domain: chim sde sdv

text:
Kiran Kumar

Mathew Sebastiao

Alexandre A Arnold

Steve Bourgault

Dror E Warschawski
email: dror.warschawski@sorbonne-universite.fr

Isabelle Marcotte
email: marcotte.isabelle@uqam.ca

IN SITU SOLID-STATE NMR STUDY OF ANTIMICROBIAL PEPTIDE INTERACTIONS WITH ERYTHROCYTE MEMBRANES

Antimicrobial peptides stand as promising therapeutics to mitigate the global rise of antibiotic resistance. They generally act by perturbing the bacterial cell membrane, and are thus less likely to induce resistance. Since they are membrane-active molecules, it is critical to verify and understand their potential action towards eukaryotic cells to help design effective and safe drugs. In this work, we studied the interaction of two antimicrobial peptides, aurein 1.2 and caerin 1.1, with red blood cell (RBC) membranes using in situ 31 P and 2 H solid-state nuclear magnetic resonance (SS-NMR). To do so, we established a protocol to integrate up to 25% of deuterated fatty acids (FAs) in the membranes of ghosts, which are obtained when hemoglobin is removed from RBCs. The FA incorporation and the integrity of the lipid bilayer were confirmed by SS-NMR and fluorescence confocal microscopy. Leakage assays were performed to assess the lytic power of the AMPs. The in situ perturbation of the ghost membranes by aurein 1.2 and caerin 1.1 revealed by 31 P and 2 H SS-NMR is consistent with a membrane perturbation through a carpet mechanism for aurein 1.2, while caerin 1.1 would act on RBCs via pore formation. These results are compatible with fluorescence microscopy images of the ghosts. The peptides interact with eukaryotic membranes following similar mechanisms that take place in bacteria, thus highlighting the importance of hydrophobicity in determining such interactions. Our work bridges model membranes and in vitro studies and provides an analytical toolbox to assess drug toxicity towards eukaryotic cells.

INTRODUCTION

Antimicrobial resistance affects the lives of many people around the world, and for this reason, the World Health Organization has recently published its first list of antibiotic-resistant bacteria that should be prioritized in the development of new treatments [START_REF]Global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics[END_REF]. New drug candidates with novel action mechanisms must be urgently proposed [START_REF]Antimicrobial resistance global report on surveillance[END_REF], and in this regard, antimicrobial peptides (AMPs) are a promising therapeutic alternative that could be submitted to advanced chemical engineering to become new antimicrobial drugs [START_REF] Koehbach | The vast structural diversity of antimicrobial peptides[END_REF]. The antimicrobial activity of AMPs involves different possible action mechanisms that cause bacterial cell death, including perturbation of the plasma membrane [START_REF] Booth | Recent progress on the application of 2 H solid-state NMR to probe the interaction of antimicrobial peptides with intact bacteria[END_REF][START_REF] Yeaman | Mechanisms of antimicrobial peptide action and resistance[END_REF]. Because these mechanisms are entirely distinct from those of current clinically-used antibiotics, there is great interest in their development as therapeutics against antibiotic-resistant bacterial infections [START_REF] Hancock | The role of antimicrobial peptides in animal defenses[END_REF]. Besides their direct cytotoxicity towards bacteria and fungi, several AMPs exhibit antiviral [START_REF] Mookherjee | Antimicrobial host defence peptides: functions and clinical potential[END_REF], antiparasitic [START_REF] Mor | Multifunctional host defense peptides: antiparasitic activities[END_REF] and anticancer properties [START_REF] Hoskin | Studies on anticancer activities of antimicrobial peptides[END_REF]. Those biologically-active candidates are not only able to kill bacteria but also to modulate host immunity [START_REF] Umnyakova | Human antimicrobial peptides in autoimmunity[END_REF] since AMPs have evolved as integral components of strategic and carefully regulated mechanisms of immunity against infection [START_REF] Yeaman | Mechanisms of antimicrobial peptide action and resistance[END_REF].

In general, the outer surface of Gram(+) and Gram(-) bacteria contain peptidoglycan, and either teichoic acids or lipopolysaccharides [START_REF] Lambert | Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria[END_REF][START_REF] Costerton | The role of bacterial surface structures in pathogenesis[END_REF], each conferring a net negative charge. In addition, the outer leaflet of the lipid bilayer in bacterial membranes often contains zwitterionic phosphatidylethanolamine (PE) as well as negatively-charged lipids such as phosphatidylglycerol (PG) and cardiolipin (CL) [START_REF] Zhao | Atomicscale structure and electrostatics of anionic palmitoyloleoylphosphatidylglycerol lipid bilayers with Na + counterions[END_REF][START_REF] Guilhelmelli | Antibiotic development challenges: the various mechanisms of action of antimicrobial peptides and of bacterial resistance[END_REF], thus promoting electrostatic interactions with cationic AMPs [START_REF] Schmidtchen | Effect of hydrophobic modifications in antimicrobial peptides[END_REF]. In contrast, the outer leaflet of most mammalian cell membranes, including the erythrocyte membrane, mainly consists of zwitterionic phospholipids such as phosphatidylcholine (PC) and sphingomyelin (SM), as well as cholesterol [START_REF] Wessels | Some aspects of the osmotic lysis of erythrocytes III. Comparison of glycerol permeability and lipid composition of red blood cell membranes from eight mammalian species[END_REF]. These fundamental differences between bacterial and eukaryotic membrane compositions should offer some specificity. Although many factors can influence this selectivity, including molecular shapes of both lipids and peptides [START_REF] Bechinger | The SMART model: Soft Membranes Adapt and Respond, also Transiently, in the presence of antimicrobial peptides[END_REF], it has been proposed that charged peptides would preferentially interact and affect the microbial membrane integrity with higher potency than they do eukaryotic membranes [START_REF] Aisenbrey | The mechanisms of action of cationic antimicrobial peptides refined by novel concepts from biophysical investigations[END_REF][START_REF] Greco | Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides[END_REF]. One major challenge is to identify AMPs for their potential therapeutic use and selectivity towards bacteria, without affecting eukaryotic cells such as erythrocytes. Many studies have shown the potential of AMPs through their interaction with model membranes [START_REF] Balla | Solid-state NMR study of antimicrobial peptides from Australian frogs in phospholipid membranes[END_REF][START_REF] Marcotte | Interaction of antimicrobial peptides from Australian amphibians with lipid membranes[END_REF][START_REF] Bechinger | The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy[END_REF][START_REF] Fernandez | Membrane defects enhance the interaction of antimicrobial peptides, aurein 1.2 versus caerin 1.1[END_REF]. However, model membranes have limited complexity and therefore overlook potentially important features of the cellular envelope. In this regard, in vivo studies are becoming more widespread, including in-cell solid-state nuclear magnetic resonance (SS-NMR) spectroscopy [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Arnold | Whole cell solid-state NMR study of Chlamydomonas reinhardtii microalgae[END_REF][START_REF] Poulhazan | Unambiguous ex situ and in cell 2D 13 C solid-state NMR characterization of starch and its constituents[END_REF]. In our previous study, aurein 1.2 (GLFDIIKKIAESF-NH 2 ) and caerin 1.1 (GLLSVLGSVAKHVLPHVVPVIAEHL-NH 2 ) -natural AMPs isolated from the skin secretions of Australian tree frogs -were investigated [START_REF] Apponyi | Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance[END_REF][START_REF] Rozek | The antibiotic and anticancer active aurein peptides from the Australian bell frogs Litoria aurea and Litoria raniformis: the solution structure of aurein 1.2[END_REF][START_REF] Wong | The solution structure and activity of caerin 1.1, an antimicrobial peptide from the Australian green tree frog, Litoria splendida[END_REF][START_REF] Wegener | Bioactive dahlein peptides from the skin secretions of the Australian aquatic frog Litoria dahlii: sequence determination by electrospray mass spectrometry[END_REF]. Their interaction with intact Gram(+) and Gram(-) bacteria was studied by in vivo 2 H SS-NMR following specific deuteration of their membrane phospholipid acyl chains [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF].

