Báo cáo khoa học: Dermaseptin DA4, although closely related to dermaseptin B2, presents chemotactic and Gram-negative selective bactericidal activities doc

Dermaseptin DA4, although closely related to dermaseptin B2, presents chemotactic and Gram-negative selective bactericidal activities Constance Auvynet1,2,*, Pierre Joanne2, , Julie Bourdais3, Pierre Nicolas2, , Claire Lacombe2,4,à and Yvonne Rosenstein1 ´ ´ Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Col Chamilpa, Cuernavaca, Morelos, Mexico ´ FRE 2852, Peptidome de la peau des amphibiens, CNRS ⁄ Universite Paris–Pierre et Marie Curie, Paris, France Independent scholar, Cuernavaca, Morelos, Mexico ´ ´ UFR Sciences et Technologie, Universite Paris 12–Val de Marne, Creteil, France Keywords antimicrobial peptide; chemotaxis; dermaseptin; frog skin; peptide–membrane interactions Correspondence Y Rosenstein, Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional ´ ´ Autonoma de Mexico, Avenida Universidad 2001, Col Chamilpa, Cuernavaca, Morelos 62270, Mexico Fax: +52 73172388 Tel: +52 55 66 22 76 63 E-mail: yvonne@ibt.unam.mx ´ C Lacombe, Laboratoire des Biomolecules, ´ Universite Pierre et Marie Curie-CNRS-ENS, Place Jussieu, 75252 Paris cedex 05, France Fax: +33 44 27 55 64 Tel: +33 44 27 51 59 E-mail: claire.lacombe@upmc.fr Present addresses ´ *INSERM UMR-S 945 Immunite et Infec´ tion, Universite Pierre et Marie Curie, Paris, France `  Biogenese des Signaux Peptidiques ´ (BIOSIPE), ER3-UPMC, Universite Pierre et Marie Curie, Paris, France Antimicrobial peptides participate in innate host defense by directly eliminating pathogens as a result of their ability to damage the microbial membrane and by providing danger signals that will recruit innate immune cells to the site of infection Dermaseptin DA4 (DRS-DA4), a new antimicrobial peptide of the dermaseptin superfamily, was identified based on its chemotactic properties, contrasting with the currently used microbicidal properties assessment The peptide was isolated and purified by size exclusion HPLC and RP-HPLC from the skin of the Mexican frog, Pachymedusa dacnicolor MS and amino acid sequence analyses were consistent with the structure GMWSKIKNAGKAAKAAAKAAGKAALGAVSEAM CD experiments showed that, unlike most antimicrobial peptides of the dermaseptin superfamily, DRS-DA4 is not structured in the presence of zwitterionic lipids DRS-DA4 is a potent chemoattractant for human leukocytes and is devoid of hemolytic activity; in addition, bactericidal tests and membrane perturbation assays on model membranes and on Escherichia coli and Staphylococcus aureus strains have shown that the antibacterial effects of DRS-DA4 and permeabilization of the inner membrane are exclusively selective for Gram-negative bacteria Interestingly, despite high sequence homology with dermaseptin S4, dermaseptin B2 was not able to induce directional migration of leukocytes, and displayed a broader bactericidal spectrum A detailed structure–function analysis of closely related peptides with different capabilities, such as DRS-DA4 and dermaseptin B2, is critical for the design of new molecules with specific attributes to modulate immunity and/or act as microbicidal agents Abbreviations DDK, dermadistinctin K; DiSC3(5), 3,3¢-dipropylthiadicarbocyanine iodide; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol; DRS-B2, dermaseptin B2; DRS-DA3, dermaseptin DA3; DRS-DA4, dermaseptin DA4; DRS-L1, dermaseptin L1; DRS-S1, dermaseptin S1; DRS-S9, dermaseptin S9; DSC, differential scanning calorimetry; ERK, extracellular signal-regulated kinase; fMLP, formyl-methionyl-leucyl-phenylalanine; FPR, formyl peptide receptor; FPRL-1, formyl peptide receptor-like 1; Gal-ONp, 2-nitrophenyl b-D-galactopyranoside; GPCR, G-protein-coupled receptor; ITC, isothermal titration calorimetry; LUV, large unilamellar vesicle; MAPK, mitogen-activated protein kinase; MIC, minimum inhibitory concentration; MLV, multilamellar liposome vesicle; PMN, polymorphonuclear; PTX, pertussis toxin; SDF1-a, stromal cell-derived factor 1a; TFA, trifluoroacetic acid FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6773 Chemotactic and antimicrobial DRS-DA4 C Auvynet et al ´ ´ àLaboratoire des Biomolecules, Universite Pierre et Marie Curie-CNRS-ENS, Paris cedex 05, France (Received 12 August 2009, accepted 21 September 2009) doi:10.1111/j.1742-4658.2009.07392.x Introduction At the interface of innate and adaptive immunity, antimicrobial peptides have been shown to enhance the overall immune response [1] The majority of these peptides are cationic, with a net charge of +2 to +7, and contain up to 50% hydrophobic amino acids This amphipathic design, consisting of spatially separated hydrophobic and charged regions, is believed to allow the insertion of the peptide into microbial membranes Until recently, direct antimicrobial activity against bacteria, fungi, parasites and viruses was considered to be the primary function of antimicrobial peptides However, there is now increasing evidence that antimicrobial peptides are multifunctional molecules of fundamental importance in host defense, modulating the innate and adaptive immune systems In addition to microbicidal activity, a large number of antimicrobial peptides, such as the human cathelicidin LL-37 and the defensins, have been found to modulate the immune response by directing the migration of immune cells to the site of injury, as well as by activating leukocytes and promoting cytokine release, wound repair, angiogenesis, and neutralization of microbial products [2,3] In particular, the chemotactic activity of antimicrobial peptides is mostly mediated through G-proteincoupled receptors (GPCRs) such as the CC-chemokine receptor 6, the formyl peptide receptor (FPR), and the formyl peptide receptor-like (FPRL-1) [4–7] Frog skin is a rich source of antimicrobial peptides, with more than half of the peptides described to date having been isolated from South American Hylidae or European, Asian or North American Ranidae; the