Characterization of a New Immunosuppressive and Antimicrobial Peptide, DRS-DA2, Isolated from the Mexican Frog, Pachymedusa dacnicolor

Inflammatory and antimicrobial diseases constitute a major burden for society, and fighting them is a WHO strategic priority. Most of the treatments available to fight inflammatory diseases are anti-inflammatory drugs, such as corticosteroids or immunomodulators that lack cellular specificity and lead to numerous side effects. In addition to suppressing undesired inflammation and reducing disease progression, these drugs lessen the immune system protective functions. Furthermore, treating infectious diseases is more and more challenging due to the rise of microbial resistance to antimicrobial drugs. Thus, controlling the inflammatory process locally without compromising the ability to combat infections is an essential feature in the treatment of inflammatory diseases. We isolated three forms (DRS-DA2N, DRS-DA2NE, and DRS-DA2NEQ) of the same peptide, DRS-DA2, which belongs to the dermaseptin family, from the Mexican tree frog Pachymedusa dacnicolor. Interestingly, DRS-DA2N and DRS-DA2NEQ exhibit a dual activity by inducing the death of leukocytes as well as that of Gram-negative and Gram-positive bacteria, including multiresistant strains, without affecting other cells such as epithelial cells or erythrocytes. We showed that the death of both immune cells and bacteria is induced rapidly by DRS-DA2 and that the membrane is permeabilized, leading to the loss of membrane integrity. We also validated the capacity of DRS-DA2 to regulate the pool of inflammatory cells in vivo in a mouse model of noninfectious peritonitis. After the induction of peritonitis, a local injection of DRS-DA2N could decrease the number of inflammatory cells locally in the peritoneal cavity without inducing a systemic effect, as no changes in the number of inflammatory cells could be detected in blood or in the bone marrow. Collectively, these data suggest that this peptide could be a promising tool in the treatment of inflammatory diseases, such as inflammatory skin diseases, as it could reduce the number of inflammatory cells locally without suppressing the ability to combat infections.


Introduction
Considering that chronic infammation plays a central role in the development of the majority of chronicdegenerative diseases, but also of some infectious diseases, including acute respiratory distress syndrome, a common complication of various viral pneumonia caused, for example, by coronaviruses, such as MERS-CoV, SARS-CoV, and recent SARS-CoV-2 [1], it is of great interest to identify new drug candidates to specifically control the infammatory process without compromising the ability of the body to combat infections.Indeed, most of the anti-infammatory drugs available treat infammatory diseases systemically and thus compromise the ability of the immune system to combat infections [2,3].Antimicrobial peptides (AMPs), produced by skin and mucosal cells, among others, are fully part of the innate immune system [4,5].Originally identifed for their microbicidal properties by killing a large scale of Gram-negative and Gram-positive bacteria, fungi, and protozoa mainly by permeabilizing their membrane, AMPs are now recognized to modulate the immune system [6].Among all the organisms producing peptides, AMPs isolated from frogs present a broad range of interesting biological activities in mammals.Notably, a number of these AMPs have been identifed to modulate infammatory processes by promoting the migration of immune cells through the activation of chemotactic receptors and/or by stimulating the production of proinfammatory or anti-infammatory cytokines [6], while exhibiting antimicrobial properties against a wide range of bacteria.
Te aim of this study was to isolate and characterize, in vitro and in vivo, a peptide that could be an alternative candidate in the treatment of infammatory diseases by regulating the pool of infammatory cells locally, rapidly, and for a determined period of time.Tus, we tested the potential of skin exudate extracted from the Mexican tree frog Pachymedusa dacnicolor to present an immunosuppressor efect.We found a fraction that regulated the pool of infammatory cells by specifcally inducing the death of immune cells.Within that fraction, we isolated three forms of the same peptide, difering only by the deletion of one or two residues from the C-terminus.Te longer peptide (DRS-DA2NEQ) corresponds to the putative sequence determined from cDNA, published in 1998 by Wechselberger [7], but the corresponding peptide was not isolated.As these sequences were rather close to a family of antimicrobial peptides, dermaseptins, we also proceeded to test their antimicrobial activity.We synthesized the two extreme forms of this peptide (DRS-DA2NEQ and DRS-DA2N) and were able to demonstrate that, in vitro, these peptides killed, at micromolar concentrations, Gram-negative and Gram-positive bacteria, including multiresistant strains, diferent Candida strains, and human and mouse leukocytes, without afecting other cells such as primary and transformed epithelial cells or erythrocytes.In all cases, the shorter peptide (DRS-DA2N) seemed to be more potent in inducing the death of immune cells and bacteria than the longer one (DRS-DA2NEQ).We showed that DRS-DA2 induced the death of both immune cells and bacteria by rapidly inducing the permeabilization of the membrane.Finally, we validated the capacity of this peptide to induce specifcally the death of infammatory cells in vivo in a murine model of infammatory disease, noninfectious peritonitis, and showed that the short form DRS-DA2N exhibited a local, and not systemic, immunoregulatory efect by lessening the number of leukocytes in the peritoneal cavity after induction of peritonitis.

