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Peptides 38 (2012) 363–370

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Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum Aline Rifflet a , Sabine Gavalda a , Nathan Téné a , Jérôme Orivel b , Jérôme Leprince c , Laure Guilhaudis d , Eric Génin e , Angélique Vétillard a , Michel Treilhou a,∗ a

Equipe VacBio EA 4357, PRES Université Toulouse, CUFR JF Champollion, Place de Verdun, 81012 Albi, France CNRS, UMR Ecologie des Forêts de Guyane, Campus Agronomique, BP 316, 97379 Kourou, France c INSERM U982, PRIMACEN, Institut de Recherches et d’Innovation Biomédicale (IRIB), Université de Rouen, 76821 Mont-Saint-Aignan, France d UMR 6014 CNRS, Equipe de Chimie Organique et Biologie Structurale, Institut de Recherche en Chimie Organique Fine (IRCOF), IRIB, Université de Rouen, 76821 Mont-Saint-Aignan, France e ThermoFisher Scientific, 16 avenue du Québec, 91963 Courtaboeuf, France b

a r t i c l e

i n f o

Article history: Received 22 December 2011 Received in revised form 29 August 2012 Accepted 30 August 2012 Available online 7 September 2012 Keywords: Tetramorium bicarinatum Ant venom Staphylococcus ESI-MS/MS Antibacterial peptide AMP Bicarinalin

a b s t r a c t A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was Cterminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens. © 2012 Elsevier Inc. All rights reserved.

1. Introduction With more than 14,700 known species and several thousands remaining to be described, ants constitute a highly diversified group of arthropods [1]. In spite of this taxonomic diversity, ant venoms have been, however, poorly studied to date in comparison with other animal venoms [3,8,26]. The minute quantities of venom secreted by ants together with the lack of high-performance analytical systems and tools, have been for a long time one of the main obstacle to their toxinological investigation. Nevertheless, several potent toxins have been already isolated from ant venoms with activities causing various health disorders in human from painful irritation to anaphylaxis in the most severe cases [4–7,10]. Ant venoms are also a rich source of antimicrobial molecules, acting in colony defense to prevent potential infections by microorganisms. Among these molecules, antimicrobial peptides (AMPs) have been identified in several species, such as

∗ Corresponding author. E-mail address: [email protected] (M. Treilhou). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.08.018

Ponericins from the neotropical ant Pachycondyla goeldii or Pilosulin from the Australian Myrmecia pilosula [9,13,20,22,27,28,33]. These AMPs demonstrate potent activities and selective antibacterial spectra against both Gram-positive and Gram-negative bacteria. Since their first discovery, a number of AMPs have been isolated, and their biological activities offer numerous potential uses in therapeutic applications [12,25]. Here we have investigated the antibacterial properties of the venom from the myrmicine ant, Tetramorium bicarinatum. The crude venom activity has been tested against Staphylococcus aureus, a coagulase-positive staphylococcus (CPS) and Staphylococcus xylosus, a coagulase-negative staphylococcus (CNS). We isolated and identified two peptides among which only one exhibiting the antimicrobial activity. Their amino acid sequences were determined by de novo sequencing and Edman degradation. The secondary structures were investigated by circular dichroism with synthetic peptides. In order to further assess their antibacterial and hemolytic properties, their activities were compared to that of widely used AMPs such as Melittin, and standard antibiotics such as Ampicillin and Tetracycline.

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2. Materials and methods

fraction C containing compounds with molecular weight less than 3 kDa (300 ␮L, about 300 ␮g of dry material).

