Structural requirements for antimicrobial versus chemoattractant activities for dermaseptin S9 Constance Auvynet 1,2 , Chahrazade El Amri 1 , Claire Lacombe 1 , Francine Bruston 1 , Julie Bourdais 2 , Pierre Nicolas 1 and Yvonne Rosenstein 2 1 UPMC Universite ´ de Paris 06, CNRS FRE 2852, Peptidome de la Peau des Amphibiens, Paris Cedex 5, France 2 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Auto ´ noma de Me ´ xico, Cuernavaca, Morelos, Mexico Antimicrobial peptides that play a role in host defence against competing or pathogenic microorganisms are small proteins, typically 10–50 residues long, interact with lipid bilayers to alter cell-membrane permeability, which often leads to cell death. Structural studies have revealed that the peptide secondary structures include a-helices, b-sheet structures stabilized by two or three disulfide bonds, and extended structures with over- representation of one or two amino acids (W, P, H or G) [1]. However, all these peptides, regardless of the secondary structure or length, are cationic and exhibit amphipathic properties upon interaction with lipid bilayers, with apolar amino acid residues segregating from the hydrophilic residues on opposite sides of the 3D structure. These structural elements are believed to play a crucial role in the binding of cationic host Keywords antimicrobial peptide; chemotaxis; dermaseptin; frog skin; infrared spectroscopy Correspondence P. Nicolas, UPMC University Paris 06, CNRS FRE 2852, Peptidome de la Peau des Amphibiens, Tour 43, 2 place Jussieu, 75251 Paris cedex 5, France Fax: +33 1 44 27 59 94 Tel: +33 1 44 27 95 36 E-mail: pnicolas@ccr.jussieu.fr Y. Rosenstein, Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Auto ´ noma de Me ´ xico, Avenida Universidad 2001, Col Chamilpa, Cuernavaca, Mor. 62270, Mexico Fax: +52 73172388 Tel: +52 5 55 66 22 76 63 E-mail: yvonne@ibt.unam.mx (Received 29 April 2008, revised 11 June 2008, accepted 16 June 2008) doi:10.1111/j.1742-4658.2008.06554.x Dermaseptin S9 (Drs S9), GLRSKIWLWVLLMIWQESNKFKKM, iso- lated from frog skin, does not resemble any of the cationic and amphipathic antimicrobial peptides identified to date, having a highly hydrophobic core sequence flanked at either side by cationic termini. Previous studies [Lequin O, Ladram A, Chabbert A, Bruston F, Convert O, Vanhoye D, Chassaing G, Nicolas P & Amiche M (2006) Biochemistry 45, 468–480] demonstrated that this peptide adopted a non-amphipathic a-helical conformation in trifluoroethanol ⁄ water mixtures, but was highly aggregated in aqueous solutions and in the presence of sodium dodecyl sulfate micelles. Circular dichroism, FTIR and attenuated total reflectance FTIR spectroscopies, combined with a surface plasmon resonance study, show that Drs S9 forms stable and ordered b-sheet aggregates in aqueous buffers or when bound to anionic or zwitterionic phospholipid vesicles. These structures slowly assem- bled into amyloid-like fibrils in aqueous environments via spherical inter- mediates, as revealed by electron microscopy and Congo red staining. Drs S9 induced the directional migration of neutrophils, T lymphocytes and monocytes. Interestingly, the antimicrobial and chemotactic activities of Drs S9 are modulated by its amyloid-like properties. Whereas spherical oligomers of Drs S9 exhibit antimicrobial activity, the soluble, weakly self- associated forms of Drs S9 act on human leukocytes to promote chemotaxis and ⁄ or immunological response activation in the same range of concentra- tion as amyloidogenic peptides Ab(1–42), the most fibrillogenic isoform of amyloid beta peptides, and the prion peptide PrP(106–126). Abbreviations ATR, attenuated total reflectance; DMPC, 1,2-dimyristoyl-sn- glycero-3-phosphatidylcholine; DMPG, 1,2-dimyristoyl-sn-glycero-3- phosphatidylglycerol; Drs B2, dermaseptin B2; Drs B4, dermaseptin B4; Drs S9, dermaseptin S9; ERK, extracellular signal-regulated kinase; fMLP, formyl methionyl-leucyl-phenylalanine; FPRL-1, formyl peptide receptor like 1; PTX, pertussis toxin; SPR, surface plasmon resonance; TFA, trifluoroacetic acid. 4134 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS defence peptides to the negatively charged outer leaflet of bacterial bilayers. Once bound, the hydrophobic face of the amphipathic peptide allows the peptide to enter the membrane interior, thereby triggering local fusion of the membrane leaflets, pore formation, cracks and membrane disruption [2–7]. Frog skin is by far the most important source of antimicrobial peptides, with more than half of the pep- tides described to date isolated from South American Hylidae or European, Asian or North American Rani- dae [1]. Among these peptides, dermaseptins S and B, from the skin of the South American tree frogs Phyllo- medusa bicolor and P. sauvagei, form a family of amphipathic, a-helical, closely related antimicrobial peptides with broad-spectrum microbicidal activities at micromolar concentrations against Gram-positive and Gram-negative bacteria, fungi, yeasts and protozoa [8]. In previous work, we have used the conservation of the preproregion sequences of the preprodermaseptin transcripts to identify a new member of the dermaseptin S family, dermaseptin S9 (Drs S9), GLRSKIWLWVLLMIWQESNKFKKM [9]. The structure of this peptide does not resemble that of any antimicrobial peptide identified