In this report, we have investigated the action of these two cationic peptides on erythrocyte ghost membranes. The term "ghost" is used to describe the discoid bodies obtained after the removal of hemoglobin from erythrocytes [START_REF] Dodge | The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes[END_REF]. As post-hemolytic remains of RBCs, their cell surface and membrane composition, including lipids, proteins, and other plasma membrane components, has been shown to be similar or even identical to that of intact RBCs [START_REF] Dodge | The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes[END_REF][START_REF] Schwoch | Preparation and properties of human erythrocyte ghosts[END_REF].

Hence ghosts can be used to study the lipid phase, dynamics and interactions in RBC membranes [START_REF] Maraviglia | Human erythrocyte membranes are fluid down to-5° C[END_REF]. Devoid of signal-perturbing hemoglobin or cytoplasmic phosphorus-containing molecules, ghosts are better-suited systems for SS-NMR studies.

We have first optimized and characterized the deuteration of erythrocyte ghosts with efficient incorporation of perdeuterated exogenous palmitic acid (PA-d 31 ) in their membranes. Palmitic acid was chosen since saturated acyl chains with 16 carbons are the most abundant type of fatty acid (FA) chains in erythrocyte lipids (see Fig. 5 in results section) and it is commercially available in its deuterated form. Similar labeling had been attempted in the past by Davis et al. Following the characterization of deuterated ghosts, we have studied the effect of caerin 1.1 and aurein 1.2. Hemolytic and leakage assays were used to determine the lytic action of AMPs on erythrocyte and model membranes. Then 31 P and 2 H SS-NMR and confocal microscopy were employed to investigate the interactions of the peptides with erythrocyte ghost membranes. By performing these experiments at various lipid-peptide molar ratios, an overall picture of peptide interactions with erythrocyte membranes is proposed. Moreover, by determining the conditions in which these peptides perturb the membrane, their relative selectivity towards bacterial membranes is established. Deionized 18.2 MΩ.cm Milli-Q water was used in all experiments (Millipore).

MATERIALS AND METHODS

Materials

Ghost sample preparation and labeling

Ghosts were prepared as previously described [START_REF] Schwoch | Preparation and properties of human erythrocyte ghosts[END_REF] with some modifications. Concentrated horse RBCs were suspended in a 40 mL round bottom centrifugation tube with isotonic HEPES buffer (20 mM HEPES, 150 mM NaCl, pH 7.4), and centrifuged at 500 g for 5 min at 4 o C.

Supplementary washes (2 to 3) in the same buffer were carried out until the supernatant became clear. After the final wash, the pellet was resuspended in 20 mL hypotonic HEPES buffer (20 mM HEPES, pH 7.4) and centrifuged at 25,000 g for 40 min at 4 o C (rotor JA-20, Beckman). The supernatant was then removed, and the pellet transferred into new centrifuge tubes, leaving behind the "red button" that contains proteases. Additional washes (3 to 4) with the same buffer were carried out until the pellet became white.

A 0.5 mM Triton X-100 / 0.

Microscopy sample preparation

Ghosts 1 % (v/v) were labeled with Fast-DiI (1/1000) using 10 rpm longitudinal rotation for 1 h at 37 o C, then pelleted at 16,000 g for 15 min. The pellet was resuspended in 500 µL HEPES buffer then transferred to Sarstedt 8-well microscopy slides (300 µL) and left to stand for 30 min to allow ghosts immobilization. NBD-PAs were incorporated to ghosts along with PA or PAd 31 as described above in section 2.2. After addition of an appropriate amount of AMPs, calcein influx experiments were performed by adding 0.5 mM calcein directly into 1 % (v/v) ghosts.

Images were acquired using a Nikon confocal microscope with a 60x oil-immersion lens and processed using ImageJ software.

Lipid and fatty acid profile

The lipid profile was determined following the procedure of Laydevant et al. [START_REF] Laydevant | Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications[END_REF]. Lipid extraction was performed in triplicate using the Folch protocol [START_REF] Pernet | Effect of ultrasonication and grinding on the determination of lipid class content of microalgae harvested on filters[END_REF], and freeze-dried sample weights were measured using XP205 Mettler Toledo Analytical Balance (up to 0.01 mg readability). Then samples were dissolved in a biphasic solution composed of 200 µL CD 3 OD, 500 µL CDCl 3 (organic phase) and 50 µL of a 200 mM EDTA (acid form) at pH 6.00 ± 0.04 (aqueous phase). Solution NMR spectra were acquired using a 600 MHz Bruker Avance III HD (Milton, Canada) spectrometer equipped with a 5-mm broad band probe, operating at 243 and 600 MHz for 31 P and 1 H, respectively. 1D 31 P NMR spectra were obtained with 1 Samples used for solution NMR analyses were then recovered and prepared for FA analysis by GCMS. Briefly, samples were filtered then transesterification was carried out in 2 mL of H 2 SO 4 (2% in methanol) and 0.8 mL of toluene for 10 min at 100°C.The GC-MS analyses were carried out as described by Laydevant et al. [START_REF] Laydevant | Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications[END_REF] using a polar HP-5MS column (30 m length  250 µm diameter  0.25 µm film thickness). The injection volume was 1 µL and the oven temperature was programmed to heat at 140 °C for 5 min followed by a 4°C/min ramp up to 300°C, prior to electron ionization and detection with a quadrupole MS. Data acquisition and processing were performed with the Chemstation software.