2.1. Chemical reagents 2.6. RP-HPLC fractionation Melittin, endoproteinase K, Ampicillin, Tetracycline and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (Saint-Quentin-Falavier, France), and Tris–tricine migration buffer, 16.5% polyacrylamide Tris–tricine gel and adapted protein standards from Bio-Rad (Marnes la Coquette, France). 2.2. Ants Tetramorium bicarinatum (Formicidae, Myrmicinae) is a widespread tramp species originating from Southeast Asia, which has spread throughout most of the tropics and subtropics via human activities [29]. Colonies are polygynous (i.e. with several queens per nest) with workers of ca. 3–3.5 mm in size [2]. The colonies were collected in Itabuna, Bahia state, Brazil and maintained in the laboratory, at 25 ◦ C in a 60–70% humidity range, fed ad libitum three times a week with fresh mealworms (Tenebrio molitor) and an aqueous honey solution (50:50, v/v). 2.3. Venom collection and preparation Ant workers were first killed by freezing and venom reservoirs were dissected under a binocular microscope (SMZ800, Nikon). The venom reservoirs were stored in sterile pyrogen-free polypropylene tubes and then disrupted by ultrasonic waves in an aqueous solution. Membranes were discarded by centrifugation at 13,000 × g for 5 min. The supernatant was dried in a vacuum concentrator and dissolved in ultra pure water (300 ␮L). These aliquots of crude venom were stored at −80 ◦ C until use. Each venom sample was prepared from 200 reservoirs, i.e. about 500 ␮g of dry crude venom in 300 ␮L of water (or PBS for endoproteinase assays). Thus, we have estimated that the average of dry crude venom per worker ant is about 2.5 ␮g. The weight of dry crude venom was determined after concentration under reduced pressure using a SpeedVac Concentrator (SPD131DDA, Thermo Fisher Scientific). For each biochemical assay, a similar representative sample of ants was used. 2.4. Proteolytic assay Crude venom was incubated with endoproteinase K, a serine protease used at 80 ␮g/mL in PBS (0.01 M Phosphate/0.154 M NaCl; pH 7.5) and incubated with a venom aliquot for 12 h at 37 ◦ C. A control sample containing only the enzyme was prepared at the same time. 2.5. Pre-fractionation Three hundred ␮L of crude venom were added on an Amicon ultra centrifugal filter (10 kDa cut-off) and centrifuged at 14,000 × g for approximately 20 min. The concentrated sample (20 ␮L) was washed ten times with 300 ␮L of pure water, diluted in ultrapure water (300 ␮L), transferred into a clean tube and stored at −20 ◦ C until use. The pooled filtrate was concentrated to 300 ␮L, transferred into a 3 kDa filter device and treated with the same conditions as described above. Three fractions were thus obtained: (i) fraction A containing compounds with molecular weight over 10 kDa (300 ␮L, about 200 ␮g of dry material), (ii) fraction B containing compounds with molecular weight between 10 and 3 kDa (300 ␮L, with only a few ␮g of dry material), and (iii)

The fractionation of fraction C was performed using a Finnigan SpectraSYSTEM HPLC (pump PC4000 and AS3000) equipped with a DAD-UV 6000LP detector and a Phenomenex C18 reversedphase column Luna (5 ␮m, particle size; 150 mm × 4.60 mm) and controlled by ChromQuest 5.0 software. The flow of the mobile phase was 1 mL/min and the solvent system was 0.1% formic acid in water (solvent A) and 80% aqueous acetonitrile, 0.1% formic acid (solvent B). The elution was carried out with a linear gradient of 10–80% of solvent B over 45 min and UV detection of the fractions was performed from 200 to 300 nm. The fractions were collected, vacuum-dried, directly analyzed by mass spectrometry (MS) as described below, or diluted in 300 ␮L of PBS to test for their antimicrobial activities. 2.7. LC–ESI-MS analysis of crude venom and fraction C Five micro liters of crude venom or sub-fraction C were analyzed with a Surveyor HPLC equipped with a Phenomenex C18 reversed-phase column Luna (5 ␮m, particle size; 150 mm × 2 mm) at a flow rate of 200 ␮L/min. The system was coupled to an ion trap LCQ Advantage mass spectrometer (ThermoFinnigan) fitted with an electrospray ionization source in the positive mode. Spray voltage was at 4.5 kV, capillary temperature set at 300 ◦ C and, sheath gas and auxiliary gas set at 50 and 5 psi, respectively. The acquisition range was from 100 to 2000 m/z. The method combined full scans and higher resolution zoom scans in data dependent mode to confirm charge states of all ions. HPLC elution conditions were the same as described above. 2.8. MS/MS sequencing We have used a linear ion-trap-orbitrap hybrid mass spectrometer (LTQ-Orbitrap Velos, Thermo Fischer Scientific) with an ESI ion source for MS/MS sequencing. The electrospray interface was set as follows: spray voltage at 4 kV, capillary temperature at 275 ◦ C, sheath gas flow at 20 (arbitrary units). The peptide sample was directly introduced at a flow of 5 ␮L/min. Scans were acquired at a high resolution (r = 100,000 at m/z 400) and the MS/MS spectra were recorded sequentially in the orbitrap after fragmentation of selected precursors in the LTQ mass analyzer. The interpretation of MS/MS spectra and the identification of peptide sequences were processed using PEAKS de novo sequencing software (version 5.2). 2.9. Automated Edman degradation The primary structures of the peptides were confirmed by automated Edman degradation using an Applied Biosystems model 494 Procise sequenator. The sequences obtained were matched to public databases of protein sequences with PATTINPROT software (http://npsa-pbil.ibcp.fr). 2.10. Peptide synthesis The syntheses of the free and amidated C-terminal peptides were achieved by solid-phase synthesis using the Fmoc strategy. Peptides were synthesized on a Liberty Microwave assisted automated peptide synthesizer (CEM, Saclay, France) using the standard manufacturer’s procedures (0.1 mmol scale) on a Rink amide MBHA resin or on 4-hydroxymethylphenoxymethyl-copolystyrene 1% divinylbenzene HMP resin. Peptides were cleaved from the resin and deprotected by adding 10 mL of an ice-cold mixture of

