPD). MIC was determined in 96-well microtitration plates by growing the microorganisms in the presence of two-fold serial dilution of the peptide, as previously described [29]. Briefly, logarithmic phase cultures of bacteria and fungi were centrifuged and suspended in MH (Muller Hinton) broth to an absorbance at 630 nm (A630) of 0.01 (106 cfu·ml−1), except for S. pyogenes, L. ivanovii, E. faecalis and Candida species which were suspended in their respective growth medium. Microtiter plate wells received aliquots of 50 μl each of the culture suspension followed by the addition of 50 μl of the diluted peptide (200 to 1 μM, final concentrations). After overnight incubation at 37 °C (30 °C for fungi), antimicrobial susceptibility was monitored by measuring the change in A630 value using a microplate reader. MIC was determined as the lowest peptide concentration that completely inhibits the growth of the microorganism and corresponds to the average value obtained from three independent experiments, each performed in triplicate with positive (0.7% formaldehyde) and negative (without peptide) inhibition control [30]. 2.3. Hemolytic assays The hemolytic activity of [I5, R8] MP was determined by incubating the peptide (final concentration ranging from 1 to 400 μM) with human erythrocytes from healthy donors. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, revised in 2000. Blood was centrifuged and the erythrocytes were washed three times with Dulbecco's phosphate-buffered saline, pH 7.4. The peptide was added to the erythrocyte suspension (1%, v:v) in a final volume of 100 μl and incubated at room temperature for 60 min. Hemoglobin release was monitored by measuring the A540 of the supernatant. The control for zero hemolysis consisted of erythrocytes suspended in the presence of Dulbecco's phosphate-buffered saline (pH 7.4), and for the positive control (100% lysis), a solution of 1% Triton X-100 dissolved in distilled water was used instead of the peptide solution, similarly to those described previously [31]. To measure the hemolytic activity the lytic concentration 50 (LC50) was used, which corresponds to the peptide concentration producing 50% cell death. LC50 corresponds to the average value obtained from three independent experiments, each performed in triplicate. 2.4. Cytotoxic activity analyses The cytotoxicity of [I5, R8] MP was determined against the human leukemia monocyte cell line THP-1, after their differentiation into macrophages by adding PMA (phorbol 12-myristate 13-acetate) in the culture medium, and also on the human embryonic kidney cell line HEK-293. THP-1 monocytes and HEK-293 cells were cultured in RPMI and DEMEM medium, respectively, and incubated at 37 °C in a humidified atmosphere of 5% CO2. The cell viability was quantified after peptide incubation using a methylthiazolyldiphenyl-tetrazolium bromide (MTT)-based microassay [32]. Briefly, cells were seeded on 96-well culture plates (5 × 105 cells·ml−1) and incubated 72 h at 37 °C with 100 μl of [I5, R8] MP (12.5 to 200 μM, final concentrations). Then, 10 μl of MTT (5 mg·ml−1) was added to each well and incubated for 4 h in the dark. In living cells, mitochondrial reductases convert the MTT tetrazolium to formazan, which precipitates. Formazan crystals were dissolved and incubated 1 h at 37 °C under shaking (150 rpm). Finally, absorbance of formazan was measured at 570 nm. The inhibitory concentration 50 (IC50), which corresponds to the peptide concentration producing 50% cell death, was determined with GraphPad Prism. 5.0

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software. Results were expressed as the mean of three independent experiments performed in triplicate. 2.5. Therapeutic index calculation The therapeutic index of [I5, R8] MP was calculated according Chen et al. [14] with minor modifications, using the following Eq. (1): sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi n

n

∏ LC50i i¼1

TI ¼ sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi m

m

ð1Þ

∏ MIC j j¼1

where n is the number of LC50 values and m is the number of MIC values. For values higher than the maximum concentration tested, it was assumed twofold the maximum tested value (e.g. if the value is N100, it was considered as 200) [14].

