Special Issue Article Received: 6 March 2014

Revised: 25 April 2014

Accepted: 28 April 2014

Published online in Wiley Online Library: 12 June 2014

(wileyonlinelibrary.com) DOI 10.1002/psc.2654

Temporin-SHa peptides grafted on gold surfaces display antibacterial activity‡ Andres Lombana,a,b Zahid Raja,c,d Sandra Casale,b,a Claire-Marie Pradier,b,a Thierry Foulon,c,d Ali Ladramc,d* and Vincent Humblotb,a** Development of resistant bacteria onto biomaterials is a major problem leading to nosocomial infections. Antimicrobial peptides are good candidates for the generation of antimicrobial surfaces because of their broad-spectrum activity and their original mechanism of action (i.e. rapid lysis of the bacterial membrane) making them less susceptible to the development of bacterial resistance. In this study, we report on the covalent immobilisation of temporin-SHa on a gold surface modified by a thiolated self-assembled monolayer. Temporin-SHa (FLSGIVGMLGKLFamide) is a small hydrophobic and low cationic antimicrobial peptide with potent and very broad-spectrum activity against Gram-positive and Gram-negative bacteria, yeasts and parasites. We have analysed the influence of the binding mode of temporin-SHa on the antibacterial efficiency by using a covalent binding either via the peptide NH2 groups (random grafting of α- and ε-NH2 to the surface) or via its C-terminal end (oriented grafting using the analogue temporin-SHa-COOH). The surface functionalization was characterised by IR spectroscopy (polarisation modulation reflection absorption IR spectroscopy) while antibacterial activity against Listeria ivanovii was assessed by microscopy techniques, such as atomic force microscopy and scanning electron microscopy equipped with a field emission gun. Our results revealed that temporin-SHa retains its antimicrobial activity after covalent grafting. A higher amount of bound temporin-SHa is observed for the C-terminally oriented grafting compared with the random grafting (NH2 groups). Temporin-SHa therefore represents an attractive candidate as antimicrobial coating agent. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: antimicrobial peptide; temporin-SHa; atomic force microscopy; surface functionalisation; scanning electron microscopy

Introduction

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* Correspondence to: Ali Ladram, Biogenèse des Signaux Peptidiques (BIOSIPE), Sorbonne Universités, UPMC, Univ Paris 06, FR 3631, Institut de Biologie Paris Seine (IBPS), F-75005, Paris, France. E-mail: [email protected] ** Correspondence to: Vincent Humblot, Laboratoire de Réactivité de Surface (LRS), Sorbonne Universités, UPMC, Univ Paris 06, UMR 7197, F-75005, Paris, France. E-mail: [email protected] ‡

This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the 1st International Conference on Peptide Materials for Biomedicine and Nanotechnology, Sorrento, October 28-31, 2013, edited by Professor Giancarlo Morelli, Professor Claudio Toniolo and Professor Mariano Venanzi.

a Sorbonne Universités, UPMC Univ Paris 06, UMR 7197, Laboratoire de Réactivité de Surface (LRS), F-75005, Paris, France b CNRS, UMR 7197, Laboratoire de Réactivité de Surface, F-75005, Paris, France c Sorbonne Universités, UPMC, Univ Paris 06, FR 3631, Institut de Biologie Paris Seine (IBPS), Biogenèse des Signaux Peptidiques (BIOSIPE), F-75005, Paris, France d CNRS, FR 3631, IBPS, BIOSIPE, F-75005, Paris, France