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TFA/TIS/H2 O (9.5:0.25:0.25, v/v/v) and then agitated at 0 ◦ C for 5 min and at room temperature for 3 h as previously described [18,19]. Crude peptides were purified by semipreparative RP-HPLC on a Vydac 218TP1022 C18 column (2.2 cm × 25 cm; Alltech, Templemars, France) using a linear gradient (10–50% over 50 min) of acetonitrile/TFA (99.9:0.1, v/v) at a flow rate of 10 mL/min. Analytical RP-HPLC analysis, performed on a Vydac 218TP54 C18 column (0.46 cm × 25 cm; Alltech), revealed that the purity of each peptide was higher than 98.6%. The authenticity of each peptide was verified by MALDI-TOF-MS on a Voyager DE-PRO (Applera-France) in the reflector mode with ␣-cyano-4-hydroxycinnamic acid as a matrix.

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(SDS) in concentrations ranging from 2 to 16 mM, i.e. below and above the critical micelle concentration (CMC) at a final concentration of 0.5 mg/mL. The accurate peptide concentrations were obtained by measuring the optical density at 280 nm. Each spectrum was recorded as an average of three scans and all spectra were corrected by subtraction of blanks. The values are expressed in terms of []T mean residue molar ellipticity (deg cm2 dmol−1 ). The ␣-helical content of all peptides was estimated by using the Forood formula: 100 × ([]222 /max []222 ) with max []222 = −40,000 [1 − (2.5/n)], where n = number of amino acid residues. 3. Results 3.1. Crude venom and fractions activities

2.11. Antibacterial activity Crude venom, HPLC fractions, synthetic peptides, Melittin, Ampicillin and Tetracycline were tested against two wild-types of Staphylococcus, S. aureus and S. xylosus obtained from the Laboratoire Départmental d’Analyse du Tarn (LDA, Albi, France), and two referenced types, S. aureus (CIP 53156) and S. xylosus (ATCC 35033). Minimum inhibitory concentrations (MIC) were measured for concentrations ranging from 0.10 to 255 ␮g/mL for the synthetic peptides and Melittin, and from 0.015 to 35 ␮g/mL for Ampicillin and Tetracycline. HPLC fractions and each tested compound were prepared in PBS (crude venom was diluted in ultrapure water), placed in 96-well microtiter cell-culture plates and completed by the bacterial inoculum (1 × 106 UFC/mL). The final volume of the wells was 50 ␮L and the microplates were incubated at 37 ◦ C for 3 h before monitoring the antibacterial activity by the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) cytotoxicity assay [21] on the bacterial strains. After incubation, 50 ␮L of MTT solution (1 mg/mL) was added to each well and incubated during 1 h at 37 ◦ C. The blue formazan crystals generated were dissolved by adding 50 ␮L of DMF/SDS 20% solution (1:2, v/v) and the absorbance was detected at 570 nm using a spectrophotometric microplate reader (Infinite 200, Tecan, Austria) as previously described [31]. 2.12. Hemolytic activity The hemolytic activity of the peptides was compared to that of Melittin against human erythrocytes from healthy donor. Fresh human erythrocytes with heparin were washed three times (10 min at 1000 × g) with PBS and resuspended in the same buffer. The hemolytic activity was quantified by incubating 20 ␮L of serially diluted peptides (2.8 × 10−3 – 5.76 mg/mL) or Melittin (0.11 × 10−3 – 0.225 mg/mL) with 80 ␮L of a 4% erythrocytes suspension in PBS for 1 h at 37 ◦ C in a 96-well microplate (Nunc Edge 96 Well Microplate). The erythrocyte suspension was then centrifuged (1000 × g for 10 min) and, 80 ␮L of the supernatant were transferred in another line of the microplate; cell lysis was determined spectrophotometrically at 540 nm. Reference samples were employed using hypotonic lysis with Milli-Q sterile water as a 100% lysis or PBS pH 7.4 (in place of the peptide) as a 0% lysis. Three replicates were performed. 2.13. Circular dichroism analysis Circular dichroism (CD) spectra were acquired on a CD6 dichrograph spectropolarimeter (Jobin-Yvon, Longjumeau, France). The instrument was calibrated using d(+)-10-camphorsulfonic acid. Spectra were collected at room temperature using a cylindrical fused quartz cell with a path length of 0.5 mm. Peptides were dissolved in water or in 2,2,2-trifluoroethanol (TFE) aqueous solutions (10%, 25% and 50%, v/v) or in sodium dodecyl sulfate