peptides are involved in the defense of the frog against predation or invading microorganisms More than 80 antimicrobial peptides have been isolated from only 12 species of the Phyllomedusinae subfamily, belonging to the genera Agalychnis, Hylomantis, Pachymedusa, and Phyllomedusa Among them, the dermaseptins, a superfamily of structurally and functionally related peptides produced by the Hylidae family, have potent microbicidal activity at micromolar concentrations against a wide range of microorganisms (Gram-positive and Gram-negative bacteria, fungi, yeasts, and protozoa), 6774 but no or little hemolytic activity [8] The microbicidal activity of these lysine-rich linear polycationic peptides, most of which are composed of 24–34 amino acids structured as an amphipathic a-helix in polar solvents, is thought to result from the interaction of the amphipathic a-helical structure with the membrane bilayer of target microorganisms Most peptides belonging to the dermaseptin superfamily have been identified primarily on the basis of their antimicrobial activity However, additional biological functions have been recognized that may, or may not, be directly associated with pathogen clearance For instance, adenoregulin [dermaseptin B2 (DRS-B2)] was first identified as a peptide able to stimulate binding of agonists to A1-adenosine receptors [9], and was further shown to enhance the binding potency of several GPCR agonists [10] Frog skin insulintropic peptide (FSIP), also a member of this superfamily, significantly stimulates insulin release in glucose-responsive BRIN-BD 11 cells [11], and dermaseptin S1 (DRS-S1) has been reported to stimulate the microbicidal activity of polymorphonuclear (PMN) leukocytes [12] and dermaseptin S9 (DRS-S9) to chemoattract PMN leukocytes [13] We report herein the isolation and characterization of a new dermaseptin-related peptide, GMWSKIKNA GKAAKAAAKAAGKAALGAVSEAM, named dermaseptin DA4 (DRS-DA4), according to the new dermaseptin nomenclature [14] DRS-DA4 was obtained by fractioning the skin exudate of Pachymedusa dacnicolor, and was first identified on the basis of its chemotactic properties rather than on the classic assessment of its antimicrobial activity Interestingly, although DRS-DA4 was found to share strong sequence homology with DRS-B2, it has distinct biological activities DRS-DA4, but not DRS-B2, induced human leukocyte migration and activation mainly through a GPCR, probably FPRL-1; moreover, it was devoid of hemolytic activity Unlike DRS-B2, which is active on Gram-positive as well as on Gram-negative bacteria, DRS-DA4 only exhibited direct antibacterial activity on Gram-negative bacteria, together with perturbation of the inner membrane against of Gram-negative FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS C Auvynet et al Chemotactic and antimicrobial DRS-DA4 RP-HPLC on a semipreparative column (Fig 1B) A peak with a retention time of 38.7 was found to have strong chemotactic activity, inducing the directional migration of human leukocytes (Fig 1B, insert) The sequence of the purified fraction determined by tandem MS (experimental monoisotopic mass of the protonated peptide: 3063.26) and Edman sequencing gave unequivocally the sequence GMWSKIKNAGKA AKAAAKAAGKAALGAVSEAM According to the new dermaseptin nomenclature [14], this peptide was named DRS-DA4 To confirm the sequence and to demonstrate that the biological activities of the purified natural peptide reflected its intrinsic properties, DRS-DA4 was synthesized by the solid-phase method After HPLC purification on a semipreparative column, synthetic DRS-DA4 was indistinguishable from the natural product, eluting exactly at the same position (38.7 min) as the natural corresponding product and giving the same monoisotopic mass to charge ratio (3063.28) by MALDI-TOF MS (data not shown) Further characterization of the conformational and biological properties was performed with the synthetic peptide bacteria The identification of a novel antimicrobial peptide on the basis of its immunomodulatory capacity broadens the panel of activities of these peptides within Hylidae frog genera In addition, our analysis of the biophysical characteristics and properties of these peptides contributes to our current knowledge regarding the different activities of antimicrobial peptides, and opens the possibility of using them as templates for the design of new molecules with specific attributes to modulate immunity and ⁄ or act as microbicidal agents Results Isolation, purification and structure of DRS-DA4 DRS-DA4 was purified to homogeneity from P dacnicolor skin exudates by a two-step protocol Specifically, 1.1 mL of skin secretions recovered by gently squeezing the parotoid glands of a single living frog was first fractionated on a Sephadex G-50 column (Fig 1A), and each fraction was tested for chemotactic activity The chemotactic fraction III was further purified by A 0.9 Absorbance (280 nm) 0.8 V0 0.7 Fraction II 0.6 0.5 Fraction III Fraction I 0.4 0.3 0.2 0.1 0.0 10 20 30 40 50 60 70 Fraction number B FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS Acetonitrile (%) Fig (A) Profile of fractionation of the P dacnicolor skin exudates on a Sephadex G-50 column The absorbance at 280 nm is represented as a solid line (B) RP-HPLC separation of the recovered fraction III using a semipreparative column Elution was achieved with a 0–60% linear gradient of solvent (dotted line) The arrow points to the elution position of synthetic DRS-DA4 under the same conditions The absorbance at 220 nm is represented as a solid line Insert: neutrophil migration induced in response to the peak indicated by the arrow The peak solution was diluted ⁄ 10 from column to column RPMI medium was used as negative control (column 0) and fMLP (100 nM) as positive control 6775 Chemotactic and antimicrobial DRS-DA4 C Auvynet et al A B Fig (A) Amino acid sequences of DRS-DA4 and DRS-B2, together with some antimicrobial peptides with nearest sequences Identical amino