to date, having a hydrophobic central core flanked by positively charged termini. This structure is similar to that of synthetic peptides originally designed as transmembrane mimetic models that spontaneously become inserted into mem- branes [10]. We have shown that Drs S9 adopted a non amphipathic a-helical conformation in the pres- ence of trifluoroethanol ⁄ water mixtures, which are known to promote helical structures, but that it aggre- gated in aqueous solutions and in the presence of SDS micelles [9]. Other antimicrobial peptides of the derm- aseptin S family, particularly Drs S4 [11], have been reported to form aggregates, leading to the proposal that the state of aggregation of a peptide in solution might be an important determinant for selective cyto- toxicity as well as other biological events [12]. In the present study, we characterized the self-organization of Drs S9 in aqueous solutions and determined how this might affect its biological activity. Drs S4 forms amor- phous aggregates, but Drs S9 is a tryptophan-rich peptide that forms ordered aggregates in aqueous solutions, two characteristics that are reminiscent of amyloid peptides. We thus determined whether Drs S9 could have amyloidogenic properties. Our data provide evidence that, similar to amyloids, Drs S9 folds into a b-sheet structure that assembles into amyloid-like fibrils via spherical oligomeric intermediates in a time- and temperature-dependent manner, and that, unlike several antimicrobial peptides that form amyloid-like fibers in the presence of acidic phospholipids [13–15], Drs S9 forms amyloid fibrils in an aqueous envir- onment. Antimicrobial peptides are fundamental components of the innate immune response; in addition to killing invading microorganisms, they modulate several immune functions such as chemotaxis of immune cells [16] through specific cell-surface receptors, such as for- myl peptide receptor-like 1 (FPRL-1) [17]. Interest- ingly, amyloid peptides, which are associated with neuroinflammatory disorders such as Alzheimer’s, prion or Parkinson’s diseases, also interact with FPRL-1 and promote chemotaxis, favouring inflamma- tion [18–21]. The neurotoxin prion peptide fragment PrP(106–126) is a chemotactic agonist for the G pro- tein-coupled receptor FPRL-1 [18]. Moreover, the vari- ous structural states of the amyloidal peptide Ab(1–42) modulate its biological activities: the low-molecular- weight form of the peptide seems to be chemotactic, while the oligomeric form seems to be neurotoxic [21,22]. With this in mind, we also tested the chemo- tactic potential of Drs S9 and determined whether the amyloidogenic properties of Drs S9 could modulate its biological activities. Maximum antimicrobial activity of Drs S9 was detected in its oligomeric form, while, similar to amyloid-like peptides, Drs S9 was chemotac- tic for human leukocytes in its soluble, low-molecular- weight, self-associated form. Together, the data pre- sented here establish that dermaseptin S9 is the first antimicrobial frog skin peptide to exhibit amyloido- genic properties with potent chemotactic and antimi- crobial activities. Results Dermaseptin S9 is highly aggregated and forms b-structures Amide H ⁄ D exchange kinetics monitored by attenu- ated total reflectance (ATR) FTIR provided informa- tion on the solvent accessibility of the peptide NH amide groups. The solid state was taken as 100% NH content for the NH ⁄ ND exchange analysis. After 15 min, lower NH ⁄ ND exchanges were observed for Drs S9 dissolved in NaCl ⁄ P i (18%) than for Drs S9 dissolved in D 2 O (50%) (Fig. 1A), which is concomi- tant with an increase in b-sheet content (see below) that underlies the self-associative propensity of the peptide in the saline buffer. The kinetics of interactions between a peptide and an adsorbing surface can be monitored in real time by surface plasmon resonance (SPR). A shift in the reso- nance signal during the adsorption step may provide information about the concentration of the peptide C. Auvynet et al. Antimicrobial and chemotactic dermaseptin S9 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS 4135 on the adsorbing surface. For the hydrophobic HPA biosensor, consisting of long chain alkanethiol mole- cules attached directly to the gold film, the SPR signals (resonance units) plotted as a function of peptide con- centration (1–300 lm) further suggest a self-associative propensity of Drs S9 (Fig. 1B). The resulting curve was biphasic, in contrast to monophasic curves obtained with another well-known dermaseptin, Drs B2 [23]. The density of Drs S9 peptide adsorbed onto HPA was quantified under continuous flow, assuming the manufacturer’s characteristics for the detection surface (1.2 mm 2 ) [24]. The surface area occupied by a 23-residue peptide molecule differs depending on its conformation. When in a-helix for- mation, its contact surface may be regarded as a rect- angle measuring 3.3 nm long by 1.5 nm wide, giving a surface area of approximately 5 nm 2 ; in the b-structure formation, the peptide has a contact area of 10– 12 nm 2 [25]. Consequently, the hydrophobic surface could bind 0.9 ngÆmm )2 of helical and 0.4 ngÆmm )2 of b-sheet peptide, respectively. The measured adsorption densities for 30 lm peptide were thus compatible with a b-structure for Drs S9 (Table 1). Figure 1B shows that, in