Hemolysis assay

The hemolytic activity of the AMPs was determined by measuring the hemoglobin released from horse RBCs as described previously [START_REF] Greco | Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides[END_REF]. A volume of 500 μL of packed RBCs was centrifuged at 500 g and 4°C for 5 min to recover the erythrocytes, and the supernatant was removed. The pellet was subsequently washed 3 times with 10 mL of PBS isotonic buffer to obtain a clear supernatant. The 30-μL erythrocyte pellet was resuspended in 270 μL of a solution containing the AMPs at the desired concentration (diluted in PBS buffer) to obtain a final concentration of 5 % RBCs (v/v) containing about 5 to 10 × 10 8 cells/mL. The sample was incubated at 37°C for 1 h. The PBS buffer and 1% Triton X-100 solution were respectively used as negative (A 0 ) and positive controls (A 100 ). After a 500 g centrifugation for 5 min, the hemoglobin concentration in the supernatant (diluted to 2% RBCs in PBS buffer) was determined using a UV-visible spectrophotometer by monitoring the optical density at 576 nm.

The percentage of hemolysis was calculated with the following equation: 1)

% hemolysis= A-A 0 A 100 -A 0 × 100 (Equation

Carboxyfluorescein leakage assays

Large unilamellar vesicles (LUVs) that mimic erythrocyte and bacterial membranes were prepared for the carboxyfluorescein (CF) leakage experiments by encapsulating CF into LUVs.

About 20 mM lipids were rehydrated in a buffer (20 mM HEPES at pH 7.4) containing 46 mM CF and multilamellar vesicles (MLV) were prepared using 5 freeze-thaw cycles and a 5-min vortex step each time. LUVs were obtained by extruding the MLV suspension through a polycarbonate filter with 100 nm pores (Avanti Polar Lipids, Alabaster, AL, USA). The CFcontaining vesicles were separated from non-entrapped CF using a Sephadex G25 Fine (20×80 mm) size-exclusion chromatographic column eluted with buffer. The lipid concentration of separated LUVs was determined by a phosphorus assay in triplicates. The 12.5 μL of the liposome suspension of each lipid system were resuspended in 87.5 μL of solution containing the AMPs at the desired concentration (diluted in HEPES buffer) and the sample was incubated at 37°C for 1 h. The buffer and 1% Triton X-100 were respectively used as negative (F 0 ) and positive controls (F 100 ). The percentage of CF released was measured using a microplate reader set at λ emission = 517 nm and λ excitation = 495 nm. The percentage of leakage (%L) was calculated with the following equation: 2)

%L= F-F 0 F 100 -F 0 × 100 (Equation
2.8 Solid-state NMR and spectral moment analysis 31 P and 2 H SS-NMR spectra were acquired using a Bruker Avance III-HD wide bore 400 MHz spectrometer (Milton, Canada), with respective resonance frequencies of 162 and 61.5 MHz.

Static 31 P SS-NMR spectra were obtained using a phase-cycled Hahn echo pulse sequence [START_REF] Rance | Obtaining high-fidelity spin-12 powder spectra in anisotropic media: Phase-cycled Hahn echo spectroscopy[END_REF], with high-power (50 kHz) 1 H decoupling during acquisition. Using a 90° pulse length of 3 µs, an inter pulse delay of 35 μs, data were collected using 1024 points with a recycle delay of 3 s and a total of 14 k scans per spectra, amounting to 12 hours of acquisition. Magic-angle spinning (MAS) 2 H SS-NMR experiments were carried out using a 10 kHz spinning frequency and a phase-cycled quadrupolar echo sequence [START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF], with 100 k data points, acquired with a 90° pulse length of 4 µs, a rotor-synchronized interpulse delay of 96 μs and a recycle time of 500 ms. A total of 44 k scans per spectra were acquired for 8 hours. A line broadening of 100 Hz was applied to all spectra, and the 31 P chemical shift anisotropy (CSA) was determined by line fitting using Bruker Topspin 4.0.6 and Sola (Solid Lineshape Analysis) softwares.

2 H spectral moment analysis was performed using MestRenova software V6.0 (Mestrelab Research, Santiago de Compostela, Spain). Second spectral moments, M 2 , were calculated using equation (3) [START_REF] Warnet | A 2 H magic-angle spinning solid-state NMR characterisation of lipid membranes in intact bacteria[END_REF].

M 2 =ω r 2 Σ N =0 ∞ N 2 A N ∑ N=0 ∞ A N = 4 Π 2 v Q 2 5 ⟨S CD 2 ⟩ (Equation 3)
where ω r is the angular spinning frequency, N is the side band number, and A N is the area of each sideband obtained by spectral integration, S 2 CD is the mean square order parameter, and ν Q is the static quadrupolar coupling constant equal to 168 kHz for a C-D bond in acyl chains [START_REF] Burnett | Deuteron quadrupole coupling constants in three solid deuterated paraffin hydrocarbons: C 2 D 6 , C 4 D 10 , C 6 D 14[END_REF]. The M 2 value provides a quantitative description of the membrane lipid ordering and is particularly sensitive to the gel-to-fluid phase transition. Despite using deuterium-depleted water, a residual HDO peak remains, which accounts for 10 to 15% of the total 2 H NMR spectral intensity. This residual peak is excluded from the spectral moment calculation.