acids between these peptides are underlined (B) Helical wheel projection of the DRS-DA4, DRS-B2, DDK, and DRS-L1 Hexagonal and round backgrounds refer to basic and acid amino acids respectively, pentagonal backgrounds to hydrophilic residues, and squares to hydrophobic residues A protein database was screened for similar sequences, using the embl-ebi fasta program, and this revealed that DRS-DA4 belonged to the dermaseptin superfamily A sequence alignment showed 6776 that the antimicrobial peptides dermaseptin DA3 [DRS-DA3 (PD-33)] [15], DRS-B2 [9], dermadistinctin K (DDK) [16] and ARP-AC1 [17], isolated from P dacnicolor, Phyllomedusa bicolor, Phyllomedusa FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS C Auvynet et al distincta, and Agalychnis callidryas, respectively, presented the highest sequence homology with DRSDA4 (87.9% identity and 93.9% similarity for DRS-DA3, 84.8% identity and 97% similarity for DRS-B2, and 84.8% identity and 93.9% similarity for DDK; Fig 2A) Both, DRS-DA3 and DRS-B2 have six positive charges (Lys), and only one negative charge (Glu) The resulting total overall net charge for DRS-DA4 is +5, whereas DRS-B2 has an overall net charge of +4, owing to the carboxyamidated end, three negative charges (Glu) and the extra positive charge of the N-terminus Figure 2A shows dermaseptin L1 (DRS-L1) [18] from the lemur leaf frog Hylomantis lemur, which showed only 48.6% identity and 22.4% similarity but whose biological properties led us to make a comparison The helical wheel projection of Edmundson showed the partial amphipathic character of the helix obtained for DRS-DA4, with hydrophobic residues on one face of the helix and polar or charged residues on the opposite face, corresponding to a hydrophobic sector that subtends a radial angle of 160° (Fig 2B) DRS-DA4 induces directional migration of human leukocytes Like natural DRS-DA4, synthetic DRS-DA4 induced the migration of human neutrophils (Fig 3A) and monocytes (Fig 3B), with a typical bell-shaped dose– response curve Maximum activity was observed at a concentration of 10 lm for both cell types Addition of various concentrations of DRS-DA4 to the upper wells of the Boyden chamber abolished the migration of the cells, suggesting that DRS-DA4 induced chemotactic movement rather than enhanced random movement (Fig 3A) As described previously [13], DRS-B2 did not induce the migration of leukocytes within the range of concentrations tested for DRSDA4 Like formyl-methionyl-leucyl-phenylalanine (fMLP), most chemotactic antimicrobial peptides induce cell migration through a seven-helix transmembrane Gia-protein-coupled receptor such as FPR or FPRL-1 [19] Pretreatment with pertussis toxin (PTX) – a specific inhibitor of Gia-protein-coupled receptors – prior to the onset of the chemotactic assay partially inhibited the motility of neutrophils induced by DRS-DA4 As expected, neutrophils preincubated with PTX before exposure to fMLP failed to migrate (Fig 4A) Preincubation of neutrophils for 30 with 100 nm fMLP, an FPRL-1 agonist, inhibited by up to 50% the migration of the cells in response to a gradient of DRS-DA4 or fMLP (100 nm) However, preincubation Chemotactic and antimicrobial DRS-DA4 A B Fig DRS-DA4 is a potent chemoattractant of leukocytes (A) Neutrophil migration induced in response to DRS-DA4 or fMLP (100 nM) as a positive control and medium as a negative control Addition of the same concentrations of DRS-DA4 to the upper and lower wells of the chemotaxis chamber abolished the chemotactic effect (B) Monocyte migration in response to DRS-DA4, DRS-B2 or SDF1-a (1 nM) as positive control The data shown represent the average cell migration of triplicate wells Similar results were obtained from three different experiments of the cells with 1.14 nm fMLP, a concentration at which fMLP is considered to be an FPR agonist, did not inhibit the motility of the cells in response to DRS-DA4 or fMLP (100 nm) As expected, preincubation of neutrophils with fMLP (1.14 nm) prior to the chemotaxis assay inhibited the migration induced by fMLP (1.14 nm) (Fig 4A) Stimulation of a GPCR by chemotactic agonist ligands can lead to the activation of mitogen-activated protein kinase (MAPK) pathways in target cells [20] Incubation of neutrophils with DRS-DA4 (10 lm) or fMLP (100 nm) for or 10 induced the phosphorylation of the extracellular signal-regulated kinase (ERK)1 ⁄ MAPK, but not that of the p38 MAPK (Fig 4B) Preincubation with PTX or PD98059 (a specific inhibitor of MEK1 and MEK2) before the addition of DRS-DA4 (10 lm) or fMLP (100 nm) inhibited the phosphorylation of ERK1 ⁄ but induced FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6777 Chemotactic and antimicrobial DRS-DA4 C Auvynet et al suggest that DRS-DA4 chemotactic activity is mediated through a GPCR, probably FPRL-1, and that it is coupled to the ERK1 ⁄ MAPK pathway A DRS-DA4, but not DRS-B2, is only microbicidal for Gram-negative bacteria B 10 ’ 10 ’ 5’ 10 ’ 10 ’ 10 ’ 10 ’ 10 ’ 10 ’ 10 ’ PTX – – – + – + – + – + PD98059 – – – – + + – – + + pErk1/2 Erk1/2 pp38 p38 DRS-DA4 exhibited good to moderate antimicrobial activity against all of the Gram-negative strains tested, as no colony was counted when the peptide–Escherichia coli (both E coli strains tested) or peptide–Pseudomonas aeruginosa mixtures were incubated on LB agar plates overnight, indicative of a bactericidal effect However, no microbicidal activity was detected against any of the Gram-positive strains tested, at concentrations up to 100 lm In comparison, DRS-B2 presented strong antimicrobial activities against both Gram-negative and Gram-positive strains (Table 1) No hemolytic or apoptotic activities were observed on rat blood cells or leukocytes with DRS-DA4 or DRS-B2, respectively, for concentrations up to 100 lm (data not shown) C DRS-DA4 interacts preferentially with 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) vesicles mimicking Gram-negative bacteria Fig DRS-DA4-induced neutrophil migration through a seventransmembrane