Se ´bastien Cardon

Emmanuelle Sachon

Ludovic Carlier

Thierry Drujon

Astrid Walrant

Estefanı ´a

Alema ´n-Navarro

Vero ´nica Martı ´nez-Osorio

Dominique Guianvarc'h

Sandrine Sagan

Yannick Fleury

Rodrigue Marquant

Christophe Piesse

Yvonne Rosenstein

Constance Auvynet
email: constance@ibt.unam.mx

Claire Lacombe
email: claire.lacombe@upmc.fr

Peptidoglycan potentiates the membrane disrupting effect of the carboxyamidated form of DMS-DA6, a Gram-positive selective antimicrobial peptide isolated from Pachymedusa dacnicolor skin

The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Grampositive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Grampositive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH 2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.

Introduction

The World Health Organization (WHO) recently reported that infectious diseases account for nearly 13% of global deaths. By 2050, the continuous rise in antimicrobial resistance could lead to the death of 10 million people every year [START_REF] Ghosh | Membrane-active small molecules: designs inspired by antimicrobial peptides[END_REF], notably due to an increase in hospitalrelated infections. Particularly, according to the WHO, Gram-positive bacteria are involved in major nosocomial infections, with 16% of these infections due to Staphylococcus aureus, one of the twelve families of bacteria considered a major threat for human health (http://www.who. int/mediacentre/news/releases/2017/bacteria-antibiotics-needed/en/). Furthermore, almost 38% of S. aureus strains are resistant to methicillin, and the number of Staphylococcus strains exhibiting resistance to vancomycin, one of the strongest available antibiotics, is increasing.

Despite the WHO considers antibiotic resistance a major issue of public health and prompts for the development of novel antibacterial molecules, major pharmaceutical companies have stopped antibiotic research due to the expanding rate of bacterial resistance and the slow approval of drugs. Over a period of twenty years , the number of antibacterial drugs approved by the United States Food and Drug Administration (FDA) has steadily decreased, and only twenty drugs have been approved for sale between 2008 and 2018 (https:// www.centerwatch.com/drug-information/fda-approved-drugs/therapeutic-area/25/infectionsand-infectious-diseases). Yet, the worldwide relentless emergence of antibiotic resistance continues to spur the search for novel anti-infectives to replace and/or supplement conventional antibiotics.

Since the discovery of the first antimicrobial peptide (AMP) in 1947, more than 2700 natural AMPs have been isolated from living prokaryotic and eukaryotic organisms (http://aps. unmc.edu/AP/main) [START_REF] Wang | APD2: the updated antimicrobial peptide database and its application in peptide design[END_REF]. Most peptides isolated to date are antimicrobial peptides with a broad spectrum against bacteria, fungi, and protozoa [START_REF] Conlon | Dermal cytolytic peptides and the system of innate immunity in anurans[END_REF][START_REF] Ponti | Expression and activity of cyclic and linear analogues of esculentin-1, an anti-microbial peptide from amphibian skin[END_REF][START_REF] Rinaldi | Antimicrobial peptides from amphibian skin: an expanding scenario: Commentary[END_REF]. The antimicrobial activity of most AMPs is related to their capacity to bind to negatively charged bacterial surface molecules, mainly phospholipids, leading to permeabilization and disruption of the cell membrane [START_REF] Epand | Bacterial membrane lipids in the action of antimicrobial agents[END_REF][START_REF] Malanovic | Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptide[END_REF]. In this context, amidation of the C-terminus is generally thought to contribute to AMP activity because the peptide is less anionic than its carboxylate analogue, thus more attracted by the bacterial membrane [START_REF] Dennison | Influence of C-terminal amidation on the efficacy of modelin-5[END_REF][START_REF] Kim | C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria[END_REF][START_REF] Nguyen | Serum stabilities of short tryptophanand arginine-rich antimicrobial peptide analogs[END_REF].

Latin American frogs synthesize and secrete a wide range of biologically active small molecules and peptides with remarkable pharmacological properties. Among Hylidae and Ranidae frog skin AMPs, the most studied are those from the Dermaseptin family, a superfamily of 27-to 34-residue AMPs. In the present study, we isolated a member of the Dermaseptin family corresponding to DMS-DA6, a cationic peptide that had been previously identified [START_REF] Meneses | Peptidomic analysis of the skin secretions of the frog Pachymedusa dacnicolor[END_REF] from the Mexican frog Pachymedusa dacnicolor. This peptide presents two naturally occurring versions, the acidic form (DMS-DA6-OH) and the corresponding amidated form (DMS-DA6-NH 2 ) (Table 1 andS1 Fig). Herein, we show that both forms have specific antibacterial activity against Gram-positive bacteria, including antibiotic-resistant strains.

Consistent with previous studies comparing carboxyamidated and carboxylated analogs [START_REF] Dennison | Influence of C-terminal amidation on the efficacy of modelin-5[END_REF][START_REF] Kim | C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria[END_REF][START_REF] Andre | Structure-Activity Relationshipbased Optimization of Small Temporin-SHf Analogs with Potent Antibacterial Activity[END_REF][START_REF] Cerovsky | Lasioglossins: three novel antimicrobial peptides from the venom of the eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae)[END_REF][START_REF] Niklison-Chirou | Microcin J25-Ga induces apoptosis in mammalian cells by inhibiting mitochondrial RNA-polymerase[END_REF][START_REF] Da Silva | The effects of the C-terminal amidation of mastoparans on their biological actions and interactions with membranemimetic systems[END_REF], we also show that the bactericidal capacity of DMS-DA6-NH 2 is higher than that of DMS-DA6-OH. However, in contrast to other studies, DMS-DA6-NH 2 does not exhibit increased hemolytic activities, making this peptide a good candidate for new drug development. Understanding the mechanism of action of such peptide, and particularly the role of the C-terminal carboxyamidation that increases its antibacterial activity but not the hemolytic activity, could be a good starting point in the development of effective antibiotics. Previous studies have proposed that carboxyamidation enhances the interaction with the negatively charged bacterial membrane, inducing its permeabilization by increasing the net positive charge of the peptide and/or by stabilizing the membrane-bound structure [START_REF] Kim | C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria[END_REF][START_REF] Mura | The effect of amidation on the behaviour of antimicrobial peptides[END_REF]. By exploring the structural properties and molecular interactions of the two analogs with Grampositive bacteria membrane mimetics, we could not explain the more significant antibacterial activity of DMS-DA6-NH 2 by (i) a more stabilized conformation, (ii) an increased positive net charge or (iii) different membrane-disrupting properties. Instead, the enhanced antibacterial activity of DMS-DA6-NH 2 could be accounted for its capacity to interact with peptidoglycan, an essential component of the membrane of Gram-positive bacteria.

Altogether our data unravel that DMS-DA6 peptides act by strong perturbation of the bacterial membrane at different levels, highlighting a role for peptidoglycan as a potentiator of antimicrobial peptide function, and underscore the potential of DMS-DA6, particularly that of DMS-DA6-NH 2 , for developing new drugs against multi-resistant bacteria.

Materials and methods

Frogs, peptide isolation, purification and identification

Specimens of Pachymedusa dacnicolor were captured in the state of Morelos (Mexico) in the private land of YR, author of this study. P. dacnicolor is an endemic frog of the state of Morelos; it is not a frog species under threat of extinction according to the IUCN Red List of Threatened Species (http://www.iucnredlist.org/). Since frog specimens can be purchased legally in local pet stores in Mexico, no specific permissions were required for capturing and housing frogs. Mexican law does not require approval of the national Bioethics committee for experimental work on amphibians. Nonetheless, housing conditions and experimental procedures were approved by the Bioethics Committee of the Instituto de Biotecnologı ´a (Universidad Nacional Auto ´noma de Me ´xico), and were undertaken by authorized investigators. Frogs were housed in a glass terrarium (70×40×40 cm) covered with a screen, close to a window, with normal daylight cycles, as previously described [START_REF] Auvynet | Dermaseptin DA4, although closely related to dermaseptin B2, presents chemotactic and Gram-negative selective bactericidal activities[END_REF]. Substrate was soil, branches provided climbing space and natural local plants were added. Clean water was always available for the frogs to soak in. The terrarium was cleaned every other week and the plants and climbing structures renewed as needed. Frogs were fed with live crickets.

A full protocol describing isolation, purification, and sequencing of DMS-DA6 from the skin exudate of Pachymedusa dacnicolor is provided in the Supporting information section (S1 Protocol). Briefly, peptides isolation and purification from the frog exudate was performed by size exclusion chromatography on a Sephadex G-50 column followed by reverse phase highperformance liquid chromatography (RP-HPLC). Peptides identification was carried out by mass spectrometry analyses (S1 Protocol) of RP-HPLC collected fractions, directly or after acetylation [START_REF] Riordan | 41] acetylation[END_REF]. Samples for MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) analysis were prepared according to the "dried droplet" method [START_REF] Karas | Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons[END_REF].

Solid-phase peptide synthesis

Peptides were synthesized by automated solid-phase peptide synthesis using Fmoc/tBu (9-Fluorenylmethoxycarbonyl/tertio-butyl) chemistry on an ABI 433A peptide synthesizer (Applied Biosystems) and purified by RP-HPLC (C18 Xbridge Waters, 5 μm 19 × 50 mm) using a solvent system composed of water containing 0.1% trifluoroacetic acid (TFA) as solvent A and acetonitrile containing 0.1% TFA as solvent B. The column was eluted at 10 mL. Peptides were dissolved in water at a concentration of 1 mM and these stock solutions were further diluted in PBS (Phosphate buffered saline) or cell culture medium for biophysical and functional experiments. [START_REF] Bugg | Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA[END_REF] were cultured as described previously [START_REF] Auvynet | Structural requirements for antimicrobial versus chemoattractant activities for dermaseptin S9[END_REF]. The minimal inhibitory concentration (MIC) of the peptides was determined by the microtiter broth dilution method. Overnight precultures were subcultured in Luria Bertani (LB) medium at 37˚C with vigorous shaking until mid-log phase (~2h) and diluted to 10 7 cfu/mL. Peptide solutions were prepared at different concentrations (from 2 to 500 μM) by successive 2-fold dilutions in water from 1 mM stock solutions. In each row of a 96-well nonbinding surface plate, 90 μL of bacteria were incubated with 10 μL of peptide solutions for 18 h at 37˚C. The final peptide concentrations ranged from 0.2 to 100 μM. Cell growth was evaluated by visual inspection and absorbance at 600 nm to determine the lowest concentration of peptide that prevented cell growth. Wells were either opaque, indicating stationary phase growth, or transparent indicating no growth. The lowest concentration showing no visible growth was considered as the MIC. After measurement of the MIC, the bactericidal activity was determined by the addition of resazurin dye (30 μL, 0.01% w/v) to each well [START_REF] Sani | Bacteria may cope differnetly to similar membrane damage caused by the Australian tree frog antimicrobial peptide maculatin1.1[END_REF]. The plates were further incubated at 37˚C for 18 h. Wells in which the dye turned pink indicated live bacteria, whereas wells in which the dye remained blue indicated dead bacteria. The minimal bactericidal concentration (MBC) is the lowest concentration with blue coloration (99.9% of bacteria were killed). Three to five independent assays were performed in triplicate. Positive controls correspond to medium plus 0.2% formaldehyde and negative controls to medium without peptide.