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2.9. SEM-FEG imaging Scanning Electron Microscopy with Field Emission Gun (SEM-FEG) was used to obtain high-resolution images of the effect of [I5, R8] MP on different microorganisms. Microbial cells in mid-logarithmic phase were collected by centrifugation, washed twice with PBS, and suspended in the same buffer at a density of 2 × 107 cfu·ml−1. 200 μl of the microorganism suspension were incubated 1 h at 37 °C with the peptide [I5, R8] MP (MIC and 2-fold above the MIC, final concentrations). As a negative control, cells were incubated in buffer without peptide. Cells were then fixed with 2.5% glutaraldehyde. SEM-FEG images were recorded with a Hitachi SU-70 Field Emission Gun Scanning Electron Microscope. The samples (gold plates where 20 μl of inoculum were deposited and dried under dry nitrogen) were fixed on an alumina SEM support with a carbon adhesive tape and were observed without metal coating. In-Lens Secondary electron detector (SE-Lower) was used to characterize our samples. The accelerating voltage was 1 kV and the working distance was around 15 mm. At least five to ten different locations were analyzed on each surface, leading to the observation of a minimum of 100 single cells.

2.6. SYTOX green uptake assay 2.10. Molecular modeling The [I5, R8] MP-induced permeation of the bacterial cytoplasmic membrane of S. aureus (ATCC 25923) and E. coli (ATCC 25922) was determined using the SYTOX green (SG) uptake assay [33]. For SG uptake assay, exponentially growing bacteria (6 × 105 cfu·ml− 1) were resuspended in PBS after centrifugation (1000 × g, 10 min, 4 °C) and three washing steps. 792 μl of the bacterial suspension was preincubated with 8 μl of 100 μM SG for 30 min at 37 °C in the dark. After peptide addition (200 μl, final concentration two-fold above the MIC), the fluorescence was measured for 1 h at 37 °C, with excitation and emission wavelengths of 485 and 520 nm, respectively. Three independent experiments were performed and results correspond to a representative experiment with negative (PBS) and positive (melittin) controls. 2.7. Membrane depolarization The cytoplasmic membrane depolarization activity of [I5, R8] MP was measured using E. coli (ATCC 25922) and S. aureus (ATCC 25923) bacteria and the membrane potential sensitive probe DiSC3(5) (3,3′-dipropylthiadicarbocyanine iodide) [34]. As previously described [35], exponentially growing bacteria were centrifuged, washed with PBS and re-suspended in the same buffer to an A630 of 0.1 (1 × 107 cells·ml− 1 ). 700 μl of bacteria were preincubated with 1 μM DiSC3(5) in the dark for 10 min at 37 °C, and then 100 μl of 1 mM KCl were added in order to equilibrate the cytoplasmic and external K + concentrations. Changes in fluorescence were recorded after addition of peptide (200 μl, final concentration: two-fold above the MIC) at 37 °C for 20 min at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. Three independent experiments were performed and results correspond to a representative experiment with negative (PBS) and positive (melittin) controls. 2.8. Time-kill studies Time-kill kinetics of [I5, R8] MP was evaluated against S. aureus (ATCC 25923) and E. coli (ATCC 25922), as previously described [36]. Exponentially growing bacteria in LB were harvested by centrifugation, washed and suspended in PBS to a final concentration of 106 cfu·ml−1. After incubation of 100 μl of this bacterial suspension with a concentration of peptide two-fold above the MIC, aliquots of 10 μl were withdrawn at different times, diluted and spread onto LB agar plates. The cfu were counted after overnight incubation at 37 °C. Two experiments were executed in triplicates and controls were run without peptide.