the range of 5–30 lm, a monolayer of Drs S9 in b-sheet formation was adsorbed (480 resonance units, see Table 1). From 30–300 lm, the resonance unit values were 2- to 4-fold greater, corresponding to various types of peptide–peptide association. Further- 0 5 10 15 0 20 40 60 80 100 D 2 O PBS NH content (%) Time (min) 0 50 100 150 200 250 300 0 250 500 750 1000 1250 1500 1750 Resonance units (RU) PS9 concentration (µM) 200 220 240 –40 –20 0 20 40 60 PBS PB H 2 O Δε (M –1 ·cm –1 ) Wavelength (nm) 1500 1600 1700 18001550 1650 1750 0.00 0.01 0.02 0.03 0.04 0.05 1624 cm –1 Absorbance (a.u.) Wavenumber (cm –1 ) 0 10 20 30 40 1624 (β-sheet) 1636 16501665 1685 (β-hairpin) (β-turn) (β-turnandbent) (disordered) A CD E B Fig. 1. Dermaseptin S9 is highly aggregated and forms b-structures. (A) NH content kinetics obtained by ATR FTIR. (B) Absorp- tion density (resonance units) versus con- centration (1–300 l M) obtained by SPR. (C) CD spectra of Drs S9 (30 l M) freshly dis- solved in water, phosphate buffer (PB) or NaCl ⁄ P i (PBS). (D) ATR FTIR spectra in the 1500–1800 cm )1 region indicating the adop- tion of b-sheet structure in NaCl ⁄ P i medium. (E) Distribution of component band contents for Drs S9 in NaCl ⁄ P i (grey) or D 2 O (open). Table 1. Adsorption potencies of Drs S9 on the hydrophobic HPA biosensor as function of peptide concentration. 50 l M HPA 100 lM HPA 300 lM HPA SPR response (resonance units) 480 750 1630 Adsorption density (ngÆmm )2 ) 0.4 0.6 1.3 Adsorbed molecule no. ⁄ mm 2 (· 10 12 ) 0.07 0.12 0.26 Complex density (ngÆmm )2 ) a 0.4 0.6 1.3 a The complex density (ng per mm 2 ) is evaluated from values of the SPR response taken at 180 s in the desorption step. Antimicrobial and chemotactic dermaseptin S9 C. Auvynet et al. 4136 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS more, for peptide concentrations between 30 and 300 lm, the density of the complex evaluated from values observed at 180 s during the desorption step were identical to adsorption densities, suggesting that, in contrast to some frog antimicrobial peptides [26], the rate of interaction between Drs S9 and the hydro- phobic support is not limited by the surface. Dichroic spectra typical of b-sheet conformation (Fig. 1C), with a negative band at 215–218 nm (amide n fi p* transition) and a positive band at 190–200 nm (amide p fi p* transition) were obtained from freshly dissolved Drs S9 in phosphate buffer or NaCl ⁄ P i . The spectrum profile of Drs S9 dissolved in H 2 O exhibited a blue shift that can be attributed to a distorted b-sheet conformation, underlining the impact of the environ- ment on peptide structure stability as well as on the degree of peptide self-association. In addition, the neg- ative band became broader in phosphate buffer at 230 nm, probably due to contributions from aromatic side chains [27]. The effect of temperature on the stabil- ity of the peptide structure was also tested by subjecting a solution of Drs S9 in 50 mm phosphate buffer to tem- peratures ranging from 5 to 70 °C. No clear transition from an ordered structure, i.e. a b-sheet conformation, to a random coil structure was observed, demonstrating that Drs S9 was quite stable, with a multimeric arrangement (supplementary Fig. S1). The 1500–1800 cm )1 region of the ATR FTIR spec- trum of Drs S9 dissolved in deuterated NaCl ⁄ P i showed a peak at 1624 ± 2 cm )1 , corresponding to an intermolecular b-sheet structure (Fig. 1D). Comparison between the distribution of the Drs S9 amide I¢ com- ponent bands in the two buffers (NaCl ⁄ P i or D 2 O) (Fig. 1E) confirmed a higher content of ordered struc- ture in NaCl⁄ P i medium than in aqueous medium as evaluated by second-derivative analysis. Moreover, Drs S9 freshly dissolved in H 2 O or in NaCl ⁄ P i migrated with apparent molecular masses of approxi- mately 6 and 12 kDa under SDS–PAGE (supplemen- tary Fig. S2), suggesting that Drs S9 could already be self-associated as dimers or tetramers. Dermaseptin S9 has b-amyloid-like properties As, similar to amyloidogenic proteins, Drs S9 exhib- ited a b-sheet-rich conformation and a high propensity to aggregate in an aqueous environment, we assessed its ability to form amyloid fibrils using classical approaches [28]: (a) detection of yellow–green bi-refrin- gence under polarized light upon staining with Congo red, (b) identification of fibrils obtained by negative stain transmission electron microscopy (TEM), and (c) amide I¢ analysis by infrared spectroscopy. After 7 days of incubation at 37 °C, Congo red-treated Drs S9 exhibited a red colour under normal light (Fig. 2Aa), and the yellow–green bi-refringence charac- teristic of amyloidogenic peptides under polarized light (Fig. 2Ab). In contrast, at day 0 or after 3 days of incubation at 37 °C, no characteristic bi-refringence was observed under polarized light (data not shown). Dried phosphate buffer, or a 7-day-old solution of Drs B2, an a-helical linear antimicrobial peptide [23], stained with Congo red in phosphate buffer did not display the characteristic red staining under normal light (Fig. 2Ac,Ae) nor yellow–green bi-refringence under polarized light (Fig. 2Ad,Af). Neither Congo red nor Drs S9 alone exhibited the red colour or the yellow–green bi-refringence. At day 3, aggregates with a granular appearance that looked like spherical oligo- mers could be