Antimicrobial assays

Hemolytic activity

The hemolytic activity of both peptides was determined using fresh erythrocytes of mice or human healthy adult donors. The blood was centrifuged (800 × g for 7 min), and the erythrocytes were rinsed three times with PBS. Peptides at two concentrations (10 μM or 50 μM) were incubated with suspended erythrocytes (4% v/v in PBS) at 37˚C for 60 min, and the cells were then removed by centrifugation at 800 × g for 5 min at 4˚C. Hemolysis was assessed by measuring the supernatant absorbance at 550 nm. Erythrocytes lysed with 1% Triton X-100 were used as a standard for 100% hemolysis. The supernatant of an untreated erythrocyte suspension in PBS was used as blank.

Stability in the supernatant of bacterial growth

Bacteria S. aureus ATCC 6538 were incubated as described for antimicrobial assays, with 6.5 μM DMS-DA6-NH 2 or 12.5 μM DMS-DA6-OH (MIC/2) for 18 h at 37˚C. After centrifugation (4,000 × g for 10 min), bacteria were discarded. The supernatant was filtered (syringe filter, pore size 0.2 μm) and incubated with 200 μM of either peptide at 37˚C for 24 h. Intact peptides (time = 0) and incubated peptides were analyzed by reverse-phase HPLC. The experiment was performed on a Dionex ultimate 3000 system using a Higgins proto 200 C18 5 μm column. The mobile phase was a mixture of Solution A (0.1% TFA in water) and Solution B (0.1% TFA in acetonitrile), with an elution gradient of 0% to 80% Solution B over 10 min. The flow rate was 1 mL/min. Effluent absorbance was monitored at both 230 and 280 nm.

Cytotoxicity experiments

A549 cells (ATCC CCL-185), a human lung carcinoma cell line, were cultivated at 37˚C, humid atmosphere and 5% CO 2 , in Petri dishes with DMEM Advanced medium (Gibco) supplemented with 5% of fetal bovine serum (FBS), 2 mM of glutamine, 100 U of penicillin and 50 μg/mL of streptomycin (Gibco). When the cells reached 80% confluence, the medium was discarded and the cells were washed with PBS 1×, before being treated with Trypsin 1× for 2 minutes to detach them form the culture dish. Cells were then resuspended in DMEM medium, seeded in a 48-well culture plate (500 000 cells/well), and either form of DMS-DA6 solubilized in PBS 1× was added and incubated for 2 hours at 37˚C, 5% CO 2 . DMSO 30% was used as a positive control to induce cell death, and DMEM medium as negative control. After incubation, cell viability was assessed by trypan blue exclusion.

Bacterial membrane permeabilization assays

Membrane permeabilization was assessed by monitoring the hydrolysis of ortho-nitrophenyl β-D-galactopyranoside (ONPG) by the β-galactosidase enzyme, using the S. aureus ST1065 strain, specifically designed for membrane permeabilization assays, through detection of cytoplasmic β-galactosidase activity leakage [START_REF] Joanne | Comparative study of two plasticins: specificity, interfacial behavior, and bactericidal activity[END_REF]. Bacteria were grown in LB to an absorbance of 0.5 at 600 nm, pelleted by centrifugation, and the pellets were washed twice with PBS. Pellets were then resuspended in 10% LB in PBS to an absorbance of 0.5 at 600 nm. Aliquots (15 μL) of bacterial suspension were mixed with 2.5 mM ONPG and incubated with various concentrations of peptide in a 96-well plate. The hydrolysis of ONPG was monitored by measuring the absorbance at 420 nm of released o-nitrophenol with a Fluostar Galaxy microplate reader (BMG LabTech, France) [START_REF] Auvynet | Dermaseptin DA4, although closely related to dermaseptin B2, presents chemotactic and Gram-negative selective bactericidal activities[END_REF].

Bacterial lipid extraction

The bacteria (E. coli K12 strain or S. epidermidis BM3302 strain) were grown in batches of 8 L of LB at 37˚C until OD 600 = 0.6. Cells were collected by centrifugation (3,000 × g, 15 min, 4˚C), and the pellets were re-suspended in 30 mL 20 mM Tris-HCl buffer, pH 8.0, centrifuged again and washed twice with the same buffer. Pellets were re-suspended in a mixture of methanol:chloroform (3 mL:1 mL/g of pellet). After agitating for 5 min, lipids were separated from the water-soluble material by diluting the extraction mixture with 1 mL/g of pellet of chloroform followed by 1 mL/g of pellet of water and agitated again for 4 min. The solution was centrifuged (4,000 × g, 12 min, 4˚C), the organic phase was separated and the aqueous solution extracted twice with 15 mL chloroform. The organic phases were combined and dried under vacuum. The residue was solubilized in 3 mL toluene, centrifuged (20,000 × g, 1 min, 20˚C) and the supernatant was dried under vacuum. The residue was dissolved in a minimal volume of chloroform and 10 volumes of acetone. The resulting phospholipid precipitate was separated through filtration on a sintered-glass filter and removed from the filter with small amounts of chloroform. The chloroform was evaporated and lipid vesicles were formed.

Preparation of lipid vesicles

Lipid vesicles were formed using bacterial lipids extracted as described above or commercially available synthetic lipids. The lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1.2-dioleyl-sn-glycero-3-phosphoglycerol (DOPG), and 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (CL), were obtained from Avanti Polar Lipids, Inc (Alabaster, AL) and used without further purification. Desired mixtures of phospholipids were first dried in glass tubes under nitrogen and then maintained under a reduced pressure for at least 60 min. Lipid films were subsequently hydrated in PBS to yield a lipid concentration of 10 mg.mL -1 for tryptophan fluorescence experiments, 1 mg.mL -1 for DSC experiments, and in PB at 1 mM for CD experiments. The resulting dispersions of large multilamellar vesicles (MLVs) were subjected to 3 to 5 freeze-thaw cycles and then extruded 15 times through polycarbonate membranes (100 nm pore size, Whatman, Maidstone, UK) on an Avanti mini-extruder apparatus (Avanti Polar Lipids, Inc., Alabaster, AL) to generate large unilamellar vesicles (LUVs). Lipid vesicles were used either alone, or in combination with S. aureus peptidoglycan (PGN) (Sigma).

Circular dichroism (CD) spectroscopy

The CD spectra of the peptides were recorded as previously described [START_REF] Carlier | Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides[END_REF]. Measurements were carried out in 10 mM phosphate buffer at pH 7.4, 10 mM detergent (DPC or SDS), or in the presence of POPC, POPG and POPG-POPC (25:75) LUVs. Peptide concentration was 20 μM in all the samples, and measurements in the presence of lipids were performed with a peptide/lipid molar ratio of 1/50.

Intrinsic tryptophan fluorescence

The fluorescence spectra of DMS-DA6 peptides were recorded from 295 to 475 nm with excitation at 280 nm, in a spectrofluorometer (Biologic Science Instruments ALX 250, MM 450, PMS 250, Software: Bio-Kine 32 V4.60, Grenoble, F). 150 μL of a 20 μM peptide solution were introduced in a 10 × 2 mm quartz cuvette (Hellman Analytics, Mu ¨llheim, GER) and increasing amounts of the lipid solution were added to obtain lipid concentrations of 10, 50, 200, 500, 1000, and 3000 μg.mL -1 . After each addition, the tryptophan residue of the peptide was excited at 280 nm and the emission spectra were recorded from 300 to 480 nm. Spectra were normalized to take into account peptide dilution. We chose to calculate Δλmax-hence the difference between λ max at any concentration of lipid and the λ max of the peptide solution without added liposomes-and to plot the values against the concentration of lipid, as described by Hawrani and coworkers [START_REF] Hawrani | Thermodynamics of RTA3 peptide binding to membranes and consequences for antimicrobial activity[END_REF]. An apparent binding constant equivalent to the concentration of lipid at which the lipid-induced blue shift was half-maximal (K L ) was thus determined.

NMR spectroscopy and structure calculations

DMS-DA6 peptides were solubilized at a concentration of 1 mM in 550 μL H 2 O/D 2 O (90:10 v/ v) in the presence of 80 mM SDS-d 25 detergent (Eurisotop, Saint-Aubin, France). NMR experiments were recorded at 40˚C on a 500 MHz Bruker Avance III spectrometer equipped with a TCI cryoprobe. 1 H and 13 C resonances were assigned from the analysis of 2D 1 H-1 H TOCSY, 2D 1 H-1 H NOESY (100 ms mixing time), and 2D 1 H-13 C HSQC spectra. NMR positioning experiments were recorded using the micellar samples of DMS-DA6 in the absence or presence of 1 mM gadodiamide. Paramagnetic relaxation enhancements were estimated from the T 1 relaxation times of Hα protons as previously described [START_REF] Carlier | Investigating the role of GXXXG motifs in helical folding and self-association of plasticins, Gly/Leu-rich antimicrobial peptides[END_REF].