One hundred molecular models for each variant were constructed by comparative molecular modeling through MODELLER 9.14 [37], using the structure of MP with detergents (PDB ID: 1D7N) [26]. The models were constructed using the default methods of automodel and environ classes from MODELLER. The final models were selected according to the discrete optimized protein energy score (DOPE score). This score assesses the energy of the models and indicates the best probable structures. The best models were evaluated through PROSA II [38] and PROCHECK [39]. PROCHECK checks the stereochemical quality of a protein structure through the Ramachandran plot, where good quality models are expected to have N90% of amino acid residues in most favored and additional allowed regions, while PROSA II indicates the fold quality. Structure visualization was done in PyMOL (http://www.pymol.org). 2.11. Molecular dynamics in water Peptides in water simulations (MD) were carried out in a water environment, using the Single Point Charge water model [40]. The analyses were performed using the GROMOS96 43A1 force field and the computational package GROMACS 4 [41]. The dynamics used the molecular models of [I5, R8] MP and [I5, R8] MP-NH2 as the initial structures, immersed in water molecules in a cubic box with a minimum distance of 10 Å between the peptide and the edges of the box. Chlorine ions were added in order to neutralize the system charge. The geometry of water molecules was constrained using the SETTLE algorithm [42]. All atom bond lengths were linked by using the LINCS algorithm [43]. Electrostatic corrections were made according to the Particle Mesh Ewald algorithm [44], with a cut-off radius of 1.4 nm in order to minimize the computational time. The same cut-off radius was also used for van der Waals interactions. The list of neighbours of each atom was updated every 10 simulation steps of 2 fs. The system underwent an energy minimization using 50,000 steps of the steepest descent algorithm. After that, the system temperature was normalized to 310 K for 100 ps, using the velocity rescaling thermostat (NVT ensemble). Then the system pressure was normalized to 1 bar for 100 ps, using the Parrinello-Rahman barostat (NPT ensemble). The systems with minimized energy, balanced temperature and pressure were simulated for 100 ns by using the leap-frog algorithm [45]. The structures were saved every 2 ps of simulation. 2.12. Molecular dynamics simulation in DOPC bilayer The molecular model of [I5, R8] MP was placed into the membrane using the CHARMM-GUI server [46]. The principal axis of the peptide

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was aligned to Z-axis and then inserted in a rectangular dioleolylphosphatidylcholine (DOPC) bilayer of 50 Å2 (X and Y axes) using the insertion method for system building. The simulation was performed similarly to peptide in water, with minor modifications. The system was simulated using the GROMOS96 53a6 force field extended for Berger lipids [47]. The topology file and the lipid parameters were downloaded from Dr. Tieleman's web site (http://wcm.ucalgary. ca/tieleman/downloads). In NVT ensemble, the system temperature was normalized to 310 K for 100 ps, using the velocity rescaling thermostat. For the NPT ensemble, the Nosé-Hoover thermostat was used, and the system pressure was normalized to 1 bar using the ParrinelloRahman barostat with semi-isotropic pressure coupling and a coupling constant of 1 ns. The systems with minimized energy, balanced temperature and pressure were simulated for 100 ns by using the leap-frog algorithm [45].

Table 1 Physicochemical properties of mastoparan L and [I5, R8] mastoparan.

Hydrophobicity Hydrophobic moment Helix propensity Net charge

Mastoparan L

[I5R8] Mastoparan

1.16 1.00 0.22 +3

0.90 1.39 0.27 +4

The sequence of [I5, R8] MP was derived from the MP sequence (1INLKALAALAKKIL14). Firstly, the MP sequence was mapped into a helical wheel diagram (Fig. 1). Ala5 and Ala8 were selected and substituted for isoleucine and arginine residues, respectively. The Arg residue was placed in position 8 since it increases the net charge from + 3 to + 4 without changing the angle subtended by the positively charged helix face (Fig. 1); besides, according to Pace-Scholtz's α-helical propensity scale [48], Arg is ranked as second residue displaying the highest α-helix propensity (the first one is Ala). The Ile residue was placed in position 5 in order to counterbalance the peptide hydrophobicity, since Ile and Arg have opposite values in Kyte-Doolittle's hydrophobicity scale: 4.5 and − 4.5, respectively [49]. The physicochemical properties of the peptides are summarized in Table 1.