RESULTS

Incorporation of fatty acids into erythrocyte ghost membranes

Plasma membranes of RBCs are significantly different from those of bacteria. Notably, they contain 30-40% cholesterol, and their phospholipid acyl chains show a higher degree of ordered phase than those of fluid bacterial membranes. While erythrocyte ghost membranes have previously been labeled with deuterated FAs [START_REF] Maraviglia | Human erythrocyte membranes are fluid down to-5° C[END_REF], here we show that the labeling can be improved to facilitate the study of peptide-membrane interactions. A first set of experiments was performed to optimize the incorporation conditions of PA-d 31 into ghost membranes. Optimal insertion was achieved by incubating ghosts in the presence of Triton X-100/PA-d 31 mixed micelles. In the presence of ghost membranes, these micelles are in a dynamic associationdissociation equilibrium, the kinetics of which depend on temperature, pressure and concentration [START_REF] Moroi | Micelles: theoretical and applied aspects[END_REF]. They were diluted such that the concentration of Triton X-100 was decreased to 0.1 mM during the incorporation step, i.e., well below the critical micelle concentration of Triton X-100 (0.23 mM) [START_REF] Preté | Multiple stages of detergent-erythrocyte membrane interaction-a spin label study[END_REF]. Nonionic surfactants such as Triton X-100 tend to undergo a slow dissociation during which the exchange of FAs between the mixed micelles and phospholipid bilayers occurs. Since the FA incorporation in the bilayer is thermodynamically favorable, the exchange equilibrium is shifted towards the bilayer, and the ghost membrane can thus be efficiently 2 H-labeled [START_REF] Patist | Importance of micellar kinetics in relation to technological processes[END_REF][START_REF] Ollivon | Vesicle reconstitution from lipid-detergent mixed micelles[END_REF]. Although a full quantitative understanding of this process is beyond the scope of this work, the relative partitioning of PA versus Triton X-100 in the membrane is likely to be a key factor for the successful labelling. Indeed, the partition coefficient of PA is two orders of magnitude higher than the one of Triton X-100 in PC membranes (10310 3 M -1 for PA versus 310 3 M -1 for Triton X-100) [START_REF] Høyrup | Lipid membrane partitioning of lysolipids and fatty acids: effects of membrane phase structure and detergent chain length[END_REF][START_REF] Heerklotz | Correlation of membrane/water partition coefficients of detergents with the critical micelle concentration[END_REF]. Palmitic acid is thus favorably inserted into the membrane, and the residual detergent, which favorably remains in the buffer, can be removed by a series of washing steps. At a concentration of 0.1 mM, the surfactant proved to be harmless to the cell membranes, as demonstrated elsewhere [START_REF] Preté | Multiple stages of detergent-erythrocyte membrane interaction-a spin label study[END_REF][START_REF] Koley | Triton X-100 concentration effects on membrane permeability of a single HeLa cell by scanning electrochemical microscopy (SECM)[END_REF]. This is supported by the 31 P SS-NMR spectrum (Fig. 1B) which is characteristic of an intact lamellar lipid phase, and comparable to PC/SM/cholesterol/PA-d 31

(5:3:7:5 molar ratio) model RBC membranes (Fig. 1C), as well as to unlabeled ghosts (Fig. 1A).

The 31 P CSA value of deuterated ghosts is in good agreement with those of unlabeled ghosts and model membranes, as well as past studies in lipid bilayers (49) (Fig. 1). As shown in Fig. 2A microscopy. Z-stack of multiple optical sections also reveals that ghosts retain the biconcave disc shape of RBCs (Fig. 2D).

To ensure that the ghost membranes were effectively labeled, 2 H SS-NMR spectra were recorded (Fig. 1D) using MAS, thus providing a high signal-to-noise (S/N) ratio in a few hours, similarly to bacteria (4). The presence of spinning sidebands proves that a significant amount of deuterated FAs are no longer incorporated in fast tumbling objects such as micelles, but are rather found in an environment with restricted motions such as a membrane bilayer. Moreover, the 2 H spectrum is very similar to the one obtained for a model membrane with the same lipid composition (Fig. 1E), confirming that PA-d 31 is indeed in a membrane environment. The incorporation of PA is further supported by fluorescence microscopy images, which locate NBD-PAs, a fluorophore structurally similar to PA-d 31 , in the ghost membranes (Fig. 2C,D).

The incorporation of PA-d 31 in ghost membranes was further ascertained by comparing the FA ordering in labeled ghosts and in model membranes with increasing composition complexity.

To do so, the second spectral moment, M 2 , from 2 H SS-NMR spectra was determined at several temperatures. Indeed, if PA-d 31 is incorporated in the ghost membranes, the evolution of its

Lipid composition of erythrocyte and ghost membranes

To characterize possible alterations to the lipid profile upon 2 H-labeling of RBC ghost membranes, phospholipids were identified and quantified using 1D 31 S1.

To identify other lipids, in particular ether-linked phospholipids, we used a 2D 1 H-31 P TOCSY experiment in solution NMR [START_REF] Balsgart | High throughput identification and quantification of phospholipids in complex mixtures[END_REF], as shown in Fig. 4. All 1 H and 31 P chemical shifts have been reported in Table S1, and two 31 P lipid columns, at -0.75 and 0.18 ppm, showed a marked shift of their g2 glycerol proton, from 5.05 ppm to 4.97 ppm. We assign this shift to originate from a change in linkage from an ester to an ether group, in ether PC (PCe) and ether PE (PEe) -the additional oxygen in ester linked lipids further deshielding the glycerol protons [START_REF] Ijare | Ex Vivo 1H NMR study of pituitary adenomas to differentiate various immunohistochemical subtypes[END_REF]. One last 31 P lipid column, at 0.22 ppm, was not assigned from the 1D 31 P solution NMR spectra. Its proton NMR pattern is similar to that of SM, and we assigned it to dihydrosphingomyelin (DHSM), which is compatible with previous assignment in a different context [START_REF] Mackenzie | Quantitative analysis of dairy phospholipids by 31 P NMR[END_REF]. While PCe is only ca. 20% of the total PC lipids, we note that around 60% of SM lipids are DHSM, and that almost all of PE lipids are actually PEe.

After the determination of lipid proportions by 31 P solution NMR, the FA proportions were determined by GCMS after lipid hydrolysis. The complete and unambiguous quantification of the phospholipid profile of native horse RBCs, ghosts and 2 H-labeled ghosts is summarized in Fig. 5 and shows that the membrane phospholipidic profile is in good agreement with the literature [START_REF] Nouri-Sorkhabi | Phospholipid composition of erythrocyte membranes and plasma of mammalian blood including Australian marsupials; quantitative 31 P NMR analysis using detergent[END_REF], and that it was unaltered during the preparation of ghosts and the incorporation of deuterated FAs. As determined by GCMS and shown in Fig. 5, the incorporation of PA-d 31 was highly successful since it represents about 25% of the total FA chains in the ghost membrane.

Consequently, a change in membrane fluidity is expected since the labeled ghosts contain about 65% of saturated FAs, as compared to 50% in native RBCs. The incorporation of PA-d 31 together

with protonated unsaturated FAs such as oleic acid (C18:1) could help reestablish a more natural saturated/unsaturated FA ratio, as done with bacteria [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Bouhlel | Labelling strategy and membrane characterization of marine bacteria Vibrio splendidus by in vivo 2 H NMR[END_REF]. However, considering the low 2 H NMR S/N ratio obtained with 2 H-labeled ghosts, the addition of protonated FAs would reduce the sensitivity of the 2 H SS-NMR experiments, thus prohibiting this approach. The lower S/N ratio obtained with 2 H-labeled ghosts, as compared to 2 H-labeled bacteria [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF][START_REF] Tardy-Laporte | A 2 H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating[END_REF], can be explained by the impossibility to prepare ghost samples with sufficient concentration through centrifugation even at 100,000 g. Moreover, exogenous PA-d 31 is used by bacteria for the phospholipid synthesis, thus increasing the membrane labeling. Nevertheless, the NMR and GC-MS results show that ghosts can be labeled and enable membrane-peptide interaction studies by SS-NMR. S1 andS2.