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The emergence of multiresistant pathogens is a real public health problem that occupies a central position in the problem of nosocomial infections. Many bacterial and fungal species can form biofilms [1] on implanted medicals devices (catheters, prostheses, etc.) and are responsible for over 60% of nosocomial infections, leading to clinical complications and death. The biofilm-forming bacteria include Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus viridans, Enterococcus faecalis, etc.) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, etc.). In these biofilms, bacteria are embedded in an extracellular polymeric matrix that makes them particularly resistant to hygiene products and antibiotics [2]. Faced with this alarming appraisal, researchers and clinicians are challenged to quickly develop truly innovative anti-infective molecules with mechanisms of action that may induce little resistance. Antimicrobial peptides (AMPs) are key effectors of innate immunity found throughout the living kingdom [3,4]. They have shown to have a rapid and straightforward killing activity against pathogens [5,6] and appear as potential alternatives to the use of conventional antibiotics. A large part of the present knowledge on the peptide innate immunity arises from pioneer studies performed with AMPs from amphibian skin secretions. The latter are known to constitute an important wealth of diverse bioactive peptides [7,8]. More precisely, the skin of several frog species has shown to be an endless source of AMPs with a wide spectrum of activity [9,10]. Temporins constitute a family of hydrophobic and low cationic amphibian AMPs that is of particular interest because of the small size of its members [11–15]. For instance,

temporin-SHf is the smallest natural linear amphibian AMP found to date (eight amino acid residues) [16]. Development of antibacterial coatings using these natural substances seems to be very attractive. Although the exact antimicrobial mechanisms of AMPs remain unclear, it has been suggested that they are able to insert into bacterial membranes and subsequently induce cell lysis [17]. The mode of action of AMPs seems to be related to their anchoring mode to the membrane and to their cationic charges [17–19]. Several recent

LOMBANA ET AL. works report the immobilisation of small peptides within polyelectrolyte layers [20,21], embedded in a polymeric matrix [22] or directly by click-chemistry and/or covalent bonding on surfaces prepared with adequate spacers [18,23–27]. All these studies demonstrated the antibacterial properties of immobilised peptides, making clear the role of the peptide mobility and accessibility to the microbial cells. We have previously characterised a small AMP, temporin-SHa (13 residues, FLSGIVGMLGKLFamide), present in the skin of the ranid frog Pelophylax saharica [11,12]. This peptide has a potent and broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and parasites of the genus Leishmania. In this work, we have analysed the antimicrobial activity of temporin-SHa once covalently grafted on a gold surface modified by a thiolated self-assembled monolayer (SAM) with carboxylic acid functionality. Furthermore, we have synthesised a C-terminal carboxylated analogue of temporin-SHa (temporin-SHa-COOH) in order to allow a different anchoring mode on a gold surface modified by a thiolated SAM containing amine groups (Figure 1). Each grafting step was characterised by IR spectroscopy [polarisation modulation reflection absorption IR spectroscopy (PM-RAIRS)], and the killing efficiency of the temporin was studied by atomic force microscopy (AFM) and scanning electron microscopy equipped with a field emission gun (SEM-FEG).

Materials and Methods Peptide Synthesis and Purification Temporin-SHa (FLSGIVGMLGKLFamide), temporin-SHa-COOH (FLSGI VGMLGKLFCOOH), [A2,6,9]temporin-SHa (FASGIAGMAGKLFamide) and [A2,6,9]temporin-SHa-COOH (FASGIAGMAGKLFCOOH) were synthesised using solid-phase FastMoc chemistry procedure on an Applied Biosystems 433A automated peptide synthesiser (Platform for Protein Engineering and Peptide Synthesis, FR 3631 UPMC-CNRS, IBPS, Paris, France) as previously described [28]. Fmoc-protected amino acids were purchased from Iris Biotech GMBH, and solvents from Carlo Erba. Briefly, carboxamidated peptides (temporin-SHa and [A2,6,9]temporin-SHa) were prepared on a Rink Amide MBHA PS resin (Iris Biotech GmBH, Marktredwitz, Germany) substituted at 0.81 mmol/g, whereas carboxylated peptides (temporinSHa-COOH and [A2,6,9]temporin-SHa-COOH) were prepared on a Fmoc-Phe-Wang resin LL (Novabiochem, Läufelfingen, Switzerland) substituted at 0.3 mmol/g. Fmoc amino acids were activated with 2(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (0.5 M HBTU) and 2 M diisopropylethylamine. After each coupling cycle, N-Fmoc protecting groups were removed with 20% piperidine. At the end of the synthesis, the peptidyl resin was cleaved and deprotected by incubation with an acidic cocktail: 94% trifluoroacetic acid (TFA), 1% triisopropylsilane, 2.5% ethanedithiol and 2.5% water. The resulting mixture was