(Pseudomonas aeruginosa) lower than [I5, R8] MP [50] (Table 2). It is probably even higher considering the fact that a bacterial suspension of 5 × 105 cfu·ml−1 was used for MP (106 cfu·ml−1 in our antimicrobial assay). MP was shown to be potent (MIC = 2.7 μM) against colistinresistant Acinetobacter baumannii clinical isolates [51]. A same activity was observed for the analog [I5, R8] MP against the sensitive strain ATCC 19606 (Table 2). Regarding Gram-positive bacteria, a MIC in the range of 12.5–25 μM was reported for MP against Staphylococcus aureus ATCC 29213 and E. faecalis ATCC 19433 (assays with 5 × 105 cfu·ml−1) [50]. Considering this lower bacterial concentration (as mentioned above) and the fact that different strains were used in our assay, the activity of [I5, R8] MP toward S. aureus appears to be similar to that of MP, with also a low activity/inactivity of MP against E. faecalis (Table 2). In order to analyze the influence of C-terminal amidation in the sequence of [I5, R8] MP, we measured the potential changes in the antimicrobial activity of [I5, R8] MP-amide against a Gram-negative (Klebsiella pneumoniae) and a Gram-positive (Staphylococcus aureus) bacterial strain. When amidated, [I5, R8] MP displayed higher activity (MIC = 3–6.25 μM), with 2-fold or 4-fold better activity against K. pneumoniae (MIC = 3 μM) and S. aureus (MIC = 6.25 μM), respectively. Interestingly, the potency of [I5, R8] MP-amide was conserved (MIC = 3 μM) against antibiotic-resistant strains of these bacterial species, such as carbapenem-resistant K. pneumoniae (3259271) and methicillin-resistant S. aureus (7133623).

3.2. Antimicrobial activities of [I5, R8] mastoparan

3.3. Cytotoxicity toward human cells

The antimicrobial activity of [I5, R8] MP was evaluated against several bacterial strains and fungi. As shown in Table 2, [I5, R8] MP was potent against Gram-positive and -negative bacteria, as well as against fungi (MIC in the range 3–25 μM). This peptide was, however, poorly active against Listeria ivanovii (MIC = 50 μM) and inactive against Enterococcus faecalis (MIC higher than 100 μM). A previous study of Moerman and collaborators indicated that MICs of MP against Gram-negative bacteria were 2-fold (Escherichia coli and Klebsiella pneumoniae) or 8-fold

We investigated the hemolytic activity of [I5, R8] MP on human erythrocytes and also its cytotoxicity toward other human cells, such as THP-1-derived macrophages and HEK-293 cells (Table 2). LC50 values, corresponding to the concentration of peptide producing 50%

3. Results 3.1. Rational design of [I5, R8] mastoparan

Table 2 Antimicrobial activity and cytotoxicity of [I5, R8] mastoparan and mastoparan L. Microorganism

MP La

Gram-negative bacteria Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853 Klebsiella pneumoniae ATCC 13883 Acinetobacter baumannii ATCC 19606

12.5 6.25 6.25 3

25 50 12.5 2.7b

Gram-positive bacteria Staphylococcus aureus ATCC 25923 Streptococcus pyogenes ATCC 19615 Listeria ivanovii Li 4pVS2 Enterococcus faecalis ATCC 29212

25 6.25 50 N100

12.5c ND ND 25d

Fungi Candida albicans ATCC 90028 Candida parapsilosis ATCC 22019 Human cells THP-1-derived macrophages HEK-293 Erythrocytes

12.5 25 IC50, LC50 (μM) 24.5 ± 12.9 N200 N200

ND ND ND ND 10e

: MICs of mastoparan L were taken from reference [50] and [51]. b,c,d: MIC values against colistin-resistant A. baumannii clinical isolates, S. aureus ATCC 29213 and E. faecalis ATCC 19433, respectively. e: Concentration causing 40% hemolysis in rat erythrocytes. ND: Not determined. MIC and inhibitory/lytic concentration 50 (IC50, LC50) are expressed as average values from three independent experiments performed in triplicate.

a

Fig. 1. Sequence alignment (above) and helical wheel diagram (below) of mastoparan L and its analog [I5, R8] mastoparan. The angle (ϕ) of the polar face is indicated. Positive charged residues are represented in white circles, polar ones in grey circles and nonpolar in black circles.