Effect of antimicrobial peptides on erythrocyte membranes

Leakage experiments

The perturbation of phospholipid bilayers by AMPs can occur through various processes [START_REF] Aisenbrey | The mechanisms of action of cationic antimicrobial peptides refined by novel concepts from biophysical investigations[END_REF], most notably via a carpet-like mechanism or pore formation [START_REF] Magana | The value of antimicrobial peptides in the age of resistance[END_REF]. At different stages of these processes, membrane disruption is such that, in the case of RBCs, it can lead to hemoglobin leakage, which can readily be monitored. The lytic activity of caerin 1.1 and aurein 1.2 on actual RBCs and model membranes has previously been determined [START_REF] Chia | Membrane binding and perturbation studies of the antimicrobial peptides caerin, citropin, and maculatin[END_REF][START_REF] Lorenzon | Dimerization of aurein 1.2: effects in structure, antimicrobial activity and aggregation of Candida albicans cells[END_REF][START_REF] Ramezanzadeh | Design and characterization of new antimicrobial peptides derived from aurein 1.2 with enhanced antibacterial activity[END_REF][START_REF] Sani | One pathogen two stones: are Australian tree frog antimicrobial peptides synergistic against human pathogens?[END_REF][START_REF] Fernandez | The antimicrobial peptide aurein 1.2 disrupts model membranes via the carpet mechanism[END_REF]. Nevertheless, we measured the lytic activity of these peptides on RBCs reporting both peptide and lipid concentrations, thus enabling the calculation of L/P molar ratios at which lysis occurs. This additional information allows direct comparison with our leakage assays on RBC ghosts and erythrocyte models with a membrane composition (POPC/SM/Chol) closer to RBCs than previously reported. For comparison with other works, data presented as a function of L/P weight ratios are also reported in the supplementary information (Fig. S3).

As shown in Fig. 6A, although caerin 1.1 seems to have a higher lytic potency on RBCs than aurein 1.2 (with a higher hemolytic activity and a plateau at a higher L/P molar ratio), differences are much smaller than in model membranes (Fig. 6B and6C) and fall within the experimental uncertainty. The threshold concentrations and L/P molar ratios at which membrane perturbation occurs are reported in Working with actual RBCs enables us to elucidate the action mechanism of AMPs on these eukaryotic membranes, but also to address the question of their selectivity towards bacterial membranes. Indeed, a striking difference is observed for aurein, for which 8 times more peptide is required to achieve a 50% lysis of the bacterial model membranes as compared to model RBC membranes, despite the negative charge of the former. On the other hand, caerin shows a strikingly lower selectivity with 50% lysis of both model membranes at roughly the same L/P ratios (126:1 and 105:1). One might thus expect aurein to show a higher toxicity than caerin for RBCs. However, the LR50s of both peptides on RBCs are very similar (2.6:1 (0.2) and 3.2:1 (0.2) for aurein and caerin, respectively) and no significant differences in the concentrations at which the peptides lyse 10% of RBCs are observed.

To quantify the selectivity of both AMPs towards bacteria, and therefore their potential toxicity, the therapeutic index (TI) was calculated. This index is defined as the ratio between the MHC and the MIC, which we obtained in our previous work on bacteria [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF]. A large TI value indicates a higher selectivity towards a given bacterium. Table 1 shows that the calculated TI values for aurein 1.2 and caerin 1.1 are low, thus revealing a poor selectivity of both peptides towards bacteria, a tendency already observed previously [START_REF] Chia | Membrane binding and perturbation studies of the antimicrobial peptides caerin, citropin, and maculatin[END_REF][START_REF] Fernandez | Membrane interactions of antimicrobial peptides from Australian frogs[END_REF]. Both peptides seem to be more selective to the Gram(+) B. subtilis with TI values three times the ones obtained for E. coli, although differences between the two peptides might be obscured by the poor accuracy on MHC values. Further chemical modifications would be required to enhance the selectivity of these AMPs towards bacteria to reduce their cytotoxicity. It should be noted, however, that it is for the moment difficult to compare TI values between different studies since they are determined with various assays and different blood-resident cells [START_REF] Greco | Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides[END_REF].

Solid-state NMR experiments

To investigate the interaction of AMPs with erythrocyte membranes, the perturbation of the phospholipid headgroups and deuterated hydrophobic core of RBC ghosts were respectively studied by static 31 P and magic-angle spinning 2 H SS-NMR (Fig. 7). Note that the 31 P SS-NMR experiments shown in Fig. 7 were carried out on non-deuterated ghosts to approach native conditions and keep the samples as fresh as possible; however, experiments performed with deuterated ghosts did not reveal significant differences (Table S3 and Fig. S4).

As seen in Fig. 7, a narrow peak appears on the 31 P NMR spectrum at ca. 0 ppm when the proportion of aurein 1.2 is increased. This type of central peak has been discussed by Yang et al. [START_REF] Yang | Distinguishing bicontinuous lipid cubic phases from isotropic membrane morphologies using 31 P solid-state NMR spectroscopy[END_REF] and can either result from phospholipids in rapidly-reorienting small objects such as micelles, or from phospholipids in high-curvature membrane regions such as buds, cubic phases or toroidal pores. Those cases differ by their relaxation times and linewidths, the narrowest lines being assigned to micelles, and the broader ones to high-curvature regions.

In the case of aurein, the breadth of this central peak (ca. 1000 Hz) gradually decreases to a value of 200 Hz, consistent with the formation of micelles that end up being released from the membrane, leading to the sharp peak at a 1.7:1 L/P molar ratio. Interestingly, the CSA value of the remaining lamellar phase is unchanged up to an L/P ratio of 3:1 (Table S4). Simultaneously, the PA dynamics in the hydrophobic core increases, as revealed by the decrease in the spinning sideband (SSB) intensities on the 2 H SS-NMR spectra, and the concomitant decrease in M 2 values (see Fig. 7A and Fig. 8B). We note that we had reported an increase in M 2 in the early stages of the interaction of aurein with B. subtilis membranes [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF]. Thus differences between the first stages of aurein association with Gram(+) bacteria and ghosts cannot be excluded. S3 andS4). The behavior of caerin is significantly different compared to aurein. As the peptide concentration increases, an isotropic peak intensifies on the 31 P spectra, and remains broad (full width at half height of 1000-1500 Hz) at all concentrations (Fig. 7B and8B), compatible with phospholipids in high-curvature membrane regions. Interestingly, and in contrast with the effect of aurein, the PA dynamics with caerin concentration evolves in two stages, as revealed by 2 H SS-NMR (Fig. 8, Table S4). In a first stage, the dynamics slightly diminishes, as seen by the increase in M 2 from 9.9 with no caerin (not shown on logarithmic scale) to 11.310 9 rad. s -2 when caerin concentration is increased up to an L/P molar ratio of 6:1. When the peptide concentration is further raised, lipid dynamics increase to return to the initial M 2 value, and remain constant up to the maximum L/P ratio studied (3:1). We should point out that this behavior is different from the one we observed in B. subtilis bacterial membranes [START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF] and could indicate subtle differences in the pore formation molecular mechanism between the two cell types.