visualized (Fig. 2Ba). Consistent with Congo red staining data, electron microscopy revealed that, at day 7, the same solutions of Drs S9 exhibited, like amyloid fibrils, a long filamentous structure of 8 nm diameter (Fig. 2Bb). As expected, at day 0, nei- ther spherical oligomers nor fibrillar structures could be observed (data not shown). Thus, three structural states can be defined for Drs S9: day 0 (D0), low molecular weight; day 3 (D3), oligomeric; day 7 (D7), fibrillar. A kinetic study of the structure evolution Drs S9 was monitored using CD (supplementary Fig. S1). The amyloid-like arrangement of the peptide was confirmed by submitting a 7-day-old solution of Drs S9 stained with Congo red and dried onto a glass slide to ATR FTIR spectroscopy. Maximum absorp- tion of the spectrum was registered at 1624 cm )1 , with shoulders at 1665 and 1685 cm )1 (Fig. 3A). Second- derivative analysis of the spectrum (Fig. 3B) showed a 15% gain in the proportion of b-structures (Fig. 3C) compared to freshly dissolved Drs S9 with 75% b-structure (Fig. 1E). In addition, 1624 cm )1 and 1685 cm )1 individual component bands were identified as fingerprints of intermolecular b-sheet aggregates and b-amyloid structures [29]. In summary, these data show that Drs S9 exhibits the biophysical and morpho- logical characteristics of amyloidogenic peptides. Dermaseptin S9 induces chemotaxis via a seven-transmembrane G protein-coupled cell surface receptor Amyloidogenic peptides have been shown to partici- pate in inflammatory processes by inducing cell migration [30]. We investigated whether, similar to amyloid-like peptides, Drs S9 was also chemotactic. We evaluated the capacity of Drs S9 to induce the migration of human peripheral blood neutrophils C. Auvynet et al. Antimicrobial and chemotactic dermaseptin S9 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS 4137 and T lymphocytes, as well as that of human acute monocytic leukaemia cells, THP-1. Drs S9 triggered the migration of human neutrophils (Fig. 4A), T lympho- cytes (Fig. 4C) and THP-1 monocytes (data not shown) with a typical bell-shaped dose–response curve at D0. For all cell types, maximal response was observed at a concentration of 50 lm. Equivalent concentrations of Drs B2 had no effect on leukocyte motility (Fig. 4C). Drs S9-induced cell migration reflected a chemotactic rather than a chemokinetic movement, as addition of various concentrations of Drs S9 to the upper wells of the chamber abrogated neutrophil migration towards the Drs S9-loaded lower wells (Fig. 4B), suggesting that the cell locomotion we detected was the result of chemotactic movement rather than enhanced random movement. To determine whether Drs S9-induced neutrophil chemotaxis was mediated through a G protein-coupled receptor, as is the case for Ab(1–42) peptides [30], we examined whether this chemotactic activity could be inhibited by pertussis toxin (PTX), a toxin that specifi- cally inhibits G ia protein-coupled receptor signalling [31]. Incubation of neutrophils with PTX for 30 min at 37 °C prior to the start of the chemotaxis assay inhib- ited the migration of neutrophils by 50% compared with freshly dissolved Drs S9 (Fig. 5A). The ability of neutrophils to migrate towards formyl methionyl- leucyl-phenylalanine (fMLP) (100 nm) was abrogated by PTX. Together, these data suggest that, in addition to a G i protein-coupled receptor, Drs S9-dependent chemotactic signals are mediated by a non-PTX- sensitive receptor. a A B b a b cd e f Fig. 2. Dermaseptin S9 forms amyloid-like fibrils. (A) Drs S9 binds Congo red and exhibits yellow–green bi-refringence; Drs S9 (500 l M) (a, b) or Drs B2 (500 lM) (c, d), both suspended in 50 m M phosphate buffer incubated for 7 days at 37 °C, or phosphate buffer alone (e, f) were stained with Congo red and observed under normal (a, c, e) or polarized light (b, d, f). (B) Negative staining electron microscopy micrograph of Drs S9 (500 l M)in50mM phosphate buffer incu- bated at 37 °C for (a) 3 days (bar, 1.1 nm) or (b) 7 days (bar, 50 nm). Antimicrobial and chemotactic dermaseptin S9 C. Auvynet et al. 4138 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS A variety of agonistic ligands have been described for the low-affinity FPRL-1 receptor, including fMLP at high concentrations (approximately 100 nm) and amyloid Ab(1–42) peptides [32–34]. To assess whether Drs S9-induced chemotaxis could be medi- ated through this receptor, experiments were per- formed in which neutrophils were pre-incubated with Ab(1–42) (50 lm) or fMLP (100 nm) for 30 min before testing their ability to respond to a gradient of Drs S9, Ab(1–42) or fMLP in a chemotaxis assay. Preincubating the cells with fMLP reduced the che- motactic activity of Drs S9 by approximately 50% (Fig. 5B), similar to what we had observed with PTX. Comparable results were observed in response to Ab(1–42) or fMLP when the cells were preincu- bated with Ab(1–42), Drs S9 or fMLP (Fig. 5B). All these data suggest that the chemotactic activity of Drs S9 could be partially mediated through a recep- tor similar to the one used by high concentrations of fMLP or by Ab(1–42) (FPRL-1). Activation of a G protein-coupled receptor by a chemoattractant can lead to activation of the MAPK pathway [35,36]. Incubation of neutrophils with freshly 0 10 20 30 40 1624 (β-sheet) 1636 16501665 1685 