Interproton distance restraints used for structure calculation were estimated from manually assigned NOEs collected on a 2D 1 H-1 H NOESY spectrum recorded at 800 MHz using a 60-ms mixing time. Three upper limit classes of 2.8, 3.8, and 5.0 Å were defined from the NOE cross-peak volumes. TALOS-N [START_REF] Shen | Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks[END_REF] was employed to derive ϕ and ψ dihedral angle restraints based on 13 Cα, 13 Cβ, 1 H N , and 1 Hα chemical shifts. A set of 100 structures was calculated by torsion angle dynamics in CNS [START_REF] Guntert | Torsion angle dynamics for NMR structure calculation with the new program DYANA[END_REF] using standard parameters. The 20 lowest energy structures were retained and refined in an explicit layer of water, including an electrostatic energy term in the energy function. After the refinement process, the 12 structures exhibiting the lowest energies were selected to represent the NMR ensemble.

Dextran leakage experiments

Rhodamine isothiocyanate (RITC)-labeled 10 kDa dextran-loaded liposomes were prepared by hydrating the dry lipid film with the buffer solution containing labeled dextran (5 mg.mL -1 ) as described by Rinaldi et al. [START_REF] Rinaldi | Effects of temporins on molecular dynamics and membrane permeabilization in lipid vesicles[END_REF] with some modifications. To achieve a better ratio of encapsulation, liposomes composed of cardiolipin (CL) and DOPG (40:60, w/w) to mimic Grampositive membranes were submitted to a series of seven freeze-thaw cycles and LUVs (400 nm) were obtained as described above. Untrapped dextran was then removed by several cycles of centrifugation (21,500 × g for 40 min) and washing with PBS, until the residual fluorescence in the supernatant was negligible. Dextran release from loaded vesicles upon interaction with DMS-DA6 peptides was followed on a spectrometer (Biologic Science Instruments ALX 250, MM 450, PMS 250, Software: Bio-Kine 32 V4.60, Grenoble, F) by measuring the fluorescence of the supernatant obtained after centrifugation at 21500 × g for 40 min, after 5 min incubation with the peptide. The excitation wavelength was 550 nm, and emission spectra were recorded from 560 to 650 nm. 100% leakage was achieved by addition of Triton X-100 to a final concentration of 0.1%.

Differential scanning calorimetry (DSC)

DSC experiments were performed on a NanoDSC microcalorimeter (TA instruments), using DMPG LUVs at a lipid concentration of 1 mg.mL -1 . Peptides were added to the DMPG vesicles suspension at concentrations that allow a peptide to lipid ratio of 1/100, 1/50, and 1/25. A minimum of five heating and cooling scans (0-50˚C, 1˚C/min) was performed for each analysis. For experiments with PGN, the appropriate amount of a PGN suspension (40 μg.mL -1 ) was added to 1 mL DMPG-LUVs. The rest of the sequence is identical. DSC experiments were analyzed using the NanoAnalyze software provided by TA instruments. When needed, peaks were deconvoluted using the Modeling tool of NanoAnalyze.

Results

The carboxyamidated form of DMS-DA6 exhibits higher antibacterial activity against Gram-positive bacteria and is not hemolytic

In the search for novel biomolecules endowed with antimicrobial activity, fresh skin exudate from the Mexican frog Pachymedusa dacnicolor was fractionated (S4 Fig) . Fraction I was further separated by RP-HPLC and RP-HPLC fractions were then tested for antibacterial activity. The fraction with a retention time of 55.7 min showed antimicrobial activity against the Gram-positive bacteria S. aureus ATCC 6538, but not against the Gram-negative bacteria E. coli ATCC 35218. The MALDI-TOF analysis of the RP-HPLC fraction revealed a peptide with a m/z ratio in the 2.0 to 3.5 kDa mass range, consistent with a Dermaseptin-like peptide (S5 Fig) . Acetylation and de novo sequencing of this RP-HPLC fraction was performed to differentiate Lys from Gln residues before tandem MS fragmentation (MALDI-TOF/TOF) of the fulllength parent ion. By combining the MS/MS information collected from the acetylated and non-acetylated parent peptides (S5 Fig) , we obtained the sequence of DMS-DA6 (GVWGIA KIAGKVLGNILPHVFSSNQS), a 26-mer peptide previously identified through a proteomic study [START_REF] Meneses | Peptidomic analysis of the skin secretions of the frog Pachymedusa dacnicolor[END_REF]. Our sequencing data unambiguously supported a free carboxyl terminus, while Meneses et al. [START_REF] Meneses | Peptidomic analysis of the skin secretions of the frog Pachymedusa dacnicolor[END_REF] found the C-terminus of DMS-DA6 in the amidated form (DMS-DA6-NH 2 ), suggesting that the two versions of DMS-DA6 may naturally co-exist in the frog exudate.

As reported in Table 2, both forms of DMS-DA6 were highly effective against a wide range of Gram-positive strains. Notably, they were active against the multidrug-resistant strains Enterococcus faecium BM4147 and Staphycoloccus aureus DAR 5829. DMS-DA6-NH 2 was consistently more active than DMS-DA6-OH. On average, the MIC and MBC values obtained for DMS-DA6-OH were two to four-fold higher in comparison with DMS-DA6-NH 2 , suggesting that amidation of the C-terminus could be associated to a systematic increase in the antibacterial potency of DMS-DA6. Noteworthy, and contrary to a number of carboxyamidated peptides [START_REF] Dennison | The role of C-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin H5[END_REF][START_REF] Chen | Synthetic magainin analogues with improved antimicrobial activity[END_REF], neither form of the peptide was hemolytic for human or murine erythrocytes at 10 μM or 50 μM (Table 2).

DMS-DA6-NH 2 has a greater capacity than DMS-DA6-OH to permeabilize the S. aureus membrane

To outline the mechanism of action of these peptides, we assessed the ability of the two forms 2). The kinetics of S. aureus membrane leakage is slow in comparison with that of Gram-negative E. coli for which significant ONPG hydrolysis is usually detected within a few minutes after AMP addition. Such slow permeabilization kinetics of S. aureus membrane induced by DMS-DA6 was previously observed with other membrane-disrupting AMP belonging to the dermaseptin family [START_REF] Joanne | Comparative study of two plasticins: specificity, interfacial behavior, and bactericidal activity[END_REF]. Together, these results indicate that both DMS-DA6 peptides induced membrane leakage of Gram-positive bacteria, a result consistent with a cell lysis process mediated through peptide-membrane interactions.

Cancer cells tend to be more negatively charged than normal cells [START_REF] Marien | Non-small cell lung cancer is characterized by dramatic changes in phospholipid profiles[END_REF] and a variety of cationic antimicrobial peptides, including dermaseptins [START_REF] Huang | Dermaseptin-PH: A Novel Peptide with Antimicrobial and Anticancer Activities from the Skin Secretion of the South American Orange-Legged Leaf Frog, Pithecopus (Phyllomedusa) hypochondrialis[END_REF][START_REF] Shi | Two Novel Dermaseptin-Like Antimicrobial Peptides with Anticancer Activities from the Skin Secretion of Pachymedusa dacnicolor[END_REF], have been reported to exhibit antitumor activity. We thus investigated whether the peptides had distinct activities on cell viability, parallel to their antibacterial activities. Taken together, our results suggest that a negatively charged membrane is not a sufficient environment to induce membrane permeation, suggesting that other components than negatively charged lipids present on Gram-positive bacterial cell wall components may participate in DMS-DA6-NH 2 higher activity.

The increased membrane permeabilization capacity of DMS-DA6-NH 2 does not result from a higher positive net charge, enhanced stability or structural differences in lipid and lipid-mimetic environments

To test the possibility that the C-terminal amidation may protect DMS-DA6-NH 2 from proteolysis, thus increasing its antibacterial activity, as previously suggested for other peptides [START_REF] Mura | The effect of amidation on the behaviour of antimicrobial peptides[END_REF][START_REF] Dennison | The effect of C-terminal amidation on the efficacy and selectivity of antimicrobial and anticancer peptides[END_REF][START_REF] Saravanan | Design of short membrane selective antimicrobial peptides containing tryptophan and arginine residues for improved activity, salt-resistance and biocompatibility[END_REF], we incubated the two versions of DMS-DA6 with bacterial culture medium for 24 hours and assessed their molecular integrity by HPLC. HPLC analysis revealed that more than 90% of either peptide remained intact after a 24-hour incubation period in the bacterial supernatant (Fig 3), thus precluding that the higher efficiency observed for the amidated version resulted from a higher proteolytic stability in culture medium.

Our next step was to determine the impact of carboxyamidation on the structural conformation of DMS-DA6 by CD and NMR. Antimicrobial peptides are generally unstructured in aqueous solution and tend to adopt a well-defined secondary structure in contact with lipid membranes. Accordingly, in phosphate buffer, the CD spectra of DMS-DA6-NH 2 and DMS-DA6-OH show a single negative band near 200 nm, evidencing a random coil conformation of the peptides (Fig 4A and4B). In contrast, in the presence of negatively charged POPG vesicles that mimic bacterial phospholipids, the two intense negative bands detected around 208 and 222 nm indicate that both peptides mostly adopted (about 80%) an α-helix conformation. In the presence of pure zwitterionic POPC vesicles, the contribution of α-helix conformation decreased to 40%. However, addition of a small proportion of negative charges, as in the mixture PC/PG (3:1) was sufficient to recover the high helical contents obtained with pure PG vesicles (Fig 4C ), indicating that electrostatic interactions between the cationic residues of DMS-DA6 and the negatively charged lipids play a major role in stabilizing the helical The solution structure of DMS-DA6-OH was calculated in SDS micelles by restrained molecular dynamics, using distance and torsion angle constraints (S1 Table ). The structure exhibits an uninterrupted helix in the central region 3-21 while terminal segments 1-2 and 22-26 are disordered (Fig 5A). The analysis of side chain distribution revealed a pronounced amphipathic character with a large hydrophobic face predominantly formed by Leu and Ile residues, along with Ala and Val amino acids (Fig 5B). The hydrophilic face is dominated by polar residues (particularly two cationic Lys) although it also contains apolar (Gly, Ile, and Pro) amino acids. The interfacial position of Trp-3 within such amphipathic topology is likely to be essential for the insertion of DMS-DA6 into bacterial membranes leading to cell disruption, as proposed for other antimicrobial peptides [START_REF] Deslouches | De novo generation of cationic antimicrobial peptides: influence of length and tryptophan substitution on antimicrobial activity[END_REF][START_REF] Li | Membrane Active Antimicrobial Peptides: Translating Mechanistic Insights to Design[END_REF][START_REF] Sun | Insights into the membrane interaction mechanism and antibacterial properties of chensinin-1b[END_REF].