GPCR (A) DRS-DA4-induced chemotaxis is mediated through a GPCR Neutrophils were preincubated with medium, PTX, fMLP (1.14 nM), or fMLP (100 nM) prior to challenge with DRS-DA4, fMLP (1.14 nM) or fMLP (100 nM) (B) ERK1 ⁄ phosphorylation is induced in response to DRS-DA4 Human neutrophils were treated with medium, DRS-DA4, or fMLP, with or without preincubation with PTX and ⁄ or PD98059 The same membrane was stripped and blotted with antibody against ERK1 ⁄ or antibody against p38 Similar results were obtained from three separate experiments (C) ERK1 ⁄ phosphorylation is necessary for DRSDA4-induced Human neutrophils were preincubated with PD98058 (ERK1 ⁄ inhibitor) or SB202190 (p38 inhibitor) prior to the chemotaxis assay, as described in (A) The data shown are representative of two independent experiments the phosphorylation of p38 Moreover, pretreatment of neutrophils with PD98059 led to decreased migration in response to DRS-DA4 or fMLP (100 nm), whereas SB202190, a specific inhibitor of the p38 MAPK, did not (Fig 4C) All together, these results 6778 When we used the membrane potential-sensitive dye 3,3¢-dipropylthiadicarbocyanine iodide [DiSC3(5)] to assess the ability of the peptide to damage, and thus depolarize, prokaryotic membranes [21,22], we found that addition of DRS-DA4 resulted in concentrationindependent increases in DiSC3(5) fluorescence within the range 10–50 lm, indicative of equivalent membrane depolarization for both strains tested, i.e E coli and Staphylococcus epidermidis (Table 1) Thus, in contrast to its Gram-negative-specific microbicidal activity, DRS-DA4 was able to depolarize the membranes of Gram-positive and Gram-negative bacteria To further characterize the bactericidal activity of DRS-DA4, we measured its ability to damage the bacterial membrane Permeabilization of the inner membrane can be assessed by monitoring the b-galactosidase substrate 2-nitrophenyl b-d-galactopyranoside (GalONp) In E coli strain ML35p and in Staphylococcus aureus strain ST1036, which lack lac permease, GalONp entry is blocked by the integrity of the inner membrane; if Gal-ONp crosses this barrier, it can be cleaved by the cytoplasmic b-galactosidase, resulting in a color change from clear to yellow, reflecting membrane damage As shown in Fig 5A, the time needed to obtain the maximum Gal-ONp hydrolysis in E coli increased as the concentration of DRS-DA4 decreased On the other hand, no permeabilization of S aureus ST1065 was FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS C Auvynet et al Chemotactic and antimicrobial DRS-DA4 Table Antimicrobial activities of DRS-DA4 and DRS-B2 Bacterial strains were considered to be resistant (R) when their growth was not inhibited by peptide concentrations up to 100 lM The data shown correspond to the MIC (lM) E coli 363 ATCC 1175 or S epidermidis BM 3302 transmembrane potential changes were induced by DRS-DA4 and assessed with the DiSC3(5) probe: membrane depolarization was monitored by an increase in fluorescence after the addition of peptide at the MIC Triton X-100 was used to fully collapse the membrane potential B, bactericidal; ND, not determined Antimicrobial activity (MIC) Gram-negative Gram-positive a DRS-DA4 DRS-B2 Membrane depolarization assay E coli ML35p E coli 363 ATCC 11775 Ps aeruginosa ATCC 27853 En faecalis ATCC 29212 S aureus ATCC 25923 S epidermidis BM 3302 5, B 5, B 40, B R R R ND 0.39, Ba 3.1, Ba ND 0.7, Ba ND ND 55 ± 9% ND ND ND 50 ± 5% See ref [26] observed for concentrations of DRS-DA4 up to 100 lm (Fig 5B and data not shown) These results indicate that the bacterial membrane of Gram-negative strains is one of the main targets of DRS-DA4 Differential scanning calorimetry (DSC) is a powerful, nondisturbing thermodynamic technique that is useful for the study of lipid–protein interactions in model membranes as well as for the evaluation of antimicrobial peptide interactions with lipid bilayer model membranes [23] We evaluated the effect of various concentrations of DRS-DA4 on the pretransition and the main transition of the zwitterionic 1,2-dimyristoyl-snglycero-3-phosphatidylcholine (DMPC) and anionic DMPG membranes used as models for eukaryotic and prokaryotic plasma membranes, respectively DSC thermograms illustrating the effect of the incorporation of increasing quantities of DRS-DA4 on the thermotropic phase behavior of multilamellar liposome vesicles (MLVs) of DMPG or DMPC are shown in Fig 5C,D In the absence of the peptide, DMPG exhibited two endothermic events: a less energetic pretransition near 12.8 °C, arising from the conversion of the lamellar phase to the rippled gel phase, and a second, more energetic, main transition at 23.2 °C, resulting from the conversion of the rippled gel phase to the lamellar liquid-crystalline phase These results, together with the enthalpy values for the pretransition ( kcalỈmol)1) and the main transition (8.8 kcalỈ mol)1), are comparable with previous data [24,25] The incorporation of DRS-DA4 into DMPG MLVs significantly altered their thermotropic phase behavior The presence of the peptide abolished the pretransition, even at the lowest concentration tested (peptide/lipid ratio : 100), which is within the same range of order as the minimum inhibitory concentration (MIC) for Gram-negative strains Increasing concentrations of DRS-DA4 broadened the DMPG main phase transition peak, probably because of a loss of cooperativity during the lipid fusion resulting from the insertion of the peptide molecules The main transition was totally abolished at : 20 peptide ⁄ lipid ratios, indicating total disorganization of lipid DMPG bilayer (Fig 5C) Like those of DMPG, aqueous dispersions of DMPC showed two endothermic transitions, a pretransition occurring at 13.1 °C with an enthalpy of about 1.1 kcalỈmol)1, and a main transition at 23.9 °C with an enthalpy of 11 kcalỈmol)1, which was again within the range previously published [24,25] At low concentrations of DRS-DA4 (1 : 100 peptide ⁄ lipid ratio), no modifications