C 1590 1620 1650 1680 1710 Wavenumber (cm –1 ) 1624 1665 1685 1675 1665 1650 1624 1685 B A (β-hairpin) (Amyloid characteristic) (disordered) (Amyloid characteristic) 2 1 0 –1 –2 –3 0.04 0.03 0.02 0.01 0.00 Absorbance (a.u.) Absorbance 10 4 (a.u.) 1590 1620 1650 1680 1710 Fig. 3. Drs S9 exhibits aggregated b -sheet structures with an amy- loid-like arrangement as demonstrated by ATR FTIR. (A) Amide I¢ band (1580–1720 cm )1 ) for Drs S9 (500 lM) incubated for 7 days and stained with Congo Red. (B) Second-derivative analysis of the spectrum in (A) indicating a strong individual band at 1685 cm )1 that is present in b-amyloid structures. (C) Distribution of the indi- vidual band content of amide I¢ from the spectrum in (A). Drs S9 day 0 Drs S9 day 3 Drs S9 day 7 Drs S9 in the lower wells Drs S9 in the lower and upper wells fMLP (100 n M) in the lower and upper wells Drs S9 day 0 Drs B 2day 0 Concentration (µM) 0 0.05 0.5 5 15 50 150 350 fMLP (100 n M) 400 A B C 350 300 250 200 150 100 50 0 400 350 300 250 200 150 100 50 0 250 200 150 100 50 0 Chemotaxis index 0 0.05 0.5 5 50 350 fMLP (100 nM) 0 0.05 0.5 5 15 30 150 250 350 500 700 fMLP (100 n M) Fig. 4. Dermaseptin S9 is a potent chemoattractant. (A) Neutrophil migration induced by freshly prepared Drs S9, or Drs S9 incubated for 3 or 7 days at 37 °C, or by fMLP as a positive control and med- ium as a negative control. (B) Abolition of the chemotactic effect by adding the same concentrations of Drs S9 or fMLP in the upper and the lower wells of the chemotaxis chamber compared with addition of Drs S9 or fMPL in the lower wells only. (C) Induction of migration of T lymphocytes by Drs S9, Drs B2 or fMLP. Similar results were obtained from three different experiments. C. Auvynet et al. Antimicrobial and chemotactic dermaseptin S9 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS 4139 dissolved Drs S9 (100 lm) for 5 or 10 min or fMLP for 5 min induced the phosphorylation of the extra- cellular signal-regulated kinase ERK1 ⁄ 2, compared to neutrophils incubated for the same time period with medium alone (Fig. 5C). Preincubation with PTX, PD98059 (a specific inhibitor of MEK1 and MEK2, the ERK MAPK kinases) or both, before adding Drs S9 or fMLP, prevented ERK1 ⁄ 2 phosphorylation. These data suggest that Drs S9 is sensed in part through a seven-transmembrane G protein-coupled receptor, probably FPRL-1, coupled to the ERK1 ⁄ 2 MAPK kinase pathway. The b-amyloid-like behaviour of dermaseptin S9 modulates its chemotactic and antimicrobial activities Taking advantage of better knowledge of the aggre- gative and amyloid properties of Drs S9, we investi- gated whether the fibrillization process influenced the chemotactic and antibacterial activities of the pep- tide. The activity of Drs S9 freshly dissolved in che- motaxis medium was compared to that of Drs S9 incubated for 3 or 7 days at 37 °C. In all cases, Drs S9 induced cell migration with a peak response at 50 lm, but with maximum efficiency for freshly dissolved Drs S9 (Fig. 4A). Interestingly, the a-helical Drs B2 freshly dissolved in the same medium or incubated for 3 or 7 days at 37 °C was not a chemoattractant for T lymphocytes (Fig. 4C), neutro- phils or THP-1 monocytes (data not shown) in the same range of concentration (0.05–350 lm), indicat- ing the importance of the peptide structure for selected biological properties. Drs S9 dissolved in NaCl ⁄ P i or RPMI-1640 exhib- ited antimicrobial activity against all Gram-negative bacteria strains tested at day 0. The antibacterial activ- ity of 3-day-old Drs S9 in NaCl ⁄ P i (Table 2) or RPMI-1640 ⁄ 1% BSA (data not shown) was stronger than that of Drs S9 at day 0. After 7 days of incuba- tion in NaCl ⁄ P i at 37 °C, Drs S9 exhibited little or no antibacterial activity. No significant differences were observed when Drs B2, a potent a-helical antibacterial peptide, was dissolved in NaCl⁄ P i or RPMI-1640 ⁄ 1% BSA and tested, or incubated for 3 or 7 days at 37 °C prior to testing its antibacterial activity. Moreover, fMLP (100 nM) 300 250 200 150 100 50 0 0 200 40 Peptides Concentrations of Drs S9 0 day aged (µ M) Chemotaxis index Neutrophils pre-incubated with medium for 30 min Neutrophils pre-incubated with PTX (200ng. mL –1 ) for 30 min Chemotaxis index 250 200 150 100 50 0 Medium Drs S9 (50 µ M)A β (1–42) (50 µm) fMLP (100 nM) Pre-incubation with Drs S9 Pre-incubation with fLMP Pre-incubation with medium Pre-incubation with Aβ(1–42) A B C RPMI Drs S9 5′ 10′ PTX PD98059 –– – – –––– –– –– +++ + ++++ Drs S9fMLP fMLP pERK1/2 ERK1/2 Fig. 5. Dermaseptin S9 induces cell migration through a seven- transmembrane G protein-coupled receptor, presumed to be the FRLP-1 receptor. (A) Pre-incubating neutrophils with PTX (200 ng mL )1 ) partially inhibits Drs S9-induced migration. fMLP was used as a positive control. (B) Pre-incubating neutrophils with Ab(1–42), Drs S9 or fMLP reduces the migration induced by freshly dissolved Drs S9, Ab(1–42) or fMLP. (C) ERK1 ⁄ 2 phosphorylation in response to Drs S9 or fMLP is abolished by pre-incubating human neutrophils with PTX and ⁄ or PD98059. The same membrane was stripped and blotted with anti-ERK1 ⁄ 2. Similar results were obtained from three separate experiments. Table 2. Effect of Drs S9 oligomerization state on the antimicrobial activity of freshly prepared Drs S9 and Drs B2 or after