Surprisingly, although known as helix breakers, the Pro residue at position 18, together with the high content of Gly in the peptide sequence (4 over 26 residues) do not interrupt the helical conformation. Indeed, in water soluble globular proteins, Gly and Pro residues exhibit the lowest helical propensities and are often proposed to induce kinks or hinges in helical structures. The long helical region of DMS-DA6-OH does not show any pronounced bending but appears slightly curved towards the hydrophobic face (Fig 5B ). A detailed inspection of the hydrogen bond networks indicates that backbone CO i -NH i+4 H-bonds, diagnostic of a αhelix, are observed throughout the helical structure, except near the Gly and Pro residues where such H-bonds are absent or show increased lengths. In particular, a CO i -NH i+3 H-bond involving the carbonyl group of Ile-16 is observed in the C-terminal region encompassing Pro-18. Interestingly, helical propensities are significantly lower at positions occupied by Gly residues and in the helical portion 15-20, as supported by decreasing CSD values (S7 Fig) . Overall, these observations suggest that the α-helical distortions of DMS-DA6-OH likely result from the presence of helix-destabilizing residues in the peptide sequence.

The position of DMS-DA6 with respect to the micelle surface was examined with gadodiamide, a water-soluble paramagnetic probe that partitions exclusively outside the micelle. The addition of gadodiamide led to differential paramagnetic relaxation enhancements (PREs) of Hα protons (Fig 5C ). Residues in the central portion of the helical region displayed the lowest PREs indicating that they are buried inside the micelle. Periodicity in the PRE values was apparent in the region 5-17 supporting a parallel positioning of this central portion with respect to the micelle surface. Apolar residues Ala-6, Ala-9, Leu-13, and Leu-17, which lie on the hydrophobic face, were the least affected by gadodiamide, demonstrating that these residues orient their side chains towards the micelle interior. Although located on the hydrophobic face, residue Val-20 is substantially affected by the paramagnetic probe and is therefore positioned close to the water-micelle interface. A higher sensitivity to gadodiamide may also result from an increased flexibility at the helix C-terminus induced by Pro-18.

The NMR structure of DMS-DA6-OH in SDS micelles is consistent with the high helical content of both peptide forms in POPG and POPC/POPG (4:1) vesicles, assessed by CD spectroscopy. The 2D NOESY spectrum of DMS-DA6-NH 2 in SDS micelles overlaid almost entirely with that of DMS-DA6-OH (Fig 6), further confirming that the C-terminal amidation does not affect the structure of the peptide.

Altogether, the CD and NMR studies uncovered the α-helix conformation of membranebound DMS-DA6 but did not reveal an influence of the C-terminal amidation on the structure of the peptide. As the central region 5-17 of both peptides can insert deeply into the hydrophobic core of micelles, we still lacked an explanation for the superior ability of DMS-DA6-NH 2 to disrupt bacterial membrane, compelling us to investigate this further.

DMS-DA6-NH 2 enhanced activity is not an effect of stronger interactions with negatively charged membranes

The fluorescence emitted by aromatic tryptophan residues of peptides and proteins varies depending on the polarity of the environment, and is thus informative of the position of tryptophan-containing peptides in lipid membranes [START_REF] Ladokhin | How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?[END_REF]. To evaluate if carboxyamidation influenced the capacity of DMS-DA6 to interact with and insert itself into membranes with different composition of phospholipids, we explored the tryptophan fluorescence spectra of DMS-DA6-OH and DMS-DA6-NH 2 in the presence of phospholipids extracted from Grampositive (S. epidermidis) or Gram-negative (E. coli) bacteria. As expected, when in aqueous buffer, the tryptophan fluorescence spectra of DMS-DA6-NH 2 and DMS-DA6-OH had a maximum emission at 355 nm. The addition of increasing concentration of LUVs prepared with E. coli or S. epidermidis phospholipids resulted in a substantial blue-shift in the emission maxima of either peptide, suggestive of the incorporation of the tryptophan aromatic ring into the hydrophobic environment of the negatively charged lipids (Fig 7A). DMS-DA6-NH 2 and DMS-DA6-OH interacted with and inserted into S. epidermidis phospholipid membranes, with a blue-shift of around 20 nm and an apparent binding constant K L around 0.02 mg.mL -1 . In contrast, a weaker interaction with E. coli phospholipid membranes was observed, with a blue-shift of around 10 nm and weaker K L values of 0.72 and 0.95 mg.mL -1 for DMS-DA6-OH and DMS-DA6-NH 2 , respectively (Fig 7A ,Table 3). These data suggest that the phospholipid composition of the membrane is crucial for the interaction and insertion of DMS-DA6 into the membrane and that both forms of DMS-DA6 have a higher affinity for Gram-positive bacteria phospholipids, consistent with the antibacterial assays results. However, the greater membrane permeabilization capacity of the carboxyamidated form of DMS-DA6 was not dependent on the nature of the lipids present in the membrane.

We next investigated membrane defects induced by the DMS-DA6 peptides using dextran leakage assays. Peptide-induced liposome leakage was monitored by measuring the fluorescence of rhodamine-labelled dextran released into the supernatant after centrifugation. Dextran leakage from the liposomes can be directly related to the lytic effect of the peptide. Release of 10 kDa dextran corresponds to pores or membrane defects larger than 2.3 nm, according to Stokes radius data provided by the manufacturer. Fig 7B shows that dextran release, calculated as a percentage of the release induced by 0.1% Triton X-100, was peptide concentration dependent, with a maximum obtained for a peptide/lipid molar ratio 1/1000. These results indicate that the pores created by DMS-DA6 peptides have an estimated radius of at least 2.3 nm, and are likely responsible for their antimicrobial activities.

We further evaluated the interaction of the two forms of DMS-DA6 with lipids by DSC. As complex lipid mixtures are not well adapted to DSC analysis, we use pure DMPG to mimic Gram-positive bacterial membranes. Thermograms illustrating the effect of incorporating increasing quantities of DMS-DA6-NH 2 and DMS-DA6-OH are presented in Fig 7C and7D, and the corresponding thermodynamic parameters are provided in Table 4. Pure DMPG vesicles display two distinct endothermic transitions: a less energetic event around 13˚C corresponding to the transition between the Lβ' gel phase to the Pβ' ripple phase, the so-called pretransition, and a more energetic event around 24˚C corresponding to the transition between the Pβ'ripple phase to the Lα fluid phase, the so-called main transition. Addition of either version of DMS-DA6 completely abolished the pre-transition, even at the lower peptide to lipid ratio (P/L) of 1/100, indicative of a strong interaction between the peptide and the lipid headgroups. At this peptide concentration, the main transition peak was minimally affected, except for some tailing towards higher temperatures, possibly explaining the slight increase in the transition enthalpy. Increasing the peptide concentration to P/L = 1/50 resulted in a clear splitting of the transition peak, suggesting that the peptides promoted the lateral segregation on the bilayer, creating peptide-rich and peptide-poor regions, as has been reported for basic arginine-rich peptides [START_REF] Walrant | Different membrane behaviour and cellular uptake of three basic arginine-rich peptides[END_REF]. Furthermore, this was accompanied by a general decrease of the transition enthalpy, pointing to an interaction between the peptides and the fatty acid chains of the lipids. The high-temperature peak, with a higher transition enthalpy closer to that of pure lipid, probably corresponds to a peptide-poor region. The low-temperature peak has a much lower transition enthalpy that would correspond to the emerging peptide-rich region. As peptide concentration increased, the two peaks were more separated, and the enthalpy The apparent binding constant K L is defined as the lipid concentration that induces 50% of maximal blue-shift.

https://doi.org/10.1371/journal.pone.0205727.t003

transition further decreased. This data indicates that both versions of DMS-DA6 equally disorganize a DMPG bilayer, consistent with the bacterial and model membrane leakage experiments, but still fails to provide an explanation for the substantially higher antibacterial activity of the carboxyamidated form of the peptide.

DMS-DA6-NH 2 interacts with peptidoglycan, a major component of Gram-positive bacteria cell wall, amplifying membrane permeabilization

Apart from phospholipids, peptidoglycan (PGN) is a major neutral molecule present on the membrane of Gram-positive bacteria. Malanovic and Lohner [START_REF] Malanovic | Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptide[END_REF] recently suggested that peptidoglycan could function as a cell wall sponge, attracting peptides to the surface of the bacterial lipid membrane, thus potentiating the action of antimicrobial peptides by increasing their local concentration at the level of the bacterial membrane. We investigated whether peptidoglycan could contribute to the greater activity of DMS-DA6-NH 2 compared to DMS-DA6-OH by using lipid vesicles containing peptidoglycan. We first defined the optimal amount of peptidoglycan to be used to avoid a complete perturbation of the DSC thermograms. Consistent with a previous report [START_REF] Malanovic | Phospholipiddriven differences determine the action of the synthetic antimicrobial peptide OP-145 on Gram-positive bacterial and mammalian membrane model systems[END_REF], even though the transition enthalpy remained unchanged, as peptidoglycan concentration augmented, the main transition peak shifted to higher temperatures and became broader, with the appearance of shoulders at higher temperatures (Fig 8A). Experiments were thus carried out with DMPG-LUVs with 0.02% (w/w) peptidoglycan since at this concentration the transition peak is still unique, though slightly broader than with pure DMPG. DMS-DA6 peptides were then gradually added to the DMPG-peptidoglycan LUVs. The corresponding data are reported in Table 5.