MIC (μM) [I5, R8] MP

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of cell death, indicate that [I5, R8] MP was neither hemolytic against human erythrocytes nor cytotoxic against HEK-293 cells (LC50 N 200 μM). By contrast, [I5, R8] MP showed cytotoxicity toward THP-1-derived macrophages, as revealed by the much lower LC50 value (24.5 ± 12.9 μM). In contrast to MP for which 40% hemolysis is already observed at a concentration of 10 μM in rat erythrocytes, our results indicate that [I5, R8] MP is not hemolytic at antimicrobial concentrations [52]. 3.4. Therapeutic index of [I5, R8] mastoparan The therapeutic index (TI) is an important parameter to evaluate the balance between the toxic and the desired effect of a drug. Taking this into account, the TI of [I5, R8] MP was calculated. We found a value of 10.29, which means that a tenfold administration of this peptide would be necessary to achieve a toxic effect. 3.5. Permeabilization and depolarization of the bacterial cytoplasmic membrane The peptide-induced permeabilization/depolarization of the bacterial cytoplasmic membrane was investigated for [I5, R8] MP using Escherichia coli ATCC 25922 and S. aureus ATCC 25923 as two reference strains of Gram-negative and -positive bacteria. We assessed the effect of this peptide on the membrane of E. coli and S. aureus at a concentration corresponding to 2-fold the MIC (25 and 50 μM, respectively). We first evaluated the ability of [I5, R8] MP to affect bacterial membrane permeability by using the SYTOX green (SG) fluorescent dye. SG is a highaffinity nucleic acid dye that is impermeant to live cells. When the cell membrane is damaged, this dye penetrates into the cell and binds to intracellular DNA, leading to an increase in fluorescence. Fig. 2 shows that [I5, R8] MP was able to permeabilize the cytoplasmic membrane of both E. coli and S. aureus cells, as indicated by the increase in the SG fluorescence compared to the negative control (PBS). However, the membrane permeabilization of S. aureus was more rapid and potent compared to

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E. coli (Fig. 2a–b), with a higher maximum of permeabilization reached. The effect of [I5, R8] MP on the bacterial membrane potential was also analyzed using the fluorescence probe DiSC3(5) (Fig. 2c – d). In the presence of intact cytoplasmic membrane, DiSC3(5) accumulates into the cytoplasmic membrane and aggregates, causing self-quenching of the fluorescence. The minimal level of fluorescence (PBS) indicated the accumulation and aggregation of the probe into the intact cytoplasmic membrane and, therefore, its quenching. After the incubation with the positive control melittin, the membrane potential was lost and the probe was released into the medium, leading to an increase of fluorescence. In the presence of [I5, R8] MP, we observed a rapid depolarization of both E. coli and S. aureus membranes (b 5 min), with a higher efficiency than melittin. 3.6. Time-kill evaluation The time-course of antimicrobial activity of [I5, R8] MP against E. coli and S. aureus was investigated at a concentration identical to that used in the permeabilization/depolarization assays (25 μM for E. coli and 50 μM for S. aureus). The time-curves revealed that [I5, R8] MP caused a rapid complete killing of E. coli within 15 min, and a slower killing of S. aureus within the first 45 min (Fig. 3). These results indicate bactericidal effects of [I5, R8] MP on both bacterial strains and confirm also the higher susceptibility of the Gram-negative E. coli to the peptide. 3.7. Visualization of the antimicrobial effect of [I5, R8] mastoparan by SEMFEG imaging SEM-FEG analysis was performed to clarify the detailed mechanisms of cell death at lethal concentrations of [I5, R8] MP against sensitive microorganisms, such as the Gram-negative bacteria Pseudomonas aeruginosa, the Gram-positive L. ivanovii and the fungus Candida albicans. High-resolution SEM-FEG images (Fig. 4) showed that all the cells were damaged after contact with [I5, R8] MP. The high activity of [I5, R8] MP against P. aeruginosa was confirmed by the observation of

Fig. 2. Effect of [I5, R8] MP on plasma membrane integrity of E. coli 25922 and S. aureus 25,923. (a–b) Time-course analysis of the SYTOX green (SG) uptake. The cytoplasmic membrane permeabilization of E. coli (a) and S. aureus (b) was analyzed by measuring the fluorescence of SG after addition (vertical dotted line) of [I5, R8] MP at a concentration 2-fold above the MIC (25 and 50 μM, respectively). (c–d) Depolarization effect of [I5, R8] MP (same concentration) on the cytoplasmic membrane of E. coli (c) and S. aureus (d) observed with the fluorescent probe DiSC3(5). The negative control (PBS) corresponds to the bacteria incubated without peptide, whereas the positive control corresponds to the incubation with melittin 5 μM.