Animals.
All experiments involving living beings were carried out according to the guidelines of the Bioethics Committee of the Instituto de Biotecnología, UNAM (bioethics approval project 334/336 Pequeños péptidos de defensa: nuevas moléculas con capacidades inmunomoduladoras y antimicrobianas), and by authorized investigators.Specimens of Pachymedusa dacnicolor were captured in the state of Morelos (Mexico) and housed as previously described [8].10-week-old male or female C57BL/6 or Balb/c mice were kept in pathogen-free conditions with food and water available ad libitum and housed in a 12 h light/12 h dark (100-500 lux) cycle.

Circular Dichroism (CD) Spectroscopy.
Te CD spectra of the peptides were recorded as previously described [10].
Measurements were carried out in 10 mM Tris bufer at pH 7.4 and in the presence of POPC, POPG, and POPG-POPC (25 : 75) LUVs or POPG-POPE (25 : 75) LUVS to mimic E. coli inner membrane composition at a peptide/lipid molar ratio of 1/50.Dichroic spectra were corrected by subtracting the background obtained for each peptide-free solution.An analysis of CD spectra was conducted after smoothing, and conversion to molar ellipticity was calculated as follows: θ = θobs/(10xnxl), where θobs is the observed ellipticity, n is the number of bonds, c is the peptide concentration in mol.L −1 , and l is the path length in cm.

NMR Spectroscopy and Structure Calculations.
Te structure of DRS-DA2N bound to SDS micelles was determined by solution-state NMR spectroscopy as previously described [11].In brief, 2D   13 C α , 13 C β , 1 H α , and 1 H N chemical shifts (Table S1) [11].Structures were calculated by torsion angle dynamics in CNS and refned in an explicit layer of water including an electrostatic energy term in the energy function [12].
2.6.Cell Preparation.Human leukocytes were provided by the « Banco de Sangre del Hospital Regional del IMSS » and the Centro Estatal de la Transfusión Sanguínea in Cuernavaca, Morelos, Mexico.Institutional committees approved the acquisition and isolation of human peripheral blood leukocytes.Human leukocytes and erythrocytes were obtained as previously described [8].
2.7.Cytotoxic Assay.5.10 5 mouse or human leukocytes or primary epithelial cells, A549 or Jurkat E6-1 cells, were incubated in 100 µl RPMI or DMEM containing 0.5% BSA and 10 nM HEPES with the peptides at diferent concentrations for 2 hours at 37 °C, 5% CO 2 .Incubation of the cells with DMSO 30% was used as a positive control.For the kinetic assay, the cells were incubated with the peptides for diferent time periods.Viability of leukocytes and Jurkat cells was determined by adding a viability dye (eFluor 780) and counted by FacsCanto fow cytometry with a predetermined number of beads (Polybead, Carboxylate Microspheres, Polysciences, Inc., Le Perray-en-Yvelines, France) added to the samples.Mouse leukocytes were stained with anti-mouse Ly6G-PE, anti-mouse NK1.1-PE, anti-mouse CD11b-PerCP, and anti-mouse F4/80-APC (BD Biosciences, Rungis, France).Human leukocytes were stained with anti-human CD45-PB, anti-human CD16-PE-Cy7, anti-human CD56-PE-Cy7, anti-human CD14-FITC, anti-human CD3V500, anti-human CD8-APC, and antihuman CD4-PE.Data were analyzed with FlowJo.For the cell line A549, viability was assessed by the CCK8 assay (DoJindo) as indicated by the manufacturer.In brief, 100 µL of cell suspension was dispensed (12 500 cells/well) in a 96-well plate.After 24-hour preincubation in a humidifed incubator (37 °C, 5% CO 2 ), 10 µL of various concentrations of peptides was added, and the plaque was incubated for 1 h in the incubator.10 µL of CCK-8 solution was then added to each well of the plate.Te absorbance at 450 nm was measured after 3 h incubation.