Similar to experiments performed in the absence of peptidoglycan, the addition of either form of DMS-DA6 to the DMPG/PGN LUVs led to the splitting of the main transition peak. However, different to what we observed without peptidoglycan, this happened even at the lowest P/L ratio (Fig 8B ). Furthermore, at a P/L ratio of 1/100, the lower temperature "peptiderich" peak was considerably bigger with DMS-DA6-NH 2 than with DMS-DA6-OH (Fig 8C ), although this difference was no longer significant at higher peptide concentrations. Collectively, this data suggests that in the presence of peptidoglycan, DMS-DA6-NH 2 is more efficiently disrupting the membrane than DMS-DA6-OH, providing a plausible explanation for the better performance of DMS-DA6-NH 2 over DMS-DA6-OH on Gram-positive bacteria.

Discussion

In the search for novel antimicrobial agents, we isolated a member of the Dermaseptin family corresponding to DMS-DA6 (GVWGIAKIAGKVLGNILPHVFSSNQS) from the Mexican frog Pachymedusa dacnicolor. Different to most Dermaseptins that share a Trp residue at Although Dermaseptins typically display a broad-spectrum activity against both Gram-positive and Gram-negative strains [START_REF] Nicolas | The dermaseptins[END_REF], we found that both forms of the DMS-DA6 peptide were highly specific against Gram-positive bacteria. Notably, they were active against the multidrug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR 582. Interestingly, AMPs with the closest sequences to DMS-DA6 (S2 Table ), i.e., the three plasticins PD36K, PD36, B1 [START_REF] El Amri | The plasticins: membrane adsorption, lipid disorders, and biological activity[END_REF], and CPF-St5 from Sirulina tropicalis [START_REF] Roelants | Origin and functional diversification of an amphibian defense Peptide arsenal[END_REF] exhibit broad-spectrum antibacterial activities, featuring DMS-DA6 as a promising peptide for the design of new antibiotics, especially against multi-resistant strains.

Amidated peptides are frequently more resistant to enzyme degradation, and also more active than their non-amidated counterparts [START_REF] Dennison | Influence of C-terminal amidation on the efficacy of modelin-5[END_REF][START_REF] Kim | C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria[END_REF][START_REF] Andre | Structure-Activity Relationshipbased Optimization of Small Temporin-SHf Analogs with Potent Antibacterial Activity[END_REF][START_REF] Cerovsky | Lasioglossins: three novel antimicrobial peptides from the venom of the eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae)[END_REF][START_REF] Niklison-Chirou | Microcin J25-Ga induces apoptosis in mammalian cells by inhibiting mitochondrial RNA-polymerase[END_REF][START_REF] Mura | The effect of amidation on the behaviour of antimicrobial peptides[END_REF]. The observation that the amidated form of the DMS-DA6 was invariably 2-to 4-fold more potent than the C-terminal acidic form compelled us to further analyze the functional and structural properties of both forms of DMS-DA6. Our structural studies showed that a small proportion of negatively charged lipids (POPG) sufficed to trigger the transition of both DMS-DA6 peptides from random coil to α-helix, in agreement with the concept that ionic interactions promote folding, as reported for magainins [START_REF] Mura | The effect of amidation on the behaviour of antimicrobial peptides[END_REF][START_REF] Saravanan | Design of short membrane selective antimicrobial peptides containing tryptophan and arginine residues for improved activity, salt-resistance and biocompatibility[END_REF][START_REF] Bechinger | Structure and orientation of the antibiotic peptide magainin in membranes by solid-state nuclear magnetic resonance spectroscopy[END_REF]. Interestingly, unlike other antimicrobial peptides with low cytotoxicity on mammalian cells [START_REF] Nguyen | The expanding scope of antimicrobial peptide structures and their modes of action[END_REF], the proline and glycine residues in DMS-DA6 do not disrupt the helical fold of the membrane-bound structure. However, there was no difference in helix formation or stability in bacterial culture medium between the two variants of DMS-DA6.

As the bacterial membrane leakage experiments pointed towards the membrane bilayer as the primary target for the DMS-DA6 peptides, we investigated whether the two forms of the peptide interacted differently with model membranes, using lipids extracted from microorganisms alongside with synthetic lipid models. The results from the dextran leakage experiments were in line with the bacterial membrane leakage experiments, suggesting that both forms of the DMS-DA6 peptide produce pores in the membrane, large enough to let 10 kDa dextran or β-galactosidase leak through, in a concentration-dependent manner. Furthermore, data of the tryptophan fluorescence done with LUVs made with Gram-positive membrane lipids indicate that the tryptophan residue transitions from a polar environment to a non-polar-one, suggesting increasing contacts of both forms of the DMS-DA6 peptide with the lipid chains. The smaller blue-shift observed in the presence of E. coli lipids LUVs (around 10 nm) is consistent with a partial insertion of DMS-DA6 into this vesicle type, further accounting for the selectivity towards Gram-positive bacteria. Overall, DSC experiments confirmed that both DMS-DA6 peptides could insert into and disorganize a membrane composed of anionic lipids. However, the greater interaction and insertion of the carboxyamidated form of DMS-DA6 was not dependent on the nature of the lipids or on the negative charge of the membrane, as supported by the low toxicity of either form of DMS-DA6 on negatively charged mammalian cancer cells.

As proposed by Brannan et al. [START_REF] Brannan | Differential scanning calorimetry of whole Escherichia coli treated with the antimicrobial peptide MSI-78 indicate a multi-hit mechanism with ribosomes as a novel target[END_REF], many AMPs must bind to non-lipid components of the cell to account for the discrepancy between the amounts of peptide needed to observe disruption in model membranes versus bacteria. Indeed, the relative leakage induced in model lipid membranes by different AMPs does not systematically correlate with their relative capacity to prevent bacterial growth [START_REF] Wimley | Describing the mechanism of antimicrobial peptide action with the interfacial activity model[END_REF], suggesting that AMPs may have alternate modes of action. Following the recent observations of Malanovic and coworkers proposing that peptidoglycan acts as a mesh that could enhance the activity of the AMP OP-145 [START_REF] Malanovic | Gram-positive bacterial cell envelopes: The impact on the activity of antimicrobial peptide[END_REF][START_REF] Malanovic | Phospholipiddriven differences determine the action of the synthetic antimicrobial peptide OP-145 on Gram-positive bacterial and mammalian membrane model systems[END_REF], we assessed the possibility that peptidoglycan may also contribute to the ability of the DMS-DA6 peptides to disrupt Gram-positive bacterial membranes. In the presence of peptidoglycan, the membrane perturbation induced by either form of DMS-DA6 was considerably more severe than in the absence of peptidoglycan, even at the lower 1/100 P/L ratio. Furthermore, at this peptide/lipid ratio, DMS-DA6-NH 2 was significantly more active than DMS-DA6-OH to induce lateral segregation. As the P/L ratio increased, the difference between the two forms of the peptide tended to diminish, with DMS-DA6-NH 2 being consistently more effective than DMS-DA6-OH at intermediate concentrations. Moreover, experiments with Enterococcus faecium BM4147, a peptidoglycan modified strain [START_REF] Bugg | Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA[END_REF], resulted in greater antimicrobial activities in comparison with a wild-type E. faecalis strain (Table 2). This is particularly interesting as the PGN modification in E. faecium BM4147 leads to vancomycin resistance. Consistent with a recent report [START_REF] Neelay | Antimicrobial peptides interact with peptidoglycan[END_REF] showing that melittin and cecropin A interact with peptidoglycan, our data strongly support a role for peptidoglycan as a potentiator of DMS-DA6-NH 2 activity, likely by trapping the peptide close to the bacterial membrane, increasing its local concentration, thus amplifying its action. Since the C-terminal part of both DMS-DA6 peptides is highly flexible, the carboxylate moiety could act as a repulsive group and curb binding of DMS-DA6-OH to PGN, a situation not occurring for DMS-DA6-NH 2 . How DMS-DA6 peptides could interact with PGN at a molecular level remains to be investigated.

In conclusion, DMS-DA6 is a promising new AMP, highly specific against Gram-positive bacteria and devoid of hemolytic activity. We demonstrated that in addition to its interaction with the bacterial membrane lipids, DMS-DA6 also interacts with peptidoglycan, a major component of the membrane of Gram-positive bacteria, possibly explaining the specificity of DMS-DA6 for Gram-positive bacteria. Notably, the carboxyamidated form of the peptide exhibited enhanced permeabilization capability resulting from a better capacity to interact with peptidoglycan, potentiating its anti-bacterial efficiency. All these particular features make this non-conventional Dermaseptin a good candidate for developing new therapeutic peptides against multiresistant bacteria.

Table 1 .

 1 Sequence, net charge at pH 7.4 and N-and C-terminus structures of DMS-DA6-OH and DMS-DA6-NH 2 /doi.org/10.1371/journal.pone.0205727.t001 min -1 with a 35-60% linear gradient of solvent B for 10 min. Peptide purity was assayed by analytical HPLC (C8 Ace, 300Å, 5 μm, 250 × 4.6 mm, Ait France). Analytical chromatograms are shown in S2 Fig. Identities of the peptides were confirmed by MALDI-TOF mass spectrometry (MS Voyager Applied Biosystems). MS spectra of the purified peptides are shown in S3 Fig. Considering the presence of one tryptophan residue in the sequence, concentrations were determined by UV spectroscopy (Nanodrop, Fischer Scientific), assuming an extinction coefficient for Tryptophan ε 280 = 5 600 M -1 .cm -1 .

  of DMS-DA6 to permeabilize the Gram-positive S. aureus membrane. As shown in Fig 1A, ONPG hydrolysis reached a plateau within 5 hours at the highest concentration (50 μM) of either form of the peptide. However, in consonance with the higher antibacterial activity of the carboxyamidated analog, ONPG hydrolysis was systematically higher for DMS-DA6-NH 2 at concentrations within the 1.75-25 μM range, as illustrated in Fig 1B. The maximal absorption intensity observed after 240 minutes for DMS-DA6-NH 2 was reached at a concentration of 12.5 μM, which corresponds to the minimal bactericidal concentration (MBC) determined for S. aureus ST 1065 (Table

  The non-small-cell lung cancer cell lines A549 were incubated in the presence of DMS-DA6-OH or DMS-DA6-NH 2 . As shown in Fig 2, even after a 2-hour incubation with concentrations as high as 100 μM, neither DMS-DA6-OH (net charge +2) nor DMS-DA6-NH 2 (net charge +3) resulted in the death of the negatively charged cancer cells.

Fig 1 .