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microorganisms (Gram-positive and -negative bacteria, fungi) according to a membranolytic killing mechanism. 3.8. Structure prediction

Fig. 3. Time-kill kinetics of [I5, R8] MP against E. coli 25922 (open triangles) and S. aureus 25923 (open circles) at a concentration 2-fold above the MIC (25 and 50 μM, respectively). Controls correspond to bacteria incubated in PBS without peptide. Data are the means ± S.E.M. of one experiment performed in triplicate.

numerous lysed cells and cell debris at 6.25 μM and 12.5 μM of [I5, R8] MP (Fig. 4b – c), in comparison to the control (Fig. 4a). For L. ivanovii, the bacterial membrane was also severely damaged after incubation with 50 μM and 100 μM of [I5, R8] MP (Fig. 4e – f, respectively) as compared to untreated cells (Fig. 4d). C. albicans treated with 12.5 μM of [I5, R8] MP presented morphological alterations, with membrane deformation and collapse leading to more flattened cells (Fig. 4h) when compared to the control without peptide (Fig. 4g). These effects were clearly visible at a peptide concentration of 25 μM (Fig. 4i). Thus, SEMFEG results confirm that [I5, R8] MP can act on a wide range of

Molecular modeling of [I5, R8] MP indicated that this peptide adopts an α-helical structure (Fig. 5), where a segregation between charged and hydrophobic amino acids is observed, as denoted by the helical wheel diagram (Fig. 1). It was possible to observe that the structure of [I5, R8] MP tends to unfold in a water environment, either with a carboxyl or a carboxamide terminal (Fig. 5). The root-mean-square-deviation (RMSD) of [I5, R8] MP along the simulations shows a variation of 2–7 Å (Fig. 5b), while for [I5, R8] MP-amide the variation is of about 2–6 Å (Fig. 5b). The residues' RMS fluctuation indicated that in the amidated peptide the N-terminal end is more stable than the C-terminal one, while for the C-terminal carboxylated peptide the whole structure showed a similar behavior (Fig. 5e). In contrast, when placed in a DOPC bilayer, [I5, R8] MP presents little variation in its structure (Fig. 5d), with a RMSD variation of about 1 Å (Fig. 5b), which is reflected in the residues' RMS fluctuation (below 1.5 Å) (Fig. 5e). 4. Discussion and conclusions AMPs assuming amphipathic α-helical structures are widespread in nature and represent one of the most successful molecules in innate defense [21,53]. Belonging to this group, mastoparans are promising small peptides with therapeutic potential. Mastoparans (MPs) are a family of tetradecapeptide components of wasp venom, rich in hydrophobic and basic residues, which adopt amphipathic α-helix structure in lipid environments. MPs and its analogs exhibit a wide variety of biological activities and several properties have been described, including the capacity to induce mast cell degranulation and formation of ion channels, affecting cell viability. The histaminereleasing capability of MP is related to the ability to directly activate G

Fig. 4. SEM-FEG visualization of the effect of [I5, R8] MP on P. aeruginosa (a–c), L. ivanovii (d–f) and C. albicans (g–i). a, d and g) Control cells without peptide. b, c) P. aeruginosa (ATCC 27853) after incubation with [I5, R8] MP at a concentration of 6.25 μM and 12.5 μM, respectively. e, f) L. ivanovii (Li 4pVS2) treated with 50 μM and 100 μM of [I5, R8] MP, respectively. h, i) C. albicans (ATCC 90028) treated with 12.5 μM and 25 μM of [I5, R8] MP, respectively. Scale bar = 1 μM.

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Fig. 5. Structural analyses of [I5, R8] MP. a) Molecular model of [I5, R8] MP. This structure showed a DOPE Score of −981.4, a Z-Score on PROSA of −2.15 and all residues in favored regions of Ramachandran Plot. The N-terminal is colored in blue, while the C-terminal is in red. b) Backbone's RMSD evolution throughout the simulation. c) Final structures of [I5, R8] MP and [I5, R8] MP-amide after 100 ns of simulation. d) Initial (0 ns) and final (100 ns) positions of [I5, R8] MP in DOPC bilayer. e) RMS fluctuation of each amino acid residue of [I5, R8] MP in the simulations.