of the thermogram were observed The pretransition was then gradually abolished as the peptide/lipid ratio increased At greater ratios only (1 : 20 peptide ⁄ lipid ratio), DRS-DA4 induced some changes to the DMPC melting profile, indicating an interaction with this type of vesicle, whereas the main transition peak was still present, without modification of its temperature CD spectra of DRS-DA4 (Fig 5E) revealed that DRS-DA4 adopted an a-helix conformation in the presence of DMPG vesicles, whereas it remained as a random coil in aqueous solution and in the presence of DMPC liposomes, whatever the peptide/lipid ratio (data not shown) In the presence of DMPG vesicles, the spectrum of DRS-DA4 showed a profile with minima at 208 and 220 nm, suggesting a major contribution of a-helix ( 40%) The conformational propensity of DRS-DA4 contrasts with that of DRSB2, which has been found to be structured as an a-helix (55%), whatever the lipidic composition of the vesicles [26] Discussion Hundreds of antimicrobial peptides have been isolated from frogs, but very few studies dealing with their immunomodulatory capacities have been published FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6779 Chemotactic and antimicrobial DRS-DA4 C Auvynet et al A B Absorbance (405 nm) 2.0 1.5 1.5 1.0 1.0 0.5 20 µM 10 µM µM 1.2 µM 0.3 µM Control 2.0 0.5 0.0 0.0 2000 4000 6000 8000 10 000 12 000 14 000 2000 4000 Time (s) C D 30 8000 10 000 12 000 30 25 Cp (kcal·k·mol–1) 25 Cp (kcal·k·mol–1) 6000 Time (s) 20 DMPG 15 10 1/100 10 15 20 25 30 DMPC 15 10 1/100 1/20 20 35 1/20 40 10 15 Temperature (°C) E 20 25 30 35 40 Temperature (°C) 16 DMPG DMPC Buffer 14 12 (M–1·cm–1) 10 –2 –4 –6 190 200 210 220 230 240 250 260 Wavelength (nm) Fig The bactericidal capacity of DRS-DA4 is linked to its interaction with DMPG (A, B) Kinetics of bacterial membrane leakage of E coli ML35p (A) and S aureus ST1065 (B) after treatment with increasing concentrations of DRS-DA4 The membrane leakage was followed by measuring Gal-ONp hydrolysis at 405 nm (C, D) DSC heating thermograms illustrating the effects of DRS-DA4 on the thermotropic phase behavior of DMPG (C) and DMPC (D) MLVs The top scan corresponds to the lipid alone, and the peptide ⁄ lipid molar ratios of the lower scans are indicated Thermodynamic parameters are given in Table (E) CD spectra of DRS-DA4 in buffer, DMPG and DMPC with a peptide ⁄ lipid molar ratio of : 50 The data shown are representative of three experiments The first antimicrobial peptide shown to exhibit immunological properties was DRS-S1, a 34 amino acid cationic antimicrobial peptide, which stimulates the microbicidal activity of PMN leukocytes [12] A few other frog peptides, temporin A, rana-6 and pLR-like 6780 peptides and, recently, another dermaseptin-related peptide, DRS-S9, have been shown to be microbicidal as well as immunomodulatory [13,27–29] Here, we report the isolation, by screening for chemotactic activity, of a new member of the dermaseptin superfamily, FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS C Auvynet et al DRS-DA4, from the defensive skin secretion of the Mexican leaf frog P dacnicolor We showed that DRS-DA4 triggered the in vitro directional migration of human neutrophils and monocytes with a typical bell-shaped dose–response curve, and a maximal response at a concentration of 10 lm Our results suggested that the chemotactic effect was mediated by the low-affinity Gia-protein-coupled receptor FPRL-1, recently reported to interact with many different ligands, such as fMLP, the a-helical antimicrobial peptides LL-37 and temporin A, and the amyloidogenic peptides Ab1–42 and, presumably, DRS-S9 [13,27,30,31] (Fig 4B) Whether FPRL-1 interacts with its various agonists through different functional domains remains to be investigated In addition, as only human leukocytes were used in this study, it remains to be discovered whether DRS-DA4 is also capable of recruiting frog leukocytes Indeed, frog peptides are secreted at the outer surface of the skin, and it is presently not known whether they also enter the blood circulation or inner tissues to act on leukocytes or to modulate other biological functions As described for other agonists [20], the interaction of DRS-DA4, presumably with FPRL-1, led to the activation of the ERK1 ⁄ MAPK pathway, but not to that of the p38 MAPK pathway The chemotaxis data suggested that ERK phosphorylation, but not that of p38, was necessary for the chemotactic process Interestingly, inhibition of FPRL-1 by PTX has been reported to activate the p38 pathway [32] Consistent with this, inhibition of ERK1 ⁄ phosphorylation by a specific inhibitor (PD98059) activated the p38 MAPK pathway in response to DRS-DA4, suggesting that DRS-DA4 may additionally bind to a non-GPCR receptor and, through a p38-dependent pathway, control other cell functions such as degranulation or cytokine ⁄ chemokine gene expression and release This is in accordance with studies indicating that inhibition of one signaling pathway could activate another one [27,33] The 32 amino acid DRS-DA4 exhibits the typical characteristics of the dermaseptin superfamily: it is a linear, Lys-rich cationic peptide with the conserved Trp at position A blast search revealed high sequence homology of DRS-DA4 with DRS-DA3 (PD-33), also isolated from P dacnicolor, and, more surprisingly, with DRS-B2 isolated from Ph bicolor, DDK from Ph distincta, and ARP-AC1 from A callidryas [17] (Fig 2A) However, unlike these other peptides, DRS-DA4 is not carboxyamidated Modeling antimicrobial peptides as idealized helices revealed their highly amphipathic nature, with hydrophobic residues on one face of the helix and polar or charged Chemotactic and antimicrobial DRS-DA4 residues on the opposite face, leading us to propose that the amphipathic a-helix structure is an important feature of these membrane-permeating peptides Secondary structure prediction methods and CD spectroscopy have also shown that dermaseptins contain 45–90% helix in structure-promoting