 1 Fig 1. Membrane permeabilization of S. aureus ST 1065 as observed by ONPG hydrolysis. (A) Kinetics of bacterial membrane leakage after treatment with increasing concentrations (μM) of DMS-DA6-OH (dashed lines) and DMS-DA6-NH 2 (solid lines). (B) The membrane leakage was followed by measuring ONPG hydrolysis at 420 nm after 240 min incubation with increasing concentrations of peptides. Data shown are representative of two independent experiments carried out in triplicate. https://doi.org/10.1371/journal.pone.0205727.g001

Fig 2 .Fig 3 .

 23 Fig 2. The DMS-DA6 peptides do not induce lysis of the negatively charged cancer cell line A549. Cells were incubated for 2 hours at 37˚C, 5% CO 2 in the presence of increasing concentrations of the peptides and the percentage of living cells was determined by trypan blue exclusion. Experiments were done in triplicate. Data represent the mean +/-SEM of 3 independent experiments run in triplicates. https://doi.org/10.1371/journal.pone.0205727.g002

Fig 4 .

 4 Fig 4. Far-UV CD spectra of DMS-DA6-OH (A) and DMS-DA6-NH 2 (B) in phosphate buffer (-), POPG LUVs (---), POPC LUVs (-), and POPG/POPC (1:4) LUVs ( . . . . . . . ). (C) Conformations of DMS-DMS-DA6-OH and DMS-DA6-NH 2 in the presence of various lipid vesicles as calculated from CD experiments. Data shown are representative of at least three different experiments. https://doi.org/10.1371/journal.pone.0205727.g004

Fig 5 .

 5 Fig 5. NMR structure of DMS-DA6 bound to SDS micelles. (A) Backbone superimposition of the 12 lowest-energy conformers over residues 3-21. (B) Ribbon representation of the central well-defined region of DMS-DA6 lowest-energy conformer. Side-chains of residues forming the hydrophobic and polar faces are represented by sticks. (C) Use of a paramagnetic reagent to probe the positioning of DMS-DA6 with respect to the micelle surface. Paramagnetic relaxation enhancements (PREs) induced by gadodiamide were measured as Hα relaxivities (differences in R 1 longitudinal rates per mM concentration of gadodiamide). https://doi.org/10.1371/journal.pone.0205727.g005

Fig 6 .

 6 Fig 6. Overlay of 2D 1 H-1 H NOESY spectra recorded at 500 MHz on DMS-DA6-OH (red) and DMS-DA6-NH 2 (blue) in the presence of SDS micelles. Additional cross-peaks corresponding to the correlations between the two protons of the Cterminus amide group in DMS-DA6-NH 2 are indicated by a black square. https://doi.org/10.1371/journal.pone.0205727.g006

Fig 7 .Table 3 .

 73 Fig 7. Perturbation of membranes or lipids induced by interaction with DMS-DA6 peptides. (A) Lipid-induced change in DMS-DA6 tryptophan fluorescence. Blue shift (Δλ max ) for tryptophan in the wavelength of maximal emission in the presence of LUVs of liposomes produced from E. coli phospholipids (black) or from S. epidermidis phospholipids (grey) in the presence of DMS-DA6-NH 2 ( __ ) or DMS-DA6-OH (---). (B) Dextran leakage from DOPG/CL LUVs after treatment with increasing concentrations of DMS-DA6. (C) and (D) DSC themograms obtained after addition of DMS-DA6-OH (C) or DMS-DA6-NH 2 (D) to DMPG LUVs. Data shown are representative of 3 different experiments. https://doi.org/10.1371/journal.pone.0205727.g007

position 3

 3 and a conserved motif (AAXKAALXA) in the central region of the molecule[START_REF] Conlon | Structural diversity and species distribution of host-defense peptides in frog skin secretions[END_REF], DMS-DA6 lacks the alanine-rich central motif. Furthermore, in contrast to a previous publication reporting that the C-terminus of DMS-DA6 was amidated (DMS-DA6-NH 2 )[START_REF] Meneses | Peptidomic analysis of the skin secretions of the frog Pachymedusa dacnicolor[END_REF], our sequencing data unambiguously supported a free carboxyl terminus, thus suggesting that these two versions of DMS-DA6 may naturally co-exist in the frog exudate.

Fig 8 .

 8 Fig 8. PGN enhances the membrane permeabilization capacity of DMS-DA6. (A) Thermograms of DMPG LUVs according to PGN concentration. (B) Thermograms showing the effect of the addition of DMS-DA6 peptides to DMPG/PGN LUVs at P/L = 1/100. (C) Transition enthalpy obtained by DSC for the "peptide-poor" (white) and "peptide-rich" (dashed) contributions to the main transition peak for the interaction of DMS-DA6 peptides with DMPG/PGN LUVs. The bars represent mean ± SEM, n � 3. Significance was tested using a t-test, � , p = 0.0042. https://doi.org/10.1371/journal.pone.0205727.g008

  Gram-negative bacterial strains, Escherichia coli ATCC 8739, Escherichia coli ATCC 35218, Escherichia coli ML 35p, Escherichia coli K12, Pseudomonas aeruginosa ATCC 27853, and Gram-positive bacterial strains, Enterococcus faecalis ATCC29212, Kocuria rhizophila ATCC 9341, Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 29213, Staphylococcus aureus ST 1065, Staphylococcus epidermidis BM 3302, and Bacillus subtilis subsp. subtilis CIP 52.65, as well as two multiresistant strains (Staphylococcus aureus DAR 5829 (http://www.ncbi. nlm.nih.gov/bioproject/228435) and Enterococcus faecium BM 4147

Table 2 . Antimicrobial and hemolytic activities of DMS-DA6 peptides.

 2

		Bacterial strain	Antimicrobial activity
			(MIC and MBC) (μM)
				DMS-DA6-OH	DMS-DA6-NH 2
			MIC	MBC	MIC	MBC
	Gram-negative	E. coli ML 35p	>100		>100
		E. coli ATCC 35218	>100		>100
		E. coli ATCC 8739	50	100	25	50
		E. coli K12	>100		>100
		Ps. aeruginosa ATCC 27853	>100		>100
	Gram-positive	S. epidermidis BM3302	25	25	12.5	12.5
		K. rhizophila ATCC 9341	6.25	12.5	3.12	3.12
		B. subtilis CIP 52.65	12.5	12.5	6.25	6.25
		S. aureus ST 1065	25	25	12.5	12.5
		S. aureus ATCC 29213	6.25	25	3.12	6.25
		S. aureus ATCC 6538	12.5	12.5	6.25	6.25
		S. aureus DAR 5829 �	50	50	25	50
		E. faecalis ATCC 29212	100	100	50	50
		E. faecium BM 4147 �	50	100	25	25
					Hemolysis (%)
				DMS-DA6-OH	DMS-DA6-NH 2
	Human	10 μM		0.9 ± 1.5		1.2 ± 1.8
		50 μM		7.4 ± 5.5		3.7 ± 3.3
	Mouse	10 μM		0 ± 2.8		0 ± 1.1
		50 μM		5.4 ± 3.5		4.7 ± 5.6

The MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration) values are averaged over three independent experiments or more performed in triplicate. Strains with � are multi-resistant strains. Hemolytic activities were obtained by incubation of human or mouse erythrocytes in PBS with two concentrations of both peptides. The hemoglobin release was monitored by measuring the absorbance at 550 nm (Mean of 2 experiments). https://doi.org/10.1371/journal.pone.0205727.t002

Table 4 . Thermodynamic parameters obtained by DSC corresponding to DMS-DA6 peptides interacting with DMPG LUVs at different peptide to lipid ratios. T m1 (˚C) ΔH m1 (kJ/mol) T m2 (˚C) ΔH m2 (kJ/mol)

 4

	Pure DMPG	24.1 ± 0.1	24.7 ± 1.3	-	-
	+ DMS-DA6-OH, 1/100	24.0 ± 0.2	28.2 ± 1.5
	+ DMS-DA6-OH, 1/50	25.9 ± 0.3	18.5 ± 2.5	23.3 ± 0.3	5.4 ± 1.2
	+ DMS-DA6-OH, 1/25	27.8 ± 0.6	11.5 ± 2.3	14.7 ± 3.3	2.6 ± 1.8
	+ DMS-DA6-NH 2 , 1/100	23.9 ± 0.2	29.6 ± 3.7	-	-
	+ DMS-DA6-NH 2 , 1/50	25.8 ± 0.1	15.7 ± 1.6	22.9 ± 0.6	5.6 ± 2.2
	+ DMS-DA6-NH 2 , 1/25	28.0 ± 0.4	9.6 ± 1.6	16.1 ± 4.0	2.0 ± 0.3
	https://doi.org/10.1371/journal.pone.0205727.t004

Table 5 . Thermodynamic parameters obtained by DSC corresponding to DA6 peptides interacting with DMPG LUVs + PGN at different peptide to lipid ratios.

 5

		T m1 (˚C)	ΔH m1 (kJ/mol)	T m2 (˚C)	ΔH m2 (kJ/mol)
	DMPG + PGN	24.61 ± 0.06	19.4 ± 0.9	-	-
	+ DMS-DA6-OH, 1/100	25.55 ± 0.05	13.9 ± 1.3	23.3 ± 0.2	3.9 ± 0.2
	+ DMS-DA6-OH, 1/50	28.7 ± 1.8	9.7 ± 0.9	18.3 ± 3.9	3.3 ± 1.5
	+ DMS-DA6-OH, 1/25	29.9 ± 3.1	5.8 ± 1.7	14.2 ± 2.9	3.0 ± 0.5
	+ DMS-DA6-NH 2 , 1/100	26.8 ± 0.2	11.3 ± 0.9	23.6 ± 0.2	7.2 ± 0.5
	+ DMS-DA6-NH 2 , 1/50	28.3 ± 0.3	10.6 ± 0.6	21.4 ± 0.5	4.2 ± 0.5
	+ DMS-DA6-NH 2 , 1/25	27.9 ± 0.2	6.6 ± 0.4	11.2 ± 0.2	3.7 ± 0.4

https://doi.org/10.1371/journal.pone.0205727.t005

PLOS ONE | https://doi.org/10.1371/journal.pone.0205727October 16, 2018

Acknowledgments

The authors are grateful to Dr. C. El Amri and Dr. L. Kemte ´mourian for help with CD experiments and interpretations, to Dr. S. Cribier for useful advice on dextran leakage assays, to Dr. N. Rodriguez for help with fluorescence experiments, and to mass spectrometry and proteomics platform Sorbonne Universite ´, IBPS FR3631. The authors thank Dr. S. Rebuffat and Dr. S. Zirah (Muse ´um d'Histoire Naturelle, Paris) for providing the K. rhizophila ATCC 9341, S. aureus ATCC 6838, B. subtilis CIP 52.65, E. coli ATCC 8739, E. coli ML35p, Ps. aeruginosa ATCC 9027 strains, and Dr. T. Masdek (Institut Pasteur, Paris) for the S. aureus ST 1065 strain. They also thank G. Tong, M. Gropp, M. Goderon, and H. Levy for technical assistance and Dr. C. Byrne for careful reading of the manuscript.