proteins, responsible for exocytosis control, causing the permeabilization of membranes in both bacteria and mast cells [54]. Furthermore, according to Silva et al. [55], when interacting with biological membranes, these peptides can penetrate via the positively charged side-chains of their amphipathic α-helical structures. Indeed, as they efficiently cross plasma membrane, mastoparans were classified as cell-penetrating peptides (CPP) whit the potential pharmaceutical application of cellular delivery vector [56,57]. Additionally, a recent study has demonstrated that a mastoparan-derived peptide is able to disrupt the lipid structure of enveloped viruses, directly inactivating their infectivity [58]. Related to its antimicrobial activity, MP has emerged as a model in the development of strategies aiming at transforming naturally occurring AMPs into therapeutically valuable anti-infective agents [59–61]. However, since several studies revealed that MP possesses toxic side effects [62–64], we have here undertaken its optimization by designing the analog [I5, R8] MP containing two substituted residues (Ala5 → Ile, Ala8 → Arg). The biological properties of [I5, R8] MP were investigated and revealed a broad-spectrum antimicrobial activity against bacteria and fungi (Table 2), besides, this peptide showed therapeutic index of 10.29, therefore, the toxic effect is only observed when [I5, R8] MP is administrated in doses tenfold higher. For evaluation of a drug by means of the therapeutic index, the higher the index, safer the drug is; however, according to the U. S. Food and Drug Administration (FDA), a therapeutic index is considered narrow when it is below two [65], which means that the toxic and the desired effect are too close, which is not the case of [I5, R8] MP. The C-terminal amidation of [I5, R8] MP (analog [I5, R8] MP-amide) led to an increase of the activity (MIC in the range 3–6.25 μM) against Gram-positive and -negative bacteria, including antibiotic-resistant bacteria. Our results are in correlation with previous studies that revealed the enhanced antimicrobial activity of carboxyamidated MPs in comparison with the deamidated peptide [55]. Killing kinetics assays performed against E. coli shows that [I5, R8] MP achieved a complete killing within 15 min. Compared with conventional

drugs, a rapid and potent non-specific mechanism of action is an asset to limit the emergence of resistant bacteria, particularly Gram-negative strains, that constitute today a growing public health concern worldwide. Furthermore, killing kinetics assays showed a slower killing of the Gram-positive S. aureus (45 min), in spite of the rapid permeabilization kinetics (Fig. 2b) observed. The rapid membrane perturbation suggests that the compromised membrane integrity of S. aureus is an important factor in the killing mechanism. However, the ability to alter the cytoplasmic membrane is not necessarily the lethal step. Additional events, such as membrane translocation and intracellular signaling might occur. Many studies have demonstrated that MP undergoes a conformational change toward an amphipathic α-helical structure in membrane environments. Detailed investigations discuss the ability of MP to rapidly pass through the cell membrane to bind intracellular targets, supporting the idea of a pore-forming mode of action [26,66,67]. The conformational behavior of MP studied by Hori and colleagues suggests the ability of the α-helical peptide to switch its orientation in a lipid bilayer (from an in-plane to a transmembrane), thus able to form pores and translocate across the membrane, in a manner similar to melittin action [26]. Supporting this idea, the study of Silva et al. (2014), described the mechanism of action of a MP analog in a way that both N- and C-termini remain positioned outside of the membrane, while the α-carbon backbone becomes partially embedded in the membrane core, assuming the pore-forming mode of action [55]. Different models have been proposed to describe the interaction between the pore-forming peptides and membranes: barrel-stave, a carpet-like or a toroidal mode of action [68]. It is well known that the propensity for helical structuring plays a dominant role in determining AMP cytotoxicity [22]. Modifications of physicochemical parameters could influence the interaction of MP with model membranes and biological cells, enhancing the antimicrobial potency and membrane selectivity [59,60,69]. Also, it is important to mention that the diverse composition of the membrane of bacteria and eukaryotic cells plays an important role in lytic activity of peptides. As eukaryotic membrane is composed of electrically neutral zwitterionic