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Figure 1. Different grafting strategies of temporin-SHa. (a) Grafting on an acidic thiol SAM (MUA) via the peptide NH2 groups (α-NH2 terminus and/or ε-NH2 of the K residue). (b) Grafting on an amino thiol SAM (MUAM) via the carboxyl group of temporin-SHa-COOH. TEMP: temporin.

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ANTIMICROBIAL PEPTIDE: TEMPORINS ON GOLD SURFACES filtered to remove the resin, and the crude peptides were precipitated with diethyl ether at 4 °C. They were recovered by centrifugation (3000 × g, 15 min, 4 °C), washed three times with cold diethyl ether, dried, dissolved in 10% acetic acid and lyophilized. The lyophilized crude peptides were purified by RP-HPLC on a Phenomenex Luna® C18(2) (Phenomenex, Le Pecq, France) semi-preparative column (10 μm, 250 × 10 mm) eluted at a flow rate of 5 ml/min by a 20–70% linear gradient of acetonitrile (ACN) (0.07% TFA) in 0.1% TFA/water (1% ACN/min). The homogeneity and identity of the synthetic peptide were assessed by MALDI-TOF MS (Voyager DE-PRO Applied Biosystems, Platform Mass Spectrometry and Proteomics, FR 3631 UPMC-CNRS, IBPS, Paris, France) and RP-HPLC on a C18 analytical column (modulocart QS uptisphere 5 ODB, 5 mm, 250 × 4.6 mm, Interchim) using the aforementioned conditions with a flow rate of 0.75 ml/min. Chemicals and Surface Preparation Mercaptoundecanoic acid (MUA), 11-mercaptoundecylamine (MUAM), 6-mercapto-1-hexanol (C6OH), hydroxysuccinimide (NHS), N-1-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), were purchased from Sigma-Aldrich (SaintQuentin Fallavier, France). All solvents were reagent-grade. Reagents were used without any further purification. Experiments were carried out under ambient conditions. The surfaces were constituted of glass substrates (11 × 11 mm), coated successively with a 5 nm thick layer of chromium and a 200-nm thick layer of gold, which were purchased from Arrandee (Werther, Germany). The gold-coated substrates were annealed in a butane flame to ensure a good crystallinity of the topmost layers and rinsed in a bath of absolute ethanol during 15 min before adsorption. Figure 1 presents the different grafting strategies for the covalent bonding of temporin-SHa on gold surfaces. The substrates were immersed in binary mixture at 0.01 M (25/75) of MUA (2.5 mM)/MUAM (2.5 mM) and C6OH (7.5 mM) in 10 ml of absolute ethanol for 3 h, in order to insure an optimal homogeneity of the adlayer [29–31], and thoroughly rinsed in ethanol and MilliQ water (EMD Millipore Corp., Billerica, MA, USA) and dried under a flow of dry nitrogen. The MUA substrates were treated with a solution of NHS (20 mM) and EDC (10 mM) in ultrapure water for 90 min, rinsed in MilliQ water and dried under a flow of dry nitrogen in order to activate the acidic groups of MUA to react with the amino groups of temporins. Immobilisation of temporin-SHa and [A2,6,9]temporin-SHa (20 mg/l in PBS) on MUA-modified gold surfaces was carried out by depositing a 150-μl drop of solution on the substrates at room temperature for 2 h. A total of 20 μl of the temporin-SHa-COOH and [A2,6,9]temporin-SHa-COOH solutions were each diluted in 980 μl of EDC/NHS solution to obtain, after 90 min, 1 ml of activated solutions at 20 mg/ml in PBS. Grafting of activated temporins was then realised by depositing 150 μl of solution of the two different solutions on MUAM modified surfaces for 2 h. After the immobilization step, the surfaces were vigorously rinsed in PBS with agitation and dried under a flow of dry nitrogen. For each step, two series of identical experiments were conducted; one series of samples was then characterised by PM-RAIRS and AFM, whereas the second one was characterised by PM-RAIRS and SEM-FEG. PM-RAIRS Measurements