This work was supported by Grant number: 2015-02-853, Fronteras de la Ciencia 2015-2 issued by CONACYT (Consejo Nacional de Ciencia y tecnologia), Mexico, https://www. conacyt.gob.mx/, to YR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All relevant data are within the paper and its Supporting Information files.

Supporting information


Alexandre Bruckert

Mona Abid

Matthieu Perreira Da Silva

Patrick Le Callet

COULD THE BUBBLEVIEW METAPHOR BE USED TO INFER VISUAL ATTENTION ON 3D GRAPHICAL CONTENT ?

Keywords: Visual attention, eye-tracking, visual saliency, 3D graphical objects

Understanding the deployment of human gaze on 3D graphical objects is of critical importance in order to propose rich and complex 3D environments without strong latency nor rendering constraints. However, the data needed to study this gaze deployment can be costly and difficult to obtain, especially in the context of the Covid-19 pandemic where in-lab experiments are strongly discouraged. In order to alleviate these issues, we propose to use the BubbleView metaphor as a way of crowdsourcing visual attention data on 3D graphical content. In this paper, we question the adequacy of this method to provide a reliable proxy for visual attention in the context of 3D graphical objects. Moreover, we show that data obtained in this manner can be used to train visual saliency models, with only a slight tradeoff in performances compared to the use of ground-truth eye-tracking data.

INTRODUCTION

Three-dimensional graphics have taken a prominent place in numerous fields of applications, including video games, digital animation or scientific simulation. However, as these graphical content become more detailed and complex, compression and simplification operations are critically needed to ensure that 3D objects are displayed to the users without rendering nor latency issues. In order to ensure an optimal quality of experience (QoE) for the user, visual attention information is often used, as it gives an indication about the perceived experience. For instance, visual attention can be used to infer the memorability of an image [START_REF] Mancas | Memorability of natural scenes: The role of attention[END_REF], or to spatially guide the compression of an image or video [START_REF] Zhu | Video saliency aware intelligent hd video compression with the improvement of visual quality and the reduction of coding complexity[END_REF].

In this context, visual saliency, which refers to the distribution of eye fixations on a given stimulus, is a particularly interesting feature to study. Indeed, predicting this distribution allows to preserve visually important areas during compression. Consequently, numerous models dedicated to predict this distribution have been proposed in the literature [START_REF] Borji | Saliency prediction in the deep learning era: Successes and limitations[END_REF]. While most of the effort has been concentrated on 2D images or videos, only a few studies explored visual saliency on 3D meshes [START_REF] Lee | Mesh saliency[END_REF][START_REF] Song | Mesh saliency via spectral processing[END_REF][START_REF] Abid | Towards visual saliency computation on 3d graphical contents for interactive visualization[END_REF] or point clouds [START_REF] Akman | Computing saliency map from spatial information in point cloud data[END_REF][START_REF] Ding | Point cloud saliency detection by local and global feature fusion[END_REF].

However, gathering visual saliency data is expensive in terms of resources, as it requires costly eye-tracking equipment and human supervision during test sessions, and thus prevents the collection of large-scale datasets needed to train deep learning models. In order to address this problem, several alternatives for in-lab eye-tracking experiments have been proposed [START_REF] Newman | Turkeyes: A web-based toolbox for crowdsourcing attention data[END_REF].

Webcam-based eye-tracking [START_REF] Hennessey | A single camera eye-gaze tracking system with free head motion[END_REF] was proposed as a direct way to collect gaze data through crowdsourcing, by following the pupil of an observer captured through their webcam. However, it appeared to be quite difficult to gather reliable data with this method, due to important variations of the quality of the equipment, lighting conditions, or the positions of the observers.

Auto-annotation methods rely on the viewer's selfassessment of where gazed-upon areas are located, usually by asking participants to paint or to click over the interesting regions [START_REF] O'donovan | Learning layouts for single-page graphic designs[END_REF][START_REF] Cheng | Social eye tracking: Gaze recall with online crowds[END_REF]. These methods however generate a lot of noise because of the intra-observer diversity in annotation style, and the top-down nature of the collected ground-truth.

Zooming interactions [START_REF] Carlier | Cozi: Crowdsourced and content-based zoomable video player[END_REF] consists in giving the observer an interactive zooming tool, and treating the zoomupon areas as areas of interest, from which visual attention heatmaps can be drawn. However, this methods is more suited to multi-scale content.

Mouse tracking methods rely on the assumption that gaze and mouse tracks are highly correlated, especially on specific stimuli where a mouse cursor is expected by the participant, such as web pages [START_REF] Rodden | Eye-mouse coordination patterns on web search results pages[END_REF][START_REF] Huang | User see, user point: Gaze and cursor alignment in web search[END_REF]. The BubbleView metaphor is another form of mouse-tracking proxy for visual attention: the image is entirely blurred, except for an area around the mouse cursor. This unblurred region can either be displayed by clicking [START_REF] Kim | Bubbleview: An interface for crowdsourcing image importance maps and tracking visual attention[END_REF], or continuously follow the mouse cursor [START_REF] Ellahi | Evaluation of the bubble view metaphor for the crowdsourcing study of visual attention deployment in tone-mapped images[END_REF]. Finally, the BubbleView metaphor shows very good correlation with eye-tracking data on various content, and allows for the collection of very large visual saliency datasets, such as SALICON [START_REF] Jiang | Salicon: Saliency in context[END_REF]. In this work, we propose to evaluate the BubbleView metaphor for crowdsourcing visual attention data on 3D graphical objects. We introduce a novel dataset composed of mouse tracks on high resolution 3D objects collected under laboratory conditions. We then compare the mouse tracks to ground truth eye-tracking data. Finally, we retrain several visual saliency models using these Fig. 1: Examples of the rendered stimuli two datasets, in order to assess the interest of the BubbleView metaphor for predicting visual attention on 3D graphical content.

DATA COLLECTION AND PROCESSING

Stimuli

We build our dataset as an extension of the 3DGC saliency dataset [START_REF] Abid | Perceptual characterization of 3d graphical contents based on attention complexity measures[END_REF]. We selected 25 high-resolution 3D objects, with various semantics and shapes, and selected four viewpoints to render them, using the same method as for the 3DGC data (i.e. considering four views corresponding to four faces of a cube), thus yielding a total of 100 images. In order to avoid any bias due to the occupancy of the objects, we applied a gray background behind them. Figure 1 shows an example of such images.

Eye-tracking data

Participants and task: In order to build an eye-tracking ground-truth, we collected data from 34 volunteers. Participants were asked to freely explore each stimulus, without any specific task. A trial session was performed, in order to ensure that the participants understood the procedure.

Apparatus and protocol conditions: We used the Eyelink 1000 Plus eye-tracker, sampling at 1000Hz, in remote mode (i.e. using no chin rest). Stimuli were displayed in a random order, at full HD resolution (1920×1080) during three seconds, on a DELL P2417H monitor. The distance between the eyes of the observers and the screen was set at 96cm (± 1cm). In this setting, one degree of visual angle amounts to roughly 64 pixels. The screen luminance was set at 200 cd/m², and the luminance for the room's walls was measured at 30 cd/m².

Data processing: In order to transform raw gaze points into fixations, we applied a threshold-based aggregation algorithm using motion, velocity and acceleration [START_REF] Salvucci | Identifying fixations and saccades in eye-tracking protocols[END_REF], and removed all fixations lasting less than 80ms, i.e. roughly the minimum amount of time to process foveal information. Visual saliency maps were obtained by aggregating the fixation points over all observers, and convolving them with a 2D Gaussian kernel, which standard deviation was set to one degree of visual angle, which is approximately the radius of the fovea.

A mask was then applied in order to only consider the surface of the rendered object, and not the background. To take into account gaze data at the border of the surface, the size of the mask was enlarged using a morphological dilation, similar to the process in [START_REF] Abid | Perceptual characterization of 3d graphical contents based on attention complexity measures[END_REF].

BubbleView data

In this study, we chose to use the BubbleView method in a continuous way, not relying on clicks, in order to grasp both bottom-up and top-down processes. This way, the whole image is blurred, except a circular region around the center of the mouse.

BubbleView parameters: Several parameters are required to set up the BubbleView metaphor. First, the size of the bubble needs to be set at approximately the size of the fovea [START_REF] Kim | Bubbleview: An interface for crowdsourcing image importance maps and tracking visual attention[END_REF]. The blur sigma also needs to be set such that some details are visible to attract the attention towards different regions, but not too many details, to force exploration using the bubble. Finally, the display time of the stimulus is also important, in order to have a balance between top-down and bottom-up processes as close as the one in the eye-tracking study.

The size of the bubble was set to 1.5°of visual angle, i.e. 96 pixels. For the blurring parameter, we applied a homogeneous blur, which kernel size is designed to match the blur in the peripheral vision at the edges of the screen when fixating the center. To do so, we use the size of the screen to measure the maximal eccentricities values (i.e. the distance, in degrees of visual angle, from the center of the screen to the edges), and we derive from it the visual acuity, expressed in the Snellen decimal [START_REF] Westheimer | Visual acuity. chapter 17[END_REF]. We can then derive the sigma for the blur necessary to approximate this acuity, 10 pixels in our case.