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phospholipids and also abundant cholesterol, MP could interact with erythrocytes and HEK-293 cells by hydrophobic interaction, binding preferentially to electrically negative bacterial membranes [62]. Here, by means of modifications in the MP physicochemical properties (Table 1), we designed a non-hemolytic peptide without cytotoxicity against HEK-293 cells and with a wide antimicrobial spectrum (Table 2) by substituting only two amino acid residues. Such modifications do not seem to alter the overall structure of MP because [I5, R8] MP can adopt an α-helical conformation (Fig. 5). Moreover, it also affects permeability of Gram-positive and -negative bacteria (Fig. 2) and induces loss of cytoplasmic membrane potential, causing the leakage of essential metabolites and cell death. Scanning electron microscopy imaging of P. aeruginosa and L. ivanovii confirmed that [I5, R8] MP causes damage in the membrane of bacterial cells. In terms of antimicrobial activity, the higher MICs observed for the Gram-positive bacteria with respect to Gram-negative bacteria (Table 2) tested indicate that [I5, R8] MP possesses differential affinity among bacterial membranes. This is in line with the faster killing kinetics of [I5, R8] MP against E. coli (15 min) compared to S. aureus (45 min). Furthermore, the observed lack of activity against E. faecalis (MIC N100 μM), a naturally more resistant strain, is not surprising since there is evidence of differences in its membrane lipid composition compared to the other Gram-positive bacteria tested [70]. It was previously observed that the structural requirements for the biological activity of amphipathic α-helical AMPs against Gram-negative bacteria are less stringent and that their bactericidal activity toward Gram-positive bacteria and fungi depends on a high cationicity combined with helix stabilization [21]. It has been postulated that the C-terminal amidation observed in many peptides of different origin increases cationicity [55,71]. Furthermore, in 2004, Sforça and colleagues analyzed how C-terminal carboxyamidation could alter the biological activity of the mast cell degranulation peptide, eumenine mastoparan-AF, isolated from the venom of the solitary wasp [72]. By dynamics analysis, they showed structural differences between this naturally carboxyamidated peptide and its carboxyl- free C-terminal form, suggesting that the reduced activity observed for the non-amidated peptide is not simply due to the decrease of the net positive charge but also to a structural perturbation of the α-helix that affects the ability to perturb the cell membrane. By means of molecular dynamics, it was possible to observe that in water environment the peptides did not achieved a stable structure since they underwent an unfolding process, while in the membrane environment the structure is maintained (Fig. 5). Despite the unfolding process in water, we could observe that the [I5, R8] MP-amide was more stable than the carboxyl-free C-terminal form (Fig. 5). Although both peptides tend to unfold in water, the α-helical portion of [I5, R8] MP-amide seems to be maintained. Thermodynamically, there is a relation between MIC and the number of residues that undergo a coil-to-α-helix transition [73]. Taking this into account, [I5, R8] MP would be more potent than [I5, R8] MP-amide since it has more unstructured residues than [I5, R8] MP-amide. However, the increase by + 1 in the net charge of [I5, R8] MP-amide may compensate and overcome the lower level of coil-toα-helix transition. In conclusion, we successfully expunged the cytotoxic effects of MP through the design of [I5, R8] MP. Such modifications seem not to alter the α-helical conformation, but rather enhance the antimicrobial activity without destroying eukaryotic cells. Therefore, single amino acid substitution is an appropriate strategy for the design of novel α-helical AMPs from the MP sequence. The data reported here demonstrated that [I5, R8] MP is a good candidate for potential clinical applications. Transparency document The Transparency document associated with this article can be found, in the online version.

Acknowledgements THP-1 and HEK-293 cells were kindly provided by Dr. B. Oury (INTERTRYP, UMR IRD 177-CIRAD 17, IRD Montpellier, France) and Dr. O. Jean-Jean (UMR 8256, IBPS, FR 3631 UPMCCNRS, UPMC, Paris, France), respectively. We thank Dr. S. André (BIOSIPE, IBPS, FR 3631 UPMC-CNRS, UPMC, Paris, France) for her assistance in the lab experiments, Dr. C. Piesse (Peptide Synthesis Platform, IBPS, FR 3631 UMPC-CNRS, UPMC, Paris, France) for help in synthesis and purification of peptides, and the Mass Spectrometry and Proteomics Platform (IBPS, FR 3631 UPMC-CNRS, UPMC, Paris, France) for MALDI-TOF analysis. The authors acknowledge IMPC (Institut des Matériaux de Paris Centre, FR2482) and the C'Nano projects of the Region Ile-de-France for SEMFEG funding.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bbamem.2016.07.001.

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