AFM Characterisation The AFM images of dried surfaces were recorded using a commercial di Caliber AFM microscope from Bruker (Nano Surfaces Corp. Santa Barbara, Ca, USA). In order to avoid tip and sample damages, topographic images were taken in the noncontact dynamic mode also known as tapping® mode. Silicon nitride tips (resonance frequency of 280–400 kHz, force constant of 40–80 N/m) have been used. Images were obtained at a constant speed of 2 Hz with a resolution of 512 lines of 512 pixels each. The raw data were processed using the imaging processing software SpmLabAnalysis v.7.0. from Bruker (Nano Surfaces Corp. Santa Barbara, Ca, USA). AFM analyses were carried out at least at three different locations on each surface, with a minimum of 100 single bacteria observed. SEM-FEG Characterisation Scanning electron microscopy images were obtained using a SEMFEG Hitachi SU-70 scanning electron microscope (Hitachi HighTechnologies Corporation, Tokyo, Japan) with an accelerating voltage of 1 kV; the working distance is around 3.1 or 3.2 mm; in lense secondary electron detector SE(U) was used. A total of 100 μl of a solution of bacteria Listeria ivanovii at 109 colonies of bacteria (cfu)/ml was deposited onto a gold substrate and dried at room temperature. The gold substrate was fixed on an alumina SEM support with a carbon adhesive tape. SEM-FEG analyses were carried out at least at four different locations on each surface, with a minimum of 100 single bacteria observed. Bacterial Strains and Adhesion of Bacteria Nonpathogenic bacteria, L. ivanovii Li4pVS2 [32], were first grown at 37 °C in brain-heart infusion (BHI, BD DIFCO, Le Pont-De-Claix, France) broth overnight. The activity of functionalized surfaces against bacteria was tested following a methodology described in a previous work [24]. A 100-μl drop, containing 108 bacteria, was deposited on gold substrates and left for 3 h at 25 °C under a wet atmosphere to avoid evaporation. Samples were then washed with 6 × 100 μl of PBS and dried under a flow of dried air. The amount of bacteria adhered on the surface was then controlled by RAIRS before the morphological tests carried out by AFM and SEM-FEG. For the antimicrobial activity in solution, 200 μl of bacteria (106 cfu/ml, A630 = 0.01) were added to 200 μl of peptide dilution (final peptide concentration: 12.5 μM). A drop (100 μl) of this solution was deposited onto a gold plate without grafted peptides. After 3 h incubation at 25 °C (wet atmosphere), samples were treated under the same conditions described in the previous text. The surfaces used for visualising the morphology of bacteria after contact in solution with the bacterial strain were nonbiocidal surfaces: either glass surfaces or bare gold surfaces for SEM-FEG experiments. For observation of bacteria after deposition on modified surfaces, the tests were conducted on the surfaces obtained after

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The gold samples were placed in the external beam of FT-IR instrument (Nicolet Nexus 5700 FT-IR spectrometer, ThermoFisher Inc., Waltham, MA, USA), and the reflected light was focused on a nitrogen-cooled HgCdTe wide band detector. The IR spectra were

recorded at 8 cm 1 resolution, with co-addition of 128 scans. A ZnSe grid polarizer and a ZnSe photoelastic modulator to modulate the incident beam between p and s polarisations (HINDS Instruments, PM90, modulation frequency = 36 kHz) are placed prior to the sample. The detector output is sent to a two-channel electronic device that generates the sum and difference interferograms. Those are processed and undergo Fourier transformation to produce the PM-RAIRS signal (ΔR/R0) = (Rp Rs)/(Rp + Rs). Using a modulation of polarisation enabled us to perform rapid analyses of the sample after treatment in various solutions without purging the atmosphere or requiring a reference spectrum.