Hemolytic
Tests.Blood from a mouse or healthy adult donor was centrifuged at 800 × g for 10 minutes at 4 °C.Te pellet containing red blood cells was then washed three times in 10 volumes of PBS and was diluted in 4% (v/v) in PBS.Blood and peptides at diferent concentrations were then incubated at 37 °C for one hour.Cells were centrifuged 1500 × g for 5 minutes at room temperature.Hemolysis was measured in the supernatant at 550 nm.Red blood cells lysed with 1% Triton X-100 was used as a control, achieving 100% hemolysis.Percent hemolysis was calculated using the following formula: % hemolysis where A represents the absorbance of the peptide sample at 550 nm and A0 and At represent 0% and 100% hemolysis determined in 10 mM PBS and 0.1% Triton X-100, respectively.
International Journal of Infammation 2.9.Antimicrobial Assays.Te minimum inhibitory concentration (MIC) was determined by a liquid test in 96-well microplates as previously described [13].100 µL of the sample to be tested was distributed in triplicate to obtain a range of fnal concentrations from 100 to 0.2 µM in the wells.90 µL of bacterial suspension (5 × 10 5 CFU/mL for Gram-negative bacteria and 5 × 10 5 CFU/mL for Gram-positive strains in Mueller-Hinton broth) were added to each well [14].Te microtiter plates were incubated at 37 °C overnight.Te optical density of the wells was measured at 620 nm, and the results were interpreted by calculating the percentage of growth in each well according to the formula: % growth = (OD 600 well − OD 600 T0)/(OD 600 of T100 − OD 600 T0) * 100, where OD 600 T100 = DO 600 growth control (bacterial suspension without the test sample, 100% growth), OD 600 T0 = OD 600 of sterility control (culture medium + bacteria + 10 µL formaldehyde, 0% growth) well, and OD 600 = OD 600 of the well which was desired to calculate the percentage of growth (+ bacterial suspension test sample).MIC is the minimum concentration of the peptide that inhibits bacterial growth after incubation at 37 °C for 20 hours.
Te minimum bactericidal concentration (MBC) is defned as the minimum concentration of the peptide killing 99.9% of bacteria after incubation at 37 °C for 20 hours.Tis test consisted of the spreading of the contents of the wells where the peptide inhibited bacterial growth on a Petri dish (MH agar), followed by incubation for 24 h at 37 °C.

Antifungal Assay: Strains and Growth Conditions.
Te fungal pathogens C. glabrata standard strain CBS138 [15] and C. albicans strain SC5314 [16] were grown in rich (YPD) media.Yeast extract-peptone-dextrose (YPD) medium was prepared, contained yeast extract at 10 g/L and peptone at 20 g/L, and was supplemented with 2% glucose.C. albicans and C. glabrata cells were grown for 48 h at 30 °C and diluted into fresh YPD media containing diferent concentrations of each peptide (DRS-DA2N and DRS-DA2NEQ): 1.5, 3, or 15 µM.Tese cultures were grown in a Bioscreen C apparatus with constant shaking for 1,500 min, and OD 600 was determined every 15 min as described previously [17].