Biological assays of crude venom have shown an antibacterial action against wild strains of S. aureus and S. xylosus. These antibacterial activities were not detected after incubation of the crude venom with proteinase K, demonstrating that the active compounds of the venom were peptides. The level of activity of the crude venom was, however, relatively low compared to reference antibiotics such as Ampicillin and Tetracycline respectively (data not shown). No activity was observed with fractions A and B and only fraction C has shown an antibacterial activity. 3.2. Characterization of the venom compounds Three spots of proteins with molecular weight ≤40 kDa were separated from the crude venom using 16.5% SDS polyacrylamide gel using Tris–tricine buffer (data not shown). Moreover, a large unresolved spot remained at the bottom of the gel indicating that low molecular weight molecules (25 kDa) cannot be detected. Thus, the TIC chromatogram of the crude venom showed the same peptides with a low size as the one of fraction C. The resolution of the peaks in the chromatogram of the venom was lower than the one of fraction C. This could be due to a matrix effect. In term of composition, it was noted that three compounds, 29–31, were absent in fraction C. The fractionation of fraction C gave 15 collected peaks (sub-fractions C1 –C15 ) and the antibacterial activity was demonstrated only for the sub-fraction C11 which contained peptides 16 and 17. 3.3. MS/MS sequencing and Edman degradation MS/MS analysis of the active sub-fraction C11 confirmed the presence of two compounds, peptide 16 (P16) and peptide 17 (P17) (Table 1 and Fig. 1). P16 was characterized by the multicharged ions m/z 1107.71 as [M+2H]2+ and m/z 738.70 as [M+3H]3+ while P17 was characterized by the ions m/z 1572.10 as [M+H]+ and m/z 787.00 as [M+2H]2+ (Fig. 2A). These two compounds co-eluted under the HPLC conditions used (compounds 16–17, Fig. 1B). For the peptide P16, the fragmentation of the triply charged species at m/z 738.93, allowed the determination of its sequence with ambiguity for leucine and

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Table 1 Main peptides, with relative intensity greater than 10, identified by LC–ESI-MS in the venom of T. bicarinatum.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Rt (min)

m/z (z)

Mass (Da)

1.6 2.83 2.83 3.61 13.15 15.31 16.36 16.36 16.36 17.85 18.01 18.71 19.84 20.81 22.71 23.37 23.77 25.35 27.44 29.67 29.67 30.16 30.16 31.5 33.30 33.70 34.86 38.52 38.38 39.37 39.89