incubation for 3 or 7 days in NaCl ⁄ P i or H 2 Oat37°C. Bacterial strains Minimal inhibitory concentration a (lM) Drs S9 (NaCl ⁄ P i or RPMI) Drs B2 (NaCl ⁄ P i ) 0 day 3 days 7 days 0, 3 and 7 days Citrobacter rodentium 12.5 6.5 50 – Salmonella typhimurium 25 6.5 100 3.1 Escherichia coli EPEC 50 12.5 100 3.1 Escherichia coli JPN15 50 25 R 0.2 a The antimicrobial activity is expressed as the minimal inhibitory concentration (l M), which is the minimal peptide concentration required for total inhibition of cell growth in liquid medium. Strains were considered resistant (R) when their growth was not inhibited by peptide concentrations > 100 l M. Antimicrobial and chemotactic dermaseptin S9 C. Auvynet et al. 4140 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS after removing the Drs S9 from the wells and replacing it with fresh LB medium overnight, none of the sensi- tive strains were capable of resuming growth (data not shown), suggesting that Drs S9 is a bactericidal agent. Together, these data indicate that Drs S9 is a more potent bactericidal agent in the oligomeric spherical form detected at 3 days of incubation at 37 °C, and is a potent chemoattractant in its low-molecular-weight oligomeric form (day 0). Dermaseptin S9 differently affects vesicle lipid assemblies Bacterial membranes contain substantial (up to 30%) amounts of negatively charged lipids such as phosphat- idylglycerol, cardiolipin and phosphatidylserine [37], thus 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) was used as a model system for determination of the relationships between antimicrobial activity, structure and membrane disturbances in the FTIR study. Phosphatidylcholine is widely recognized as representative for mammalian cell membranes, thus 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) was used as the lipid model to assess the inter- actions of Drs S9 with neutral eukaryotic membranes. We examined the conformation of Drs S9 in the presence of DMPG or DMPC vesicles, and evaluated the impact of freshly dissolved Drs S9 on the lipid bilayer assembly by transmission FTIR spectroscopy. Maximum absorbance of the amide I¢ bands was observed at 1624 ± 1 cm )1 in NaCl ⁄ P i , DMPC and DMPG vesicles (Fig. 6A). Analysis of the amide I¢ bands using a decomposition procedure described recently [25] allows band-to -band c omparisons (Fig. 6B). The coil ⁄ helix contents (25%) were the same in aque- ous NaCl ⁄ P i buffer and in the presence of DMPG and DMPC, but phospholipid vesicles promoted intercon- version from b-hairpin to b-sheet structure with better efficiency. The stretching vibration mode of the DMPG carbonyl groups [m(CO)] was used to check the hydra- tion and conformational changes of the membrane interface region (Fig. 7A). As shown in Table 3, Drs S9 caused redistribution of the two [m(CO)] components (hydrogen bond at 1726 cm )1 and dehydration at 1743 cm )1 ), characterized by a higher value (2.1) of the 1726 ⁄ 1743 ratio compared to pure DMPG (1.8). Thus, the interaction of Drs S9 with DMPG vesicles decreased the water content associated with phospholipid head- groups (CO), probably due to hydrogen bonding inter- actions between the lipid head groups and the peptide [26,38,39]. To evaluate the perturbations generated by Drs S9 in the hydrocarbon core of the DMPG bilayer, we analysed spectra in the 2800–3000 cm )1 region (Fig. 7B). The symmetric m S (CH 2 ) stretching band at 2852 cm )1 and the antisymmetric m AS (CH 2 ) stretching band at 2923 cm )1 were shifted towards higher wave numbers. These displacements and redistributions could be due to a gel to liquid crystalline-phase transition [39]. Drs S9 strongly affected the antisymmetric m AS (CH 3 ) stretching modes at 2956 cm )1 , indicating enhancement of the alkyl chain flexibility, as confirmed by the deter- mination of the bilayer core disruption (Table 3). In contrast, Drs S9 did not perturb the DMPC bilayer as neither the m(CO) lipid carbonyl groups nor the m S (CH 2 -CH 3 )orm AS (CH 2 -CH 3 ) stretching vibration- mode were affected (Fig. 7B). This suggests that no noticeable interactions were established between the zwitterionic vesicles and the peptide. These data are corroborated by adsorption density measurements by SPR on hybrid bilayers of DMPC and HPA, the results DMPG PBS DMPC 0 20 40 60 80 100 β -hairpin β -hairpin β -hairpin coil/helix coil/helix coil/helix β -sheet β -sheet β -sheet Component content (%) Absorbance (a.u.) 0.002 0.015 0.010 0.005 0.000 Buffer A B DMPC DMPG 1500 1600 17001550 1650 Wavenumber (cm –1 ) Fig. 6. Conformations of dermaseptin S9 in the presence of anionic DMPG and zwitterionic DMPC vesicles. (A) Transmission FTIR spectra in the 1500–1700 cm )1 region for Drs S9 in NaCl ⁄ P i or DMPC or DMPG vesicles. (B) Individual band contents (± 1%) resolved in the amide I¢ domain (lipid : peptide ratio = 10). C. Auvynet et al. Antimicrobial and chemotactic dermaseptin S9 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS 4141 of which were found to be very low and inferior to those obtained on hydrophobic surfaces (Table 1). The polar head groups of DMPC prevent strong peptide adsorption. Discussion The dermaseptin superfamily includes peptides with very different structural characteristics: (a) the derm- aseptins stricto sensu (dermaseptins S and B) from Phyllomedusa sauvagei