Microscopy experiments

Confocal microscopy results further support our SS-NMR findings. With both Fast-DiI and NBD-PA labeling, both the labeled and unlabeled ghosts appear to be destroyed by aurein 1.2 at a L/P molar ratio of 1.7:1 (Fig. 9 center column and Fig. S5). Aurein 1.2, with its relatively short 13 amino-acid sequence, has been shown to perturb both bacterial and mammalian model membranes through a carpet-like mechanism [START_REF] Fernandez | The antimicrobial peptide aurein 1.2 disrupts model membranes via the carpet mechanism[END_REF]. Both our SS-NMR and fluorescence microscopy results support a similar action towards RBC ghosts.

A contrario, caerin 1.1, with its 25 amino-acid sequence that can span the membrane, triggers hemolysis at lower concentrations than aurein (Fig. 6). The fluorescence confocal microscopy images in Fig. 9 recorded using FAST DiI entrapped in ghosts show intact membranes in the presence of caerin at concentrations where hemoglobin was shown to leak from the cells (Fig. 6).

Interestingly, the NBD-PA labeled ghosts (Fig. 9 and Fig. S6) show intense spots on the membrane where the dye appears to concentrate. Since the hemolysis assays were carried out with RBCs while SS-NMR measurements were performed with labeled ghosts, we thus imaged the calcein influx on ghosts by fluorescence microscopy. The results (shown in Fig. S7) not only prove that the influx in the ghosts membrane is possible but also that the membrane can be crossed by molecules as large as calcein.

DISCUSSION

These results, analyzed together with those of the hemolysis assay, can be interpreted in terms of membrane perturbation mechanisms. The fact that aurein 1.2 exerts its hemolytic action at later stages than caerin 1.1, that the lipid dynamics in the hydrophobic region increases when the peptide concentration increases, and that small rapidly-reorienting objects are formed, all indicate a carpet-like mechanism, as schematized in Fig. 10A. This mechanism is also supported by the more diffuse appearance of fluorescence labeled ghosts as seen by confocal fluorescence microscopy (Fig. 9).

In the case of caerin 1.1, the leakage results, 31 P SS-NMR spectra and confocal microscopy images are compatible with a pore-forming mechanism (represented in Fig. 10B), where the broad isotropic contribution would result from high-curvature regions in the membrane such as those expected in a toroidal pore [START_REF] Li | Membrane active antimicrobial peptides: translating mechanistic insights to design[END_REF]. It is also tempting to assign the bright NBD-PA fluorescence spots to pore locations although an unambiguous explanation for the concentration of the fluorescent tag in the pores remains elusive. Nonetheless, other mechanisms have been described that cannot be ruled out [START_REF] Bechinger | The SMART model: Soft Membranes Adapt and Respond, also Transiently, in the presence of antimicrobial peptides[END_REF][START_REF] Aisenbrey | The mechanisms of action of cationic antimicrobial peptides refined by novel concepts from biophysical investigations[END_REF]. In particular, dynamic models in which the membrane only transiently becomes permeable could be considered. A full proof of pore formation can only be obtained by more detailed spectroscopical measurements which are currently under way.

It should be noted that 2 H SS-NMR spectra monitor the acyl chain order profile of the PA-d 31 molecules inserted into the ghost membrane, and not of the endogenous phospholipids' chains, although this "reporter" molecule is very likely to reflect the overall state of the membrane. As a result of the presence of PC, SM and cholesterol in ghost membranes, liquid-ordered (L o ) and liquid disordered (L d ) domains can coexist, a delicate phase equilibrium that can potentially be perturbed by the addition of PA-d 31 [START_REF] Shimokawa | Formation of modulated phases and domain rigidification in fatty acid-containing lipid membranes[END_REF]. The preferential interaction of an AMP with membrane domains of lower thickness or at domain boundaries has recently been predicted and reported [START_REF] Lee | The impact of antibacterial peptides on bacterial lipid membranes depends on stage of growth[END_REF][START_REF] Pinigin | Amphipathic Peptides Impede Lipid Domain Fusion in Phase-Separated Membranes[END_REF]. A preferential interaction of caerin with the L d domain could expel the PA into the L o phase or at the interface, resulting in a stiffening of the rest of the membrane. This would explain the different dynamical behavior of PA-d 31 upon caerin addition as compared to aurein. It is noteworthy that the effects of AMPs observed by SS-NMR on labeled ghost membranes occur at L/P molar ratios similar to those observed in the hemolytic assays on intact RBCs. We thus consider that despite the changes in the FA chain profile due to labeling (an unavoidable drawback required to perform experiments on whole cells rather than model membranes), the mechanisms of action that we have elucidated are closely related to those taking place in intact RBCs. Altogether, our results indicate that aurein 1.2 and caerin 1.1 would interact with erythrocyte membranes via similar mechanisms that take place with bacterial membranes, and that their relative potency is maintained [START_REF] Fernandez | Membrane interactions of antimicrobial peptides from Australian frogs[END_REF]. Both peptides have been shown to at least partially adopt an α-helical structure when interacting with membranes [START_REF] Sani | One pathogen two stones: are Australian tree frog antimicrobial peptides synergistic against human pathogens?[END_REF]. These α-helices are amphipathic with all charged and polar residues found along one side of the helix, and amino acids with non-polar side chains on the opposite side (Fig. S8) [START_REF] Gautier | HELIQUEST: a web server to screen sequences with specific α-helical properties[END_REF]. The interaction with the membrane results from electrostatic and hydrophobic interactions, as well as from the respective shapes of lipids and peptides [START_REF] Aisenbrey | The mechanisms of action of cationic antimicrobial peptides refined by novel concepts from biophysical investigations[END_REF][START_REF] Greco | Correlation between hemolytic activity, cytotoxicity and systemic in vivo toxicity of synthetic antimicrobial peptides[END_REF]. The fact that the AMPs maintain similar action mechanisms on RBC and bacteria membranes despite differences in surface charge seems to indicate that hydrophobic interactions play an important role in dictating this mechanism. Electrostatic interactions are likely to play a greater role in the "capture" of the AMPs, thus explaining their higher potency towards bacteria.

CONCLUSIONS AND PERSPECTIVES

In this study, we have taken a new approach in understanding the action mechanism of AMPs on erythrocyte membranes. Preparing ghosts by removing the contents of erythrocytes preserves the cell surface and membrane composition whilst ensuring minimal interference with 31 P and 2 H SS-NMR analysis. The successful non-biochemical labeling of erythrocyte ghost membranes with deuterated FAs opens the possibility for other types of lipid labeling, such as with 13 Clabeled FAs for example.