891.25 (3); 1316.63 (2) 779.32 (2); 1557.65 (1) 968 (2); 1934.88 (1) 616.1 (1) 798.18 (3); 1063.87 (3); 1595.87 (2) 568.06 (3); 851.54 (2) 858.64 (2); 1715.84 (1) 527.41 (2); 1053.39 (1) 649.09 (2); 1296.75 (1) 810.09 (2), 1618.95 (1) 869.27 (3); 1304.34 (2) 925.81 (3); 1388.34 (2) 952.16 (3), 1427.46 (2) 1006.07 (4); 1341.32 (3) 670.66 (3); 1005.32 (2) 554.22 (4); 738.96 (3); 1107.22 (2) 786.66 (2); 1572.67 (1) 940.25 (1) 831.95 (4); 1109.01 (3); 1664.08 (2) 968.75 (2), 1936.20 (1) 646.05 (2); 1291.26 (1) 971.91 (2); 1943.20 (1) 648.88 (2); 1295.38 (1) 788.60 (3); 1182.04 (2) 826.64 (2); 1652.57 (1) 826.33 (2); 1652.53 (1) 660.31 (4); 880.16 (3); 1319.83 (2) 829.11 (3); 1243.32 (2) 604.25 (2); 1207.58 (1) 705.66 (2); 1409.65 (1) 705.66 (2); 1409.69 (1)

2671 1556.64 1933.99 615.1 3189.01 1701.12 1715.56 1052.60 1295.96 1618.56 2605.74 2774.52 2853.20 4020.62 2008.81 2213.06 1571.49 939.25 3324.66 1935.35 1290.18 1942.01 1295.07 2362.44 1651.42 1651.09 2637.46 2484.48 1206.54 1408.98 1408.82

isoleucine (Fig. 3). Similarly, the doubly charged species m/z 786.70 was selected for the sequencing of P17. The MS/MS spectrum suggested the presence of several leucine/isoleucine residues in the sequence of P17. In addition, the theoretical monoisotopic masses of each peptide were one mass unity higher than the measured experimental masses, demonstrating that P16 and P17 were amidated. Edman sequencing led to the exact assignments for each leucine/isoleucine ambiguity in P16 and P17 sequences. The primary sequence of the two peptides was determined as: KIKIPWGKVKDFLVGGMKAV-NH2 and LFKEILEKIKAKL-NH2 for P16 and P17, respectively. 3.4. Synthetic peptides Sequence confirmation and purity of the synthetic peptides were controlled before the biological assays. The HPLC analysis showed that the purity was greater than 98.8% mass spectra of both natural and synthetic peptides were similar (Fig. 2). 3.5. Antibacterial activity P16 and its carboxylated counterpart noted P16 COOH , showed antibacterial activities against both Staphylococcus strains (Table 2). P16 was 20 and 6 times more potent than P16 COOH on referenced and wild (LDA) S. aureus strains, respectively. It should be noted that, the antibacterial activity of P16 against both Staphylococcus strains, were comparable to that of Melittin and standard antibiotics, Ampicillin and Tetracycline. Because of its potent antibacterial activity, we propose to name P16 Bicarinalin. Concurrently, P17 and its carboxylic form, P17 COOH , did not display any

Fig. 1. Total ion current (TIC) chromatogram of Tetramorium bicarinatum crude venom (A) and fraction C (B) recorded by mass spectrometry during the HPLC elution. Each peak corresponds to one or several of peptides, which are listed and numbered in Table 1.

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Fig. 2. (A) LC–ESI-MS (ion trap) mass spectrum of the C11 active fraction containing the two natural peptides P16 and P17. (B and C) Mass spectra of synthetic peptides P16 and P17 achieved by direct infusion.

Table 2 Antimicrobial activity of crude venom and synthetic peptides. All assays were performed at least in triplicate. Micro-organisms

S. aureus (LDA) S. aureus CIP 53156 S. xylosus (LDA) S. xylosus ATCC 35033 a

MIC (␮g/mL)a Crude venom

P16

P16

>500 nt 23.22 nt

6.3 6.4 1.3 1

34.01 126.32 3 4.6

COOH

P17

P17

na na na na

na na na na

COOH

Melittin

Ampicillin

Tetracycline

8.3 10 6.7 7.4

nt 0.1 nt 0.3

nt 0.5 nt 6

MIC: minimum inhibitory concentration; na: no activity under 250 ␮g/mL; nt: not tested.

antibacterial activity (Table 2). Interestingly, P16 was more active than Tetracycline on referenced S. Xylosus. Finally, P16 and P17 did not act in synergy since no improvement of antibacterial activity was observed (data not shown).

3.6. Hemolytic activity At low concentrations, Bicarinalin showed only a weak hemolytic effect on human erythrocytes (