and P. bicolor, amphipathic a- helical peptides that all have a conserved tryptophan residue at position 3 and a positive net charge attribut- able to the presence of lysine residues between alter- nating hydrophobic and hydrophilic sequences [8], (b) the plasticins, which are rich in glycine residues arranged in regular pentamer motifs GXXXG (where X is any amino acid residue) and are characterized by a high structural malleability [40], and (c) dermaseptin S9, from Phyllomedusa sauvagei, a highly aggregated and non-amphipathic peptide that has a hydrophobic core sequence flanked at both termini by several posi- tively charged residues [9]. Taking advantage of earlier observations by Lequin et al. [9] regarding the aggre- gative properties of Drs S9, we evaluated the peptide self-organization properties in aqueous solutions. We found that Drs S9 self-assembled via spherical interme- diates into amyloid-like fibrils as evidenced by elec- tronic microscopy and Congo red staining (Fig. 2). Antimicrobial peptides and amyloids are known to play a role in chemotaxis and to ultimately promote inflammation [16]. We therefore evaluated the antimi- crobial and chemotactic potential of the various struc- tural forms of Drs S9, i.e. monomeric and⁄ or weakly self-associated, oligomeric or protofibrillar, and fibril- lar forms. Interestingly, these structural intermediates resulted in various biological properties (Fig. 8). Amyloid-like properties of dermaseptin S9 Assembly of proteins or peptides into amyloid-like fibrils is a multistep process initiated by conforma- tional changes, during which intermediate aggregation states such as oligomers, protofibrils and filaments are seen [41]. Here we have shown that Drs S9 possesses most amyloidogenic characteristics [28]. Drs S9 has a b-sheet-rich structure as shown by ATR FTIR, shows 1680 1700 1720 1740 1760 1780 1800 –0.01 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 1739 1733 A B Absorbance (a.u.) Wavenumber (cm –1 ) DMPG + Drs S9 DMPG 2800 2850 2900 2950 3000 DMPC DMPD + Drs S9 DMPG DMPG + Drs S9 Wavenumber (cm –1 ) Fig. 7. Dermaseptin S9 interacts with anionic DMPG and zwitter- ionic DMPC vesicles. (A) DMPG CO ester spectra with or without Drs S9. (B) Normalized FTIR spectra (2800–3000 cm )1 ) of Drs S9 with zwitterionic DMPC or anionic DMPG vesicles. Table 3. Disturbance of anionic lipid assembly by Drs S9; assignment and distribution of component bands (± 1%) in lipid m(CO) and m(CH) stretching vibration modes in FTIR spectra obtained with 2 m M Drs S9 in the presence of 20 mM DMPG or DMPC vesicles suspended in NaCl ⁄ P i . Interface: peptide ⁄ DMPG lipid CO (%) Bilayer core: peptide ⁄ DMPG alkyl chain (%) 1726 cm )1 hydrogen bond 1743 cm )1 dehydrated 1726 ⁄ 1743 ratio 2852 cm )1 m S (CH 2 ) 2875 cm )1 m S (CH 3 ) 2923 cm )1 m AS (CH 2 ) 2956 cm )1 m AS (CH 3 ) Without peptide 65 35 1.8 21 6 68 4 With Drs S9 68 32 2.1 21 1 63 15 Antimicrobial and chemotactic dermaseptin S9 C. Auvynet et al. 4142 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS green bi-refringence as observed by microscopy after Congo red staining, and produces spherical oligomers and fibrils visualized by electron microscopy after 3 and 7 days of incubation at 37 °C, respectively (Fig. 2). FTIR allowed clear distinction between amy- loidogenic peptides and simple b-aggregated peptides by the observation of specific 1665 and 1685 cm )1 bands as reported previously [29]. Multiple b-structures were observed, and were consistent with the formation of oligomeric species (3 days) and long fibrils (7 days) (Fig. 3). Measurement of aggregation by physicochem- ical methods is a difficult task. We have demonstrated that the SPR technique can provide information on the bound peptide structure through determination of its adsorption density on the sensor chip surface [25,42]. Calculated adsorption densities on the HPA hydrophobic surface as a function of peptide concen- tration were in agreement with a b-structure for Drs S9 and suggested b-oligomer elongation (Table 1). This is in contrast with neutral plasticins, which also aggregate and form b-structures, but differ in their adsorption mode on the HPA hydrophobic support, which is governed by the surface-limited rate [25]. Our data are in agreement with previous observations indi- cating that the rate of adsorption of amyloidogenic Ab(1–42) peptide to lipid and sensor chip surfaces is a strictly diffusion-limited process [43]. Furthermore, the self-associating properties of Drs S9 were also corrobo- rated by slow NH ⁄ ND exchange kinetics monitored by ATR FTIR (Fig. 1A). Amyloidogenic peptides self-assemble, probably through specific molecular interaction patterns, gener- ating ordered mature fibrils. It is thought that, due to the multiple aromatic residues present in amyloid-like peptides, p–p stacking plays a crucial role in the fibril- lization process, by reducing the energetic barrier [44,45]. As Drs S9 has a hydrophobic core sequence (residues 8–15) that is rich in aromatic residues, the peptide can potentially establish p–p interactions [46] that could be at the origin of its amyloid-like behav- iour. The structural properties of most of the amyloi- dogenic peptides are environment-dependent. For example, Ab(1–42) is a-helical in the presence of triflu- oroethanol [47], but is poorly structured