LOMBANA ET AL. each steps of the functionalization; thus, bacteria were deposited on Au, Au-MUA25 (or MUAM25) surfaces and on Au-MUA-Temp modified surfaces [Figure S1 (see Supporting Information)]. MIC Determination The MIC values of temporin-SHa and [A2,6,9]temporin-SHa were determined using a liquid growth inhibition assay, as previously described [33]. Briefly, L. ivanovii bacteria were cultured in BHI medium. A logarithmic phase bacterial culture was diluted in BHI to a A630 = 0.01 (106 cfu/ml). Diluted bacteria (50 μl) were mixed with 50 μl of twofold serial dilutions of synthetic peptide (200 to 1 μM, final concentrations). The bacterial growth was monitored after overnight incubation at 37 °C by measuring the change in A630 value using a microplate spectrophotometer. MIC was expressed as the lowest concentration of peptide that completely inhibited bacterial growth, and as the average value from three independent experiments, each performed in triplicate with positive (0.7% formaldehyde) and negative (without peptide) inhibition control.

Results and Discussion Activity of Temporins Against L. Ivanovii The determination of the MIC revealed that temporin-SHa displays a potent antibacterial activity against L. ivanovii (MIC = 6.25 μM). When the MIC well content was spread on agar plates and incubated overnight at 37 °C, no bacterial growth was observed, indicating that the peptide is bactericidal. No activity was found for the analogue [A2,6,9]temporin-SHa (MIC > 200 μM) that displays a lower hydrophobicity (HeliQuest, http://heliquest.ipmc.cnrs.fr/), = 0.63, compared with temporin-SHa ( = 0.91). Therefore, temporin-SHa and [A2,6,9]temporin-SHa were used as positive and negative controls, respectively, for the determination of antibacterial activity after covalent grafting of the peptide on the Au-MUA surface. Temporin-SHa-COOH (positive control) and [A2,6,9]temporin-SHa-COOH (negative control) were used for the grafting on Au-MUAM surface. The MIC of theses two carboxylated temporins were not determined because these analogues display a C-terminal amide bond once grafted on an amino thiol SAM (MUAM), and therefore, they are considered as carboxyamidated, such as their parent peptides (temporin-SHa and [A2,6,9]temporin-SHa). Surface Functionalization

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Figure 2 shows the PM-RAIRS spectra recorded after the successive steps of gold surface functionalization. The Au-MUA spectrum (a) presents a rather intense νC=O band at 1722 cm 1, characteristic of carboxylic groups [30,34], showing the presence of the carboxylic acid-terminated thiol. The νCH band of the CH2 chains are recorded at 2850 and 2928 cm 1, and the IR feature at 1247 cm 1 is attributed to the OH deformation of the mercaptohexanol. The broad massif between 1400 and 1550 cm 1 likely includes contributions from the scissor mode of CH2 groups and from the symmetric and asymmetric COO . When comparing the IR intensities of pure MUA and pure C6OH SAMs elaborated under similar conditions [31], the lower intensities of each bands in this experiment confirm the presence of both thiols at the surface.

Figure 2. PM-RAIRS spectra of the gold functionalized surface without (a and d) and with covalently grafted temporins (b–c and e–f). (a) 2,6,9 Au-MUA25; (b) Au-MUA25-SHa; (c) Au-MUA25-[A ]-SHa; (d) Au-MUAM25; (e) 2,6,9 Au-MUAM25-SHa-COOH; (f) Au-MUAM25-[A ]-SHa. Corresponding amides I and II integrated IR area (a.u.): (b) 1.38, (c) 0.58, (e) 1.52 and (f) 0.95.