Finally, the viewing time was set at 4.5 seconds. Between each image, participants were asked to position their mouse cursor on a target at the center of the screen.

Participants: The dataset was split into two playlists, of 50 images each. The 60 participants were split into two group, each group viewing one of the two playlists. Participants were asked to explore freely each stimulus, without any specific task. This experiment was done in a laboratory setting, under supervision, in order to avoid spammers or outliers. The adjustment of this protocol for crowdsourcing settings, i.e. the design of efficient filters to ensure trustworthy data, in a context where the experiment is proposed to a large panel of online participants, will be explored in future works.

Data processing: To infer visual saliency maps from the collected BubbleView data, we followed the work in [START_REF] Ellahi | Evaluation of the bubble view metaphor for the crowdsourcing study of visual attention deployment in tone-mapped images[END_REF]. For each pixel of a stimulus, we compute the total aggregated time during which the mouse cursor was at this specific location. The resulting map was then convolved with a 2D Gaussian kernel, emulating the size of the fovea, i.e. 1°of visual angle. Similarly to the eye-tracking saliency maps, a mask was applied and dilated to only account for the surface of the object. Figure 2 shows an example of this process, for both eyetracking and BubbleView data.

EXPERIMENTS

In this section, we propose two different evaluations of the BubbleView metaphor for 3D graphical objects. First, we evaluate how similar the saliency maps generated with the BubbleView paradigm are to the eye-tracking ground-truth; then, we evaluate and compare how visual saliency models trained with BubbleView or eye-tracking perform.

Similarity of the BubbleView metaphor with eyetracking

In order to assess how close BubbleView saliency maps are to the eye-tracking ground-truth, we relied on four metrics commonly used in the context of visual saliency [START_REF] Bylinskii | What do different evaluation metrics tell us about saliency models?[END_REF]: Pearson's correlation coefficient (CC), Normalized Scanpath Saliency (NSS), Kullback-Leilbler Divergence (KLD) and Similarity (SIM). As recommended in [START_REF] Newman | Turkeyes: A web-based toolbox for crowdsourcing attention data[END_REF], we also compared these scores as a percentage of a baseline score. To compute this baseline for the NSS, we used the inter-observer congruency, as described in [START_REF] Bruckert | Deep learning for inter-observer congruency prediction[END_REF]: for each observer, we create a fixation map based only on their fixations, and compared it to the saliency map created by aggregating all of the other observers. Scores were then averaged over observers and images. For CC, KLD and SIM metrics, the IOC baseline is computed by randomly spliting the observers into two groups, and comparing the saliency maps of both groups. The percentage of the IOC is then given by normalizing the scores with the baselines [START_REF] Newman | Turkeyes: A web-based toolbox for crowdsourcing attention data[END_REF]. Table 1 shows these similarity scores. Overall, we observe very high similarity compared to scores usually obtained on natural images [START_REF] Newman | Turkeyes: A web-based toolbox for crowdsourcing attention data[END_REF][START_REF] Ellahi | Evaluation of the bubble view metaphor for the crowdsourcing study of visual attention deployment in tone-mapped images[END_REF]. This can be explained by two factors: the simplicity of the considered stimuli, which do not present any background to explore, thus focusing the mouse of the participant on the content, and the relatively small size of the objects, allowing for a fast first exploration, and time to come back to interesting or salient areas afterwards.

Inter-observer congruency (IOC) is a measure of the similarity of gaze patterns between several observers watching at the same stimulus. In the context of 3D graphical objects and QoE, inter-observer visual congruency is a particularly useful metric. Indeed, it can be used to characterize the visual attention complexity of a rendered view of a 3D object [START_REF] Abid | Towards visual saliency computation on 3d graphical contents for interactive visualization[END_REF], and such views can then be compared together, for instance to infer a preferred point of view.

Therefore, we are interested in knowing whether or not the BubbleView metaphor preserves this congruency. As explained in the previous section, we can compute IOC scores using a leave-one-out approach, comparing each observer with the aggregation of the others. However, while this approach can be replicated on BubbleView tracks, the comparison with eye-tracking scores might be uncertain due to the different nature of data. Since our work here is focused on visual saliency, we use a heuristic by computing Shannon's entropy of the saliency map. This way, when entropy is high, salient areas are dispersed over the whole surface of the object, and thus IOC scores are low, and vice versa.

We computed the entropy of both BubbleView and eyetracking saliency maps for each view of the objects, and compared them together. We obtain a Pearson's correlation coefficient between entropies of 0.886 (p -value ≪ 10 -5 ), showing that the BubbleView metaphor preserves relatively well information about the dispersion of gaze tracks on 3D graphical objects.

Visual saliency models

In order to evaluate the usefulness of BubbleView data for training visual saliency models on 3D graphical content, we selected and retrained three models that are representative of the state-of-the-art: MSINet [START_REF] Kroner | Contextual encoder-decoder network for visual saliency prediction[END_REF], SAM-Resnet [START_REF] Cornia | Predicting Human Eye Fixations via an LSTM-based Saliency Attentive Model[END_REF] and EML-Net [START_REF] Jia | Eml-net: An expandable multi-layer network for saliency prediction[END_REF].

Those three models were evaluated in three different configurations: using their base weights (i.e. pretrained on the SALICON dataset [START_REF] Jiang | Salicon: Saliency in context[END_REF]), fine-tuned on the eye-tracking data, and fined-tuned using the BubbleView data. In each of the fine-tuning conditions, 85 images were randomly chosen to fine-tune the model, and the 15 left were used for validation. Each training was repeated 10 times, with a new random split each time, and the performances averaged, to ensure consistency in the results. The images were resized to fit the original input size of each model, using padding to conserve the aspect ratio. For the SAM-Resnet model, considering the high number of parameters of this model, and the low number of stimuli, we froze the weights of the Resnet-50 feature extractor.

To test these models, we used the 84 images of the 3DGC eye-tracking dataset [START_REF] Abid | Perceptual characterization of 3d graphical contents based on attention complexity measures[END_REF]. As described in Section 2.2, we apply a dilated mask to only keep the saliency values on the surface and on the border of the object.

The predicted saliency maps are then scored against the eye-tracking ground-truth using the same four metrics as in Section 3.1. The results for each metric, model and training setting are summarized in Table 2.

Overall, EML-Net gives the best results, while SAM-Resnet exhibits the lowest performances. All of the models, when used in their basic settings, show relatively poor performances (under 1.5 in NSS, and under 0.7 in CC). This is easily explained by the nature of the SALICON dataset, which is composed of natural images, and not 3D graphical content.

As expected, fine-tuning on the eye-tracking dataset shows a significant improvement in performances compared to the base model, even with the low amount of training data that we used. This procedure allows the models to adjust their weights to the specific features of the considered content, but also to the properties of the ground-truth saliency maps, which can slightly differ due to the diversity of experimental conditions in which eye-tracking data are collected.

Interestingly, we observe that for the three models, finetuning on the BubbleView dataset also significantly improves the performances, although not as much as the eye-tracking data. Nonetheless, it seems that BubbleView data can be successfully used to improve the prediction of visual saliency on 3D graphical objects.

Similarly to Section 3.1, we also evaluated the performances of the models in each setting to predict the visual attention complexity. We compared the entropy of the predicted saliency map to the ground truth using Pearson's correlation 3 compiles these scores. The EML-Net model shows the highest correlation, especially when finetuned using eye-tracking data (CC = 0.88). We also observe a systematic improvement of the correlation when any kind of fine-tuning is used, including with the BubbleView data.

CONCLUSION

In this work, we explored the BubbleView metaphor's ability to be a reliable proxy for gathering visual attention data on 3D graphical objects. Based on attention data gathered with both eye-tracking and BubbleView experiments, we showed strong similarities between both collecting methods in the considered features, namely the visual saliency and the visual attention complexity. It should be noted that 3D graphical objects, due to their nature, are particularly good candidates for the BubbleView metaphor: indeed, their simplicity and the absence of background allows the observers to explore the stimuli very quickly and easily, which is not always the case with natural images, for instance. We also showed that Bub-bleView data can be reliably used to fine-tune visual saliency models in this context, and can improve the prediction of visual attention complexity.

Considering the high cost of eye-tracking experiments, it appears that BubbleView can be a good cost-effective alternative for 3D graphical content. However, it remains to evaluate how this metaphor would perform outside of the lab, with different devices and an increased probability of spammers.

Fig. 2 :

 2 Fig. 2: Example of eye-tracking and BubbleView tracks transformed into saliency maps. (a) Original stimulus, (b) Eye fixation points, (c) Ground-truth visual saliency map, (d) Bub-bleView tracks, (e) BubbleView saliency map.

Table 1 :

 1 Average similarity scores (± standard deviation) between BubbleView saliency maps and ground-truth eyetracking data. First line is the score between the BubbleView map and the eye-tracking ground-truth, and second line is the same score as a percentage of the IOC. CC ↑ NSS ↑ KLD ↓ SIM ↑ 0.90 ± 0.05 2.90 ± 0.57 0.19 ± 0.09 0.79 ± 0

Table 2 :

 2 Scores of the three visual saliency models on the test dataset. Best performances are bolded. For each model, three settings are compared: base, BubbleView fine-tuned (BV) and eye-tracking fine-tuned (ET). CC ↑ NSS ↑ KLD ↓ SIM ↑

		Base 0.582 1.023	0.527 0.621
	SAM-Resnet	BV	0.679 1.215	0.463	0.632
		ET	0.711 1.320	0.401 0.649
		Base 0.641 1.289	0.368	0.633
	MSINet	BV	0.690 1.587	0.303	0.650
		ET	0.724 1.613	0.287	0.675
		Base 0.684 1.432	0.239	0.645
	EML-Net	BV	0.741 1.721	0.207	0.678
		ET	0.758 1.932	0.196	0.691

Table 3 :

 3 Pearson's correlation coefficient between the en-

	tropies of ground-truth and predicted saliency maps, for base
	model (B), BubbleView (BV) and eye-tracking (ET) fine-
	tuned.
	SAM-Resnet		MSI-Net			EML-Net
	B	BV	ET	B	BV	ET	B	BV	ET
	0.52 0.63 0.68 0.75 0.81 0.85 0.82 0.86 0.88
	coefficient. Table