and aggre- gated in water and shows b-sheet formation in phosphate buffer solutions [28,48]. Similarly, Drs S9 was shown to fold into a non-amphipathic a-helical conformation in trifluoroethanol⁄ water mixtures [9]. Amyloid peptides and proteins can be divided into two main categories: those associated with pathological D3 D7 Amyloid-like fibrils R E C E P T O R G-PROTEIN Chemotactic activity on eukaryotes D0 Antimicrobial activity on prokaryotes Fig. 8. Summary of the main oligomeriza- tion states adopted by Drs S9 in relation to its biological activities. D0, freshly dissolved Drs S9 is mainly dimeric or tetrameric and exhibits maximal chemotactic activity. D3, after 3 days, Drs S9 has formed self-associ- ated oligomers with potent antimicrobial activity. D7, after 7 days, Drs S9 has formed amyloid-like fibrils. C. Auvynet et al. Antimicrobial and chemotactic dermaseptin S9 FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS 4143 [...]... interaction of Drs S9 with the immune cell membrane through a receptor (Fig 7) Antimicrobial activity of dermaseptin S9 and interaction with anionic membranes The antimicrobial activity spectrum of Drs S9 has been previously reported for peptide samples freshly dissolved (D0) with or without trifluoroacetic acid (TFA) counter-ions [9] The minimal inhibitory concentrations were in the range 3–10 lm for the trifluoroacetic... the same concentrations of Drs S9 present in the lower wells were added simultaneously to the upper wells For some experiments, neutrophils were pre-incubated with PTX (200 ng mL)1) (Sigma, St Louis, MO, USA), Ab(1–42) Antimicrobial and chemotactic dermaseptin S9 (50lm), Drs S9 (50 lm) or fMLP (100 nm) for 30 min at 37 °C prior to evaluation of their migration towards Drs S9 or Ab(1–42) Cell activation... data provided here indicate out that antimicrobial peptides represent interesting lead molecules that could boost innate immune responses and selectively modulate pathogen-induced inflammatory responses [52] Interestingly, our data indicate that structural requirements for chemotactic effects of Drs S9 and Ab(1–42) peptide are similar Analogues of Drs S9 retaining antimicrobial activity but with no effect... form at day 3 than in its low-molecular-weight, self-associated form, with a potency similar to that of Drs B2, one of the most potent frog antimicrobial peptides [56] In contrast, the antimicrobial activity of fibrillar Drs S9 was strongly reduced (Table 2) Thus, the amyloidogenic properties of Drs S9 influence its antibacterial activity, suggesting different modes of membrane permeabilization Drs S9. .. (m)1Æcm)1) Drs S9 was freshly dissolved in 50 mm phosphate buffer (pH 7), 50 mm (NaCl ⁄ Pi, pH 7) or H2O at a final peptide concentration of 30 lm If required, each FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS 4145 Antimicrobial and chemotactic dermaseptin S9 C Auvynet et al dilution was incubated at 37 °C for 3 or 7 days Thermal denaturation of Drs S9 in phosphate... room temperature were used as reference and subtracted For the amide H ⁄ D exchange kinetics experiments, lyophilized Drs S9 was hydrated using D2O or deuterated NaCl ⁄ Pi for 15 min, and spectra were recorded after 1, 5, 10 and 15 min [63] For amyloid structure experiments, 30 lL of a 500 lm Drs S9 suspension in phosphate buffer, pH 7, was incubated for 7 days at 37 °C with or without Congo red and dried... grids (200-mesh) covered by a carbon-stabilized Formvar film were hydrated with bacitracin for 10 min FEBS Journal 275 (2008) 4134–4151 ª 2008 The Authors Journal compilation ª 2008 FEBS C Auvynet et al Excess fluid was removed and the grids were placed in 10 lL Drs S9 samples for 10 min After wiping the grids, samples were fixed with paraformaldehyde (2% v ⁄ v) for 2 min and rinsed with 0.2 m ammonium acetate... peptide sample, and 12–50 lm for trifluoroacetic acid-free peptide (Table 2) Trifluoroacetic acid counter-ions slow down b-sheet structure formation of several amyloidogenic peptides (supplementary Fig S3 and Doc S1), and this may explain discrepancies between the minimal inhibitory concentration results In our study, performed using a trifluoroacetic acidfree form of Drs S9, the antimicrobial activity of.. .Antimicrobial and chemotactic dermaseptin S9 C Auvynet et al processes, and those with functional implications in vivo [49] For example, both human Pmel17, which plays an important role in the biosynthesis of the pigment melanin, and the factor XII protein of the haemostatic system are activated by amyloid structures [50] Drs S9 does not seem fall into either these... fall into either these two categories However, several examples of amyloidogenic peptides with biological activities in vitro, such as salmon calcitonin, have been reported [51] Drs S9 could thus be a member of this third group of amyloidogenic peptides Chemotactic properties of dermaseptin S9 Many antimicrobial peptides are multifunctional endogenous effectors of host innate defences against pathogenic . Structural requirements for antimicrobial versus chemoattractant activities for dermaseptin S9 Constance Auvynet 1,2 , Chahrazade. 1,2-dimyristoyl-sn-glycero-3- phosphatidylglycerol; Drs B2, dermaseptin B2; Drs B4, dermaseptin B4; Drs S9, dermaseptin S9; ERK, extracellular signal-regulated kinase; fMLP, formyl methionyl-leucyl-phenylalanine;