For MUAM (spectrum b of Figure 2), bands in the lower wavenumber region at 1650–1640 cm 1 can be attributed to the N–H deformation vibration, thus confirming the presence of primary amine groups. As previously observed for MUA, one can distinguish on the IR spectrum the presence of peaks assigned to CH2 groups of the alkyl chain at ~1460, 2928 and 2854 cm 1, respectively, for CH2 scissor and stretching vibration modes. Finally, the peak around 1550 cm 1 can be due to the deformation vibration of protonated amine groups, and one can also notice the presence of a very small and broad feature at ~1250 cm 1, assigned to the δOH vibration of the diluting mercaptohexanol thiol, thus confirming that both thiols are present at the surface in a mixed layer. The PM-RAIRS bands assignments could be found in Figure S2 and Table S1 (see Supporting Information). The successful binding of temporins (temporin-SHa, temporinSHa-COOH, [A2,6,9]temporin-SHa, [A2,6,9]temporin-SHa-COOH) is indicated by the appearance of two new intense bands on spectra (b–c and e–f) arising at 1662 and 1548 cm 1 and assigned respectively to the amide I (νC=O) and amide II (δNH + νC–N) vibrations of the peptide backbone. However, the quantity of adsorbed peptides varies as a function of the anchoring SAMs or depends on the temporin analogues. For instance, temporins-SHa (spectra b and e) are present in higher quantity (roughly one and a half more) than the [A2,6,9]temporin corresponding analogues (spectra c and f), regardless of the binding mode (i.e. via NH2 or COOH groups). When looking at the PMRAIRS signal for temporins-SHa, one can notice that grafting via the COOH C-terminal function induces a small increase of adsorbed peptides (spectra b and e, 1.38 a.u. vs 1.51 a.u.), which could be explained by a better accessibility of the peptides towards the aminothiol-functionalized SAMs coupled to only one binding mode, ensuring a better packing of the peptide layers (Figure 1 (b)). This phenomenon, also observed for [A2,6,9] temporin-SHa analogues, shows even higher differences with a ratio of almost 2 (spectra c and f). Few other parameters could induce difference in the amount of adsorbed peptides. For temporin-SHa-COOH analogues, the activation in solution of the acidic group by EDC/NHS without preliminary protection of the amino group can induce intermolecular

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ANTIMICROBIAL PEPTIDE: TEMPORINS ON GOLD SURFACES polymerisation, with the creation of dimers, trimers, … that will still be covalently grafted, as such, on the thiol SAMs, thus increasing the amount of grafted peptide. Another important parameter driving the amount of adsorbed peptide will be the hydrophobic trends of the SAMs; amino-terminated thiols are known to be more hydrophilic than acidic-terminated ones [35,36], and it is verified in the present case, with Au-MUA surfaces exhibiting a water contact angle of ~64° for only ~44° for Au-MUAM. Because all temporin analogues are composed of hydrophobic and polar residues [11,12], they could interact more with NH2-terminated surfaces, showing higher IR intensities for these layers (spectra e and f). Finally, when synthesising [A2,6,9]temporin-SHa and [A2,6,9] temporin-SHa-COOH analogues, Alanine (A) residues replace Leucine (L) and Valine (V) residues, and this new sequence could possibly induce perturbations in the helical propensity of the peptide that will influence again the accessibility of the peptides towards the surface, explaining why, both Ala-containing analogues are grafted in lower quantity (Figure 2, spectra c and f). Visualisation by AFM and SEM-FEG of Antimicrobial Activity of Temporins Before testing the antimicrobial activity of the temporins immobilised on a gold surface, a first test was carried out in solution to visualise the antibacterial activity of temporin towards the Gram-positive bacteria, L. ivanovii. Two sets of bacterial solutions were prepared, one bearing only bacteria and the second one covered with a solution containing bacteria preincubated 3 h at 25 °C with a concentration of temporin-SHa twofold above the MIC (12.5 μM). The first solution was deposited on bare clean gold surfaces (Figure 3(a) and (b)). The AFM and SEM data are presented in Figure 3 for both intact and damage bacteria. Figure 3(a) represents a 3D AFM image of one isolated alive bacterium presenting a well-defined oval shape as expected for Listeria [37]. On the 2D SEM image (Figure 3(b)), one can distinguish a white plain line following the shape of the bacteria, corresponding to the plasma membrane of the Gram-positive bacteria and confirming the integrity of the bacteria with no damage to the membrane. On the contrary, when L. ivanovii was in contact with temporin-SHa, SEM data (Figure 3(d)) reveals membrane damages, causing leakage of the cytoplasmic content (right part of the image). 3D AFM image (Figure 3(c)) also shows the drastic morphological changes of the damage bacteria (the