Using hemolysis assays, 31 P static and 2 H MAS SS-NMR, we have confirmed the poor therapeutic quality of both aurein 1.2 and caerin 1.1 in their current version [START_REF] Chia | Membrane binding and perturbation studies of the antimicrobial peptides caerin, citropin, and maculatin[END_REF][START_REF] Fernandez | Membrane interactions of antimicrobial peptides from Australian frogs[END_REF]. While both antimicrobial peptides lyse bacteria at low concentrations, they present poor selectivity between Gram(+) and Gram(-) bacteria, and we showed that their hemolytic potency is too high for a safe use in the clinics. We have also shown that higher concentrations of these peptides are required to perturb intact RBCs as compared to model membranes. This may be explained by the fact that RBC membranes have not only lipids but also glycocalyx on their surface, which is rich in carbohydrates, and many transmembrane proteins embedded. Although our experimental protocols are established in order to compare similar L/P ratios in erythrocytes, ghosts and model membranes, we want to stress that in some medical cases, the ratio of bacteria to red blood cells, or other mammalian cells, may be such that a small concentration of AMPs may be enough to kill all bacteria while leaving most mammalian cells alive. Despite these concentration considerations, AMPs' mechanisms of interaction on RBCs are shown to be similar to those adopted in bacteria. Such a similarity may be specific to the peptides studied here, as many factors need to be taken into account when comparing peptidemembrane interactions with different cell membrane types. Comparison between erythrocytes and bacteria highlights the importance of hydrophobic effects in determining the mode of peptide interaction with membranes. A detailed understanding of the interaction of AMPs with mammalian cells constitutes an essential step for their use as therapeutics in the clinics, and for their improvement before reaching the market. It should be noted that the methodology described here does not rely on cell metabolism and can, at least in principle, be transposed to other eukaryotic cells, or those of other organisms.

( 34 )

 34 who intercalated PA-d 31 molecules into lyophilized human erythrocytes and rehydrated them prior to NMR experiments. They obtained a 5 % deuteration of the membranes and monitored the second moment of 2 H SS-NMR spectra as a function of temperature, showing no detectable phase transition down to 2°C, suggesting that PA-d 31 molecules are present in all lipid regions of the membrane. In this work, we have improved the FA incorporation in the ghost membranes, reaching a level of 25% deuteration. We have assessed changes in the lipid headgroup profile by 31 P solution NMR, and in the FA chain composition by gas chromatography coupled to mass spectrometry (GCMS). We have also validated the PA-d 31 insertion by 2 H SS-NMR and GCMS, and the membrane integrity by 31 P SS-NMR, and confocal fluorescence microscopy.

Caerin 1 .

 1 1 and aurein 1.2 were synthesized by GenScript Corporation (Piscataway, NJ, USA) with >95% purity. Sphingomyelin (SM) (egg, chicken), 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoylsn-glycero-3-phospho-L-serine (DOPS), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-glycerol) (POPG) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). Ethylenediaminetetraacetic free acid (EDTA) was purchased from Fisher Scientific (Fair Lawn, NJ, USA), while deuterated palmitic acid (PA-d 31 ), cholesterol, deuteriumdepleted water, Triton X-100, fatty acid methyl ester mix C4-C24 (FAME mix), [16-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] palmitic acid (NBD-PA), 1,1'-dilinoleyl-3,3,3',3'tetramethylindocarbocyanine 4-chlorobenzenesulfonate (Fast DiI) and all other solvents and chemicals were purchased from Sigma Aldrich (Oakville, ON, Canada). Fresh horse RBCs packed 100% were purchased from Cedarlane Laboratories (Burlington, ON, Canada).

  H decoupling and employed 384 scans at 25 °C with a 10 s relaxation delay to obtain quantitative data. To identify the lipid profile, commercially available pure synthetic lipids (DOPC, DOPE, DOPS and SM) solutions were spiked in the sample. Remaining ambiguities were resolved by 2D 31 P-1 H TOCSY NMR experiments as described by Balsgart et al. (39), at 25 °C with 12k data points in the direct dimension and 200 data points in the indirect dimension, a repetition delay of 2 s, a TOCSY mixing time of 70 ms. The acquisition time in the direct dimension was 2.8 s, for a total acquisition time of 22 h per 2D spectrum.

Figure 1 .

 1 Figure 1. Static 31 P SS-NMR spectra with spectral fitting and average CSA of (A) unlabeled, (B) deuterated ghosts and (C) DOPC/SM/Chol/PA-d 31 (5:3:7:5) model membranes. 2 H SS-NMR spectra with MAS (10 kHz) of (D) deuterated ghosts and (E) DOPC/SM/Chol/PA-d 31 (5:3:7:5) model membranes with M 2 values. All spectra were recorded at 293 K. The residual HDO peak in deuterium-labeled ghosts accounts for 10 to 15 % of the total 2 H intensity and is not integrated in the M 2 calculation.

Figure 2 .

 2 Figure 2. Characteristic confocal fluorescence microscopy images of prepared erythrocyte ghosts indicating that the membranes are intact, the incorporation of PA-d 31 does not appreciably alter the overall morphology, and PA molecules are evenly dispersed throughout the plasma membrane. (A) Non-deuterated ghosts labelled with the lipophilic tracer FAST-DiI (red). (B) Ghosts with PA-d 31 incorporated into the plasma membrane, labelled with the lipophilic tracer FAST-DiI (red). (C) Deuterated ghosts prepared in the presence of fluorescent NBD palmitic acid (green). (D) Z projection of multiple planes of the deuterated ghosts prepared with fluorescent NBD palmitic acid (green).

2 H

 2 second spectral moment -which reflects the fluidity of the membrane -with temperature is expected to be similar to the one in a model membrane which closely matches the lipid composition of ghosts Fig.1 D-E. As shown in Fig. 3, the ordering of PA-d 31 in DOPC/Chol/PAd 31 and, to a lesser extent, in DOPC/PA-d 31 , shows a reduction with increasing temperature. The addition of sphingomyelin appears to cancel this temperature dependence. Interestingly, PA-d 31 ordering in labeled ghosts as a function of temperature closely follows the one of the model membranes which composition is the closest to erythrocyte membranes (DOPC/SM/Chol/PAd 31 ) (Fig. 3). The temperature dependence of 2 H M 2 values thus appears to confirm the incorporation of FAs into the ghost membrane. Moreover, as shown by 31 P SS-NMR and confocal fluorescence microscopy, the membrane integrity of these labeled ghosts is preserved.

Figure 3 .