solvents [16,26, 34,39] We illustrated here by CD (Fig 5E) that DRSDA4 was randomly coiled in water and, unexpectedly, also in the presence of DMPC vesicles In contrast, DRS-DA4 adopted an a-helical structure in the presence of DMPG vesicles, consistent with its cidal action against prokaryotic cells Thus, DRS-DA4 fits the model proposed by Khandelia et al [35], in which the composition of the target membrane (zwitterionic or anionic) modulates the extent of helical content induced in antimicrobial peptides CD results are consistent with data from the calorimetric tests and isothermal titration calorimetry (ITC) experiments with DMPC large unilamellar vesicles (LUVs) (data not shown), again suggesting very weak or no interaction with this lipid In contrast, strong perturbations of the pretransition and the main phase transition (Fig 5C and Table 2) were recorded in the presence of DMPG, suggesting that electrostatic interactions participate in the peptide–lipid interaction, and that DRS-DA4 is able to penetrate the acyl chain region A highly cationic charge such as that of DRS-DA4 favors the accumulation of peptides on negatively charged DMPG bilayers via electrostatic interactions, suggesting that the bactericidal activity of DRS-DA4 towards Gram-negative bacteria results from the preferential binding of the peptide to the negatively charged lipopolysaccharides of the outer membrane, and that the subsequent membrane damage occurs through hydrophobic interactions with the inner target membrane, which is rich in neutral phosphatidylethanolamine This hypothesis is supported by a study showing that helical amphipathicity prevails over hydrophobicity in interfacial binding, underlining the Table Thermodynamic parameters obtained by DSC for the interaction of DRS-DA4 with MLVs of either DMPG or DMPC –, no pretransition observed Pretransition Lipid DMPG DMPC FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS Transition Peptide ⁄ lipid ratio T (°C) DH (kcalỈmol)1) T (°C) DH (kcalỈmol)1) 1 1 12.8 – – 13.0 14.1 13.5 – – 1.1 1.2 0.1 23.2 23.3 23.9 23.9 24.0 23.9 9.6 10.6 4.3 11.8 10.2 5.0 : 100 : 20 : 100 : 20 6781 Chemotactic and antimicrobial DRS-DA4 C Auvynet et al importance of amphipathicity as a driving force for cell lytic activity In addition, conformational constraints and appropriate positioning of aromatic residues for the formation of hydrophobic clusters have been shown to be critical for antimicrobial activity and selectivity [36] Moreover, the presence of regions with different order and polarity within the membrane has shown the existence of domains enriched in phosphatidylethanolamine or phosphatidylglycerol, localized in highly curved regions of the bacterial membrane [37] Segregation of the membrane lipid components, leading to clustering of anionic lipids through an induced lateral-phase separation, and the subsequent perturbation of existing domains of the membrane has been proposed as a mechanism contributing to the antimicrobial activity of numerous antimicrobial peptides In agreement with this, as they have significant amounts of both anionic and zwitterionic lipids, most Gram-negative bacteria are more susceptible to this membrane-disrupting mechanism Consistent with this, and as reported for oligo-acyl lysine [38], we found that Gram-negative bacteria were killed by DRS-DA4 It has been suggested that an uninterrupted section of five hydrophobic residues, as identified on the helical wheel, is sufficient for good antimicrobial activity, with reduced hemolysis [39] This is the case for DRS-DA4 and DDK However, despite strong sequence homology with DRS-B2, DRS-DA4 exhibits distinct biological activities Contrasting with the wide microbicidal spectrum of DRS-B2, DRS-DA4’s bactericidal capacity was selective for Gram-negative bacteria (Table 1), and, whereas DRS-B2 did not induce leukocyte motility, DRS-DA4, at equivalent concentrations, was a potent chemotactic agent for PMN leukocytes (Fig 4B) A comparison of the helical wheel projections of DRS-B2, DDK, ARP-AC1 and DRS-L1 (a dermaseptin with a similar selectivity for Gramnegative bacteria [18]) revealed that, if the last three residues – which are rarely involved in the a-helix formation – are disregarded, these peptides exhibit an amphipathic distribution (Fig 2B) The main information provided by the Edmundson projection is that DRS-DA4 and DRS-L1, the two peptides that are active only on Gram-negative strains, not expose a negative residue on the apolar face, as DRS-B2 and DDK, which have a Glu at position 31, or ARP-AC1, with an Asp at position 27 In agreement with this, the position of acidic residues seems to be a critical parameter for the antibacterial activity against Staphylococcus strains [40] However, the ability of a peptide to depolarize the cytoplasmic membrane does not necessarily correlate with bactericidal activity Indeed, our 6782 data show that, like plasticins, DRS-DA4 was able to depolarize the membrane of Gram-positive (S epidermidis ST1065) as well as of Gram-negative (E coli ML35p) strains of bacteria, but it was toxic only for Gram-negative strains (Table 1) In contrast to the situation with clinically used antibiotics, resistance to natural antimicrobial peptides is not frequent, raising interest in the use of antimicrobial peptides to fight antibiotic-resistant microbes Moreover, recent data have suggested that antimicrobial peptides participate actively in preventing the appearance of resistant mutants, and are thus the last line of defense dealing with persistent infections [41] Our data showing the antibacterial potency of DRS-DA4, resulting from its ability to interact with anionic model membranes and to induce directional locomotion of mammalian cells through a receptor, presumably FPLR-1, highlight the multifunctionality of antimicrobial peptides as antibiotics and immunomodulatory molecules [3,42,43] Modification of DRSDA4 to enhance its direct bactericidal effect and to extend its antibacterial activity