perfect oval shape has disappeared and the single bacterium looks like it has collapsed on itself). Thus, these data confirm the results obtain in our study from the MIC determination, i.e. the killing mode of action of temporin-SHa in solution. Moreover, they indicate that this killing activity is due to a membrane permeabilization/disruption of the bacterial cells, as suggested by previous studies on other temporins [10,11,14]. The antibacterial activity of the grafted temporins was analysed by adding a solution of L. ivanovii bacteria (3 h incubation) onto the differently functionalized surfaces containing the covalently grafted peptides. First, tests were conducted on the surfaces at each step of the grafting process. AFM data [Figure S1 (see Supporting Information)] show alive and well-shaped bacteria on Au, Au-MUA and Au-MUAM-modified surfaces, thus showing that the thiolated linkers do not have any antibacterial effect towards L. ivanovii strain. Turning now to the temporin-modified surfaces, the AFM results of Figure 4 show that L. ivanovii bacteria have been damaged after contact with temporin-SHa and temporin-SHa-COOH AMPs, compared with Figure 3(A). The SEM-FEG data (Figure 4(c) and (d)) confirm damaging of the bacteria; the bacterial membrane is disrupted after contact with the AuMUA-temporin-SHa surface (Figure 4(c)), whereas bacteria became swollen when incubated with the Au-MUAM-temporin-SHa-COOH surface (Figure 4(d)). From these results, it appears that temporin-SHa covalently bound, either via its NH2 groups or its C-terminal end to a gold surface, still have an antimicrobial effect. However, the killing efficiency of the grafted temporins seems to be lower than in solution, where the bacterial membrane is destroyed and the bacterium has collapsed (leaking of intracellular material). An estimation of the killing efficiency of the modified surfaces has been calculated over observation of ~100 bacteria by SEM-FEG, and the results are presented in Table 1 [more bacteria images can be found in Figure S3 (see Supporting Information)]. One can clearly see from these results that both temporin-SHa and temporin-SHa-COOH-modified surfaces exhibit killing efficiency over 75%, whereas the control surfaces (Ala-containing peptides) display, nonetheless, a small killing activity (15–30%). This efficiency difference can be explained by at least two reasons. First, temporins bound to a surface are losing some freedom, and one can imagine that the killing mechanism in solution involves several temporin molecules that have to be in contact with the whole bacteria to completely destroy it. Following the

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Figure 3. AFM and SEM-FEG visualisation of Listeria ivanovii bacteria deposited on gold surface after preincubation or not with temporin-SHa (solutions recovered from MIC determination experiments). (a) and (b): AFM and SEM-FEG, respectively, of alive bacteria. (c) and (d): AFM and SEM-FEG, respectively, of bacteria preincubated 3 h with a concentration of temporin-SHa twofold above the MIC (12.5 μM). For visualisation of bacteria morphology after the in-solution assay, the bacterial suspensions were deposited on clean and nonbiocidal surfaces (either glass of bare gold, respectively, for AFM and SEM-FEG imaging).

LOMBANA ET AL.