 3 Figure 3. Membrane fluidity of model membranes and ghosts incorporating PA-d 31 , as a function of temperature, as reported by the second spectral moment, M 2 , calculated from 2 H SS-NMR spectra. The similarity between the M 2 values obtained for labelled ghosts and model membranes with the closest lipid composition are a strong indication that PA-d 31 is incorporated in the ghost membrane.

Figure 4 :

 4 Figure 4: 2D 1 H-31 P TOCSY spectrum of RBC membrane phospholipids extracted by the Folch method and solubilized in CD 3 OD:CDCl 3 solution to which and small amount of EDTA aqueous solution is added. This eliminates ionic species from the organic phase and improves lipid solubility (see Materials and Methods section for more details).

Figure 5 .

 5 Figure 5. Phospholipid headgroup (left panel) and fatty acyl chain profile (right panel) of horse red blood cells, ghosts and deuterated ghosts obtained from 31 P NMR and GCMS analyses, respectively. Crosshatched areas correspond to ether linked or DHSM lipids. Complete data are reported in TablesS1 and S2.

Figure 6 .

 6 Figure 6. Lytic activity of antimicrobial peptides on erythrocytes and model membranes as determined by (A) hemoglobin leakage of RBCs with a phospholipid concentration of 200 µM, (B) carboxyfluorescein leakage of POPC/SM/Chol LUVs with a lipid concentration of 100 µM and (C) carboxyfluorescein leakage of POPE/POPG with a lipid concentration of 150 µM. Results are presented as the mean of triplicate assays. Dashed and dotted lines correspond to the best fit to a sigmoidal curve. Note that in order to compare the relative effects of the AMPs on whole cells and lipid model membranes, molar L/P ratios are indicated. Details of the experimental estimation of RBC lipid concentration are presented in the supplementary information.For comparison with other works, data presented as a function of L/P weight ratios are also reported in the supplementary information (Fig.S3).

Figure 7 .

 7 Figure 7. 2 H MAS (10 kHz) SS-NMR spectra (left) and 31 P static SS-NMR spectra (right) of ghosts, acquired at 293 K, with different concentrations of (A) aurein 1.2 and (B) caerin 1.1. The corresponding lipid-to-peptide molar ratios (L/P) are indicated. 31 P static and 2 H MAS SS-NMR spectra were respectively obtained with unlabeled and deuterated ghosts. For 31 P SS-NMR spectra, dotted lines represent spectral fitting of both the broad anisotropic component and the central peak (numerical values are in TablesS3 and S4).

Figure 8 .

 8 Figure 8. Effect of aurein 1.2 and caerin 1.1 on erythrocyte ghosts as a function of the peptideto-lipid (P/L) molar ratio, at 293 K. (A) Isotropic contribution to the 31 P SS-NMR spectra of unlabeled ghosts. (B) Second spectral moment (M 2 ) of deuterated ghosts determined from 2 H MAS SS-NMR spectra. Phospholipid concentrations range from 4 mM to 9 mM. Dashed and dotted lines correspond to the best fit to a sigmoidal curve or a simple guide to the eye when fitting was not possible (caerin 1.1 panel B).

Figure 9 .

 9 Figure 9. Confocal fluorescence microscopy images of (A) FAST DiI entrapped in the unlabeled ghosts with addition of AMPs and (B) NBD-PAs entrapped in the deuterated ghosts with addition of AMPs. The corresponding lipid-to-peptide molar ratios (L/P) are indicated.

Figure 10 .

 10 Figure 10. Cartoon representation of AMPs' potential action mechanisms on ghost membranes incorporating deuterated palmitic acid. (A) Carpet mechanism with aurein binding on the surface and then disrupting the membrane by micellization. (B) Pore formation by caerin after binding on the surface.

  P solution NMR after lipid extraction (see Fig. S2 in the Supplementary information).

31

P resonances of the main phospholipids (PE, PC, SM and PS) were identified by comparison with the chemical shifts of standards and the literature

[START_REF] Laydevant | Growth-phase dependence of bacterial membrane lipid profile and labeling for in-cell solid-state NMR applications[END_REF]

. Lipid proportions were determined by integrating those resonances and are reported in Table

Table 1 .

 1 Characteristic literature values are also indicated in order to compare our results with those of previously published antimicrobial assays. By examining the results in Table1, it appears that caerin systematically shows a higher permeabilization potency than aurein in all membrane systems. Indeed, our results show that caerin has lower minimum inhibitory concentrations (MIC) on both E. coli and B. subtilis, and higher LR50s on model membranes and, to a lesser extent, RBCs. It is noteworthy that lysis of 50% of the RBCs occurs at L/P molar ratios that are one to two orders of magnitude lower than in the model eukaryotic (POPC/SM/Chol) or bacterial (POPE/POPG) membranes. This result indicates that RBCs are significantly more resistant than model membranes to AMP-induced lysis, and highlights the importance of assessing AMP activities on intact cells.

Table 1 .

 1 Comparison of effect of the antimicrobial peptides on red blood cells (RBCs), model RBCs and bacteria membranes, as well as different bacteria. The leakage assays were performed on intact RBCs. The minimum hemolytic concentrations causing 10% leakage of RBCs (MHC) and the lipid-to-peptide molar ratio at which 50 % leakage occurs (LR50) are obtained by fitting of the leakage assays shown in Fig.6A.

		RBCs	POPC/SM/ Chol	POPE/POPG	RBCs		E. coli	B. subtilis
	AMPs
		LR50 a	LR50 a	LR50 a	MHC b (μM)	MIC c (μM)	TI d	MIC c (μM)	TI d
	Aurein 1.2	2.6:1 (0.2) 54.3:1 (2.4)	7.2:1 (0.1)	15.8 (2.7)	68	0.23 (0.04)	20	0.79 (0.13)
	Caerin 1.1	3.2:1 (0.2) 124.7:1 (2.0) 100.3:1 (10.2) 12.1 (3.6)	39	0.31 (0.09)	12	1.00 (0.30)

a LR50: L/P molar ratio at which 50% of RBCs or membranes are lysed. b MHC: minimum hemolytic concentration causing 10% hemolysis of RBCs. c MIC: minimum inhibitory concentration

[START_REF] Laadhari | Interaction of the antimicrobial peptides caerin 1.1 and aurein 1.2 with intact bacteria by 2 H solid-state NMR[END_REF]

d TI: therapeutic index, i.e., MHC/MIC.

ACKNOWLEDGMENTS

This research was funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada (grant RGPIN-2018-06200 to I.M.) and the Centre National de la Recherche Scientifique (UMR 7203 to D.E.W.). K.K. would like to thank the Quebec Network for Research on Protein Function, Engineering, and Applications (PROTEO) -strategic cluster of the Fonds de recherche du Québec -Nature et technologies (FRQNT) -for the award of a scholarship.

M.S. is grateful to the NSERC for the award of a scholarship.