to Gram-positive strains, without compromising its immunomodulatory potency, could be achieved, as for dermaseptin S4, through acylation [44], or, as for magainin analogs, amidation [45] This new class of peptides will be useful for therapeutic application purposes Experimental procedures Frogs Male and female specimens of P dacnicolor were captured in the state of Morelos (Mexico) and housed in a nonsterile environment, in a large plastic container covered by a fence phyllodendron, potos and dracena were used as perches, and a water bowl was provided for nocturnal baths Once a week, the frogs were fed with crickets Purification of the peptide Fresh skin exudate was recovered by gently squeezing the latero-dorsal portion of a frog skin, resuspended in de-ionized water, and centrifuged for 15 at 400 g The supernatant was first fractionated by size exclusion chromatography on a Sephadex G-50 fine column (60 · 0.75 cm) eluted with 10% acetic acid Absorbance was monitored at 280 nm Three main fractions were obtained and tested for their chemotactic activity Fraction III was further fractionated by RP-HPLC on a semipreparative column (Nucleosil lm C18, 250 · 10 mm), using a solvent system composed of water containing 0.1% trifluoroacetic acid (TFA) as solvent A, and acetonitrile containing 0.07% FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS C Auvynet et al TFA as solvent B The column was eluted at mLỈmin)1 with a 0–60% linear gradient of solvent B for 65 ESI-MS, and peptide sequencing by ESI-MS/MS and Edman degradation ESI-MS and tandem MS (MS ⁄ MS) experiments were performed on a Q-TOF II mass spectrometer (Waters, Guyancourt, France) equipped with its standard ESI source and operated under the control of masslynx 3.5 software (Waters) [46] Solid-phase peptide synthesis and purification Synthesis and purification of the peptides were performed ´ ´ ` by the platform ‘Ingenierie des Proteines et Synthese Peptidique of IFR83’ (UPMC, Paris, France) The synthesis of DRS-DA4 and DRS-B2 was carried out by a solid-phase Fastmoc chemistry procedure on an Applied Biosystems 433A Automated Peptide Synthesizer (Applera, Courtaboeuf, France) Resins and Fmoc-protected amino acids were purchased from Merck Chemicals (Novabiochem, Nottingham, UK) and solvents from Carlo Erba, SDS Val de Reuil, France (Aix-en-Provence, France), as previously described [47] Fmoc-Met-NovasynTGA (100–200 mesh) was used for DRS-DA4 Briefly, synthesis products were cleaved from the resin with a mixture of TFA (94%), H2O (2.5%), ethanedithiol (2.5%), and tri-isopropylsilan (1%), precipitated in ether, centrifuged at 5000 g for 10 min, and lyophilized Peptides were purified by semipreparative HPLC (C18 reverse-phase column, PrepLC 25 mm module, 250 · 100 mm, 15 mm particle; Waters) with a Waters 1252 Binary HPLC pump (flow rate mLỈmin)1) Purity was assessed by MALDI-TOF MS (Voyager DE P60; Applied Biosystems, Courtaboeuf, France) Membrane polarization assay The cytoplasmic membrane depolarization was determined with the membrane potential-sensitive dye DiSC3(5) (Molecular Probes–Invitrogen, Cergy Pontoise, France) and E coli 363 ATCC 11775 or S epidermidis BM3302, as described by Zhu et al [48] Briefly, bacteria were harvested in the midlogarithm phase by centrifugation at 1000 g 10 at °C, washed twice in mm glucose and mm Hepes (pH 7.2), resuspended to an absorbance of 0.2 at 630 nm in the same buffer, and incubated with lm DiSC3(5) at 37 °C until a stable reduction of fluorescence was achieved; once the dye uptake was maximal, as indicated by a stable reduction in fluorescence due to the quenching of the accumulated dye in the bacteria, 0.1 m KCl was added to maintain a high membrane potential gradient Membrane depolarization was monitored by measuring the change in the intensity of fluorescence emission of the membrane potential-sensitive Chemotactic and antimicrobial DRS-DA4 dye DiSC3(5) (kex = 622 nm, kem = 670 nm) after addition of the peptides, on a Perkin-Elmer LS 50B spectrophotometer with spectro winlab software (Perkin-Elmer, Courtabeouf, France) Full dissipation of the membrane potential was obtained by adding Triton X-100 (final concentration, 0.1%) The membrane potential-dissipating activity of the peptides was calculated as follows: percentage membrane depolarization = 100 · [(Fp ) F0) ⁄ (Fg ) F0)], where F0 is the stable fluorescence value after addition of the KCl, Fp is the fluorescence value 10 after addition of the peptide, and Fg is the fluorescence signal after addition of Triton X-100 Peptide-induced permeabilization of the cytoplasmic membrane of E coli Permeabilization of the cytoplasmic membrane of E coli ML35p (generously provided by S Rebuffat, Laboratoire ´ Molecules de Communication et Adaptation des microorganismes, Museum National d’Histoire Naturelle, Paris) or of S aureus ST1065 by DRS-DA4 was assayed by measuring the b-galactosidase activity with the chromogenic substrate Gal-ONp Bacteria were grown in 10% LB broth, washed twice with NaCl ⁄ Pi (0.15 m phosphate, 0.2 m NaCl, pH 7.4), and diluted in 10% LB in NaCl ⁄ Pi to an absorbance of 0.5 at 630 nm The assay was carried out in a 96-well microtiter plate Aliquots (15 lL) of bacterial suspension were mixed with 2.5 mm Gal-ONp and incubated with various concentrations of peptide The hydrolysis of Gal-ONp was monitored by measuring the absorbance at 405 nm of released o-nitrophenol with a Fluostar Galaxy (BMG LabTech, Champigny-sur-Marne, France) Antimicrobial assays and hemolytic activity The Gram-negative bacterial strains E coli ATCC ML35p, E coli ATCC 11775, Ps aeruginosa ATCC 27853, S aureus ATCC 25923, S aureus ST1065 (generously given by M Falord and T Msadek, Biology of Gram Positive Pathogens, CNRS URA 2172, Institut Pasteur, Paris), S epide´ rmidis BM3302 (generously given by O Chesneau, Unite ´ Membranes Bacteriennes CNRS URA 2172, Institut Pasteur, Paris) and Enterococcus faecalis ATCC 29212 were used Strains were cultured and antimicrobial assays were