Figure 4. AFM images of Listeria ivanovii on (a) Au-MUA-temporin-SHa and (b) Au-MUAM-temporin-SHa-COOH surfaces. (c) and (d): corresponding 2,6,9 2,6,9 SEM-FEG images. (e) and (f): SEM-FEG images of L. ivanovii on Au-MUA-[A ]temporin-SHa and Au-MUAM-[A ]temporin-SHa-COOH, respectively.

Table 1. Percentage of alive Listeria ivanovii from SEM-FEG imaging Surface

Au

SHa

% Alive

92

23

SHa-COOH [A2,6,9]-SHa 17

85

[A2,6,9]SHa-COOH 69

Over 100 bacteria were observed; the percentage was calculated as % = alive/(alive + dead). The value for dead bacteria also includes the damaged bacteria.

same frame, the conditions used in our antibacterial tests do not allow us to control the contact time between bacteria and surface, even if the tests are run for 3 h, compared with the few minutes needed in solution for the killing to be effective [10–12,14]. Finally, the surface concentration of adsorbed temporins could also be a determining parameter in the killing efficiency. Indeed, the concentrations used for MIC determination in solution are such that the number of AMPs per cfu is close to 108 molecules [38]. When looking at the surface concentration of grafted peptides in our tests conditions, the equivalent amount of temporins per Listeria cfu is reduced to ~106 peptides, which may be a possible explanation for a lower killing activity. Finally, the same experiments were carried out with both negative control peptides, [A2,6,9]temporin-SHa and [A2,6,9]temporinSHa-COOH, and the results are presented in Figure 4(e) and (f). As expected from MIC determination of [A2,6,9]temporin-SHa (MIC > 200 μM), there is no or very little activity on adhered bacteria.

Conclusions

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Temporin-SHa, a small 13-residue AMP, has been successfully grafted on a mixed alcohol/acidic thiol SAM on gold surfaces. The efficacy of the coated surface was tested towards the

nonpathogenic Gram-positive L. ivanovii after determining the antimicrobial activity of temporin-SHa in solution (MIC = 6.25 μM). AFM and SEM-FEG imaging have been performed to assay the mode of action of temporin-SHa. Results show that when this peptide is covalently grafted to the surface, the adhering bacteria are most probably permeabilized (killed or at least damaged), thus unable to grow, whereas bacteria integrity is not affected on a nonfunctionalized surface. The temporin-SHa-COOH analogue was designed and synthesised in order to change the anchoring mode of temporinSHa (oriented grafting via its C-terminal end onto an amino thiol SAM). This analogue has proven to have at least the same efficiency as the parent peptide with nonetheless a higher density of peptides present at the surface, due to a better accessibility to the reactive modified surface. Finally, the analogue [A2,6,9] temporin-SHa, not active in solution (MIC > 200 μM), have been proven also inactive towards L. ivanovii once grafted on the gold surface via its NH2 groups or its COOH-terminal group (analogue [A2,6,9]temporin-SHa-COOH). The efficacy of the killing has now to be more accurately calculated and probably improved, but such temporin-coated surfaces are of potential importance when looking for new ways of protecting surfaces towards biofilm growth.

Acknowledgements This work was supported by French state funds managed by the ANR within the Investissements d’Avenir programme under reference ANR-11-IDEX-0004-02, and more specifically within the framework of the Cluster of Excellence MATISSE. This work was also supported by funds of the Convergence MECV 2011 programme of UPMC. We thank Dr C. Piesse (Platform for Protein Engineering and Peptide Synthesis) and Dr G. Bolbach and G. Clodic (Platform Mass Spectrometry and Proteomics) for their expertise and technical assistance in peptide synthesis and MS analysis, respectively (FR 3631

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ANTIMICROBIAL PEPTIDE: TEMPORINS ON GOLD SURFACES UPMC-CNRS, IBPS, Paris, France). Finally, the authors acknowledge IMPC (Institut des Matériaux de Paris Centre, FR2482) and the C’Nano projects of the Region Ile-de-France for SEM-FEG funding.

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