performed as described previously [13] The hemolytic activity of the peptide was determined using fresh rat erythrocytes, as previously described [47] CD spectroscopy The CD spectra of the peptide were recorded on a JobinYvon CD6 dichrograph linked to a PC microprocessor, as previously described [13] Spectra were scanned at room temperature in a quartz-optical cell with a mm path length Spectral measurements were obtained at wavelengths FEBS Journal 276 (2009) 6773–6786 ª 2009 The Authors Journal compilation ª 2009 FEBS 6783 Chemotactic and antimicrobial DRS-DA4 C Auvynet et al of 190–260 nm with a bandwidth of nm Typically, five scans were accumulated and averaged The CD spectrum of the peptide was measured in phosphate buffer (10 mm phosphate, pH 7.0), and in the presence of DMPC or DMPG vesicles, at a peptide ⁄ lipid molar ratio of : 50 The relative a-helix content was estimated from De (m)1 cm)1) at 220 nm, according to Zhong and Johnson [49] some experiments, neutrophils were preincubated with PTX (200 ngỈmL)1) (Sigma-Aldrich, St Louis, MO, USA), fMLP (1.14 nm), fMLP (100 nm), PD98059 (30 lm) (Calbiochem) or SB202190 (10 lm) (Calbiochem, Los Angeles, CA, USA) for 30 at 37 °C, prior to evaluation of their migration towards DRS-DA4 Cell activation and immunoblot DSC and ITC One milligram of DMPC or DMPG (Avanti Polar Lipids) was dissolved in 100 lL of chloroform or 100 lL of chloroform ⁄ methanol (1 : 1), respectively Lipid films were obtained by evaporating solvents under a nitrogen stream, desiccated for several hours under vacuum at 40 °C, and resuspended in NaCl ⁄ Pi to obtain MLVs at a final concentration of mgỈmL)1 The peptide was then added to this solution to achieve lipid ⁄ peptide ratios of 100 : 1, 50 : 1, or 20 : NaCl ⁄ Pi and this mixture were then degassed under vacuum for 15 before being loaded into the reference cell of a Nano-Differential Scanning Calorimeter III (Calorimetry Sciences Corp., Lindon, UT, USA) and in the sample cell, respectively Scans were recorded between and 40 °C at scan rates of 0.5 °CỈmin)1 for the heating and °CỈmin)1 for the cooling MLVs were used rather than LUVs for DSC experiments, because they exhibit much more cooperative lipid phase transitions As the thermotropic phase behavior was not completely reproducible, we performed at least 10 scans Thermogram analysis, using the last scans once equilibrium had been reached, was performed with cpcalc, the software provided with the calorimeter Final figures were plotted with origin 6.0 (Microcal, Inc., Northampton, MA, USA) Cell preparation and chemotaxis assays Neutrophils and monocytes were obtained as previously described [13] The chemotactic activity was tested on calceinlabeled human neutrophils or monocytes, using a 48-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD, USA) as described previously [13] Briefly, different concentrations of DRS-DA4 or DRS-B2, each diluted in chemotaxis medium, were placed in the bottom wells of the chamber; fMLP (1.14 nm), fMLP (100 nm) or stromal cellderived factor 1a (SDF1-a) (1 nm) was used as positive control, and chemotaxis medium as negative control The chamber was incubated for 45 (neutrophils) or h (monocytes) at 37 °C in a 5% CO2 humidified atmosphere Cell migration was assessed by measuring the fluorescence of the lower face of the membranes with an Alpha Innotech image analyzer, using fluorchem 8800 software from Alpha Innotech To differentiate between chemotaxis and chemokinesis, experiments were performed in which the same concentrations of DRS-DA4 present in the lower wells were added simultaneously to the upper wells For 6784 Purified human neutrophils (2 · 106 per sample) resuspended in RPMI-1640 were incubated with DRS-DA4 (10 lm), fMLP (100 nm) or chemotaxis medium for the indicated periods of time at 37 °C in 5% CO2 In some cases, neutrophils were pretreated for 30 at 37 °C with PTX and ⁄ or with PD98059 Phosphorylation of MAPK ERK1 ⁄ ERK2 and p38 were detected with an antibody against phospho-ERK1 ⁄ (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and an antibody against phospho-p38 (Cell Signaling Technology, Inc., Danvers, MA, USA) by chemiluminescence with ECL (Amersham Biosciences Inc., Piscataway, NJ, USA) Membranes were reprobed for ERK1 ⁄ (Santa Cruz Biotechnology) and p38 (Santa Cruz Biotechnology) Acknowledgements ` This work was supported by grants from the Ministere ` des Affaires Etrangeres (France), the Centre National ´ de la Recherche Scientifique (CNRS), the Universite Pierre et Marie Curie, the Secretaria de Relaciones Exteriores (Mexico), and CONACyT (Mexico) The ´ authors are grateful to C El Amri (Universite Pierre et ´ Marie Curie, UR4-Enzymologie Moleculaire et Fonctionnelle, Paris, France) for help with ITC experiments and fruitful discussions, R Thai (Laboratoire de Bio´ chimie des Proteines, CEA, Saclay, France) for peptide ´ sequencing, F Bruston (Universite Denis Diderot, ´ Unite EA4413, Paris, France) for helpful advice, especially in CD, A Alagon, G Corzo, H Clement and E Melchy (Instituto de Biotecnologica, Universidad Nac´ ´ ´ ional Autonoma de Mexico, Mexico) for suggestions and technical help with HPLC purification, F Abassi ´ ´ (Laboratoire de Biochimie, Faculte de Medecine de Sousse, Sousse, Tunisia) for help with antimicrobial ´ assays, I Alves (Laboratoire des Biomolecules, Univer´ 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DRS -DA4, DRS -B2, DDK, and DRS-L1 Hexagonal and round backgrounds refer to basic and acid amino acids respectively, pentagonal backgrounds to hydrophilic residues, and squares to hydrophobic residues... peptides as antibiotics and immunomodulatory molecules [3,42,43] Modification of DRSDA4 to enhance its direct bactericidal effect and to extend its antibacterial activity to Gram-positive strains,... herein the isolation and characterization of a new dermaseptin- related peptide, GMWSKIKNA GKAAKAAAKAAGKAALGAVSEAM, named dermaseptin DA4 (DRS-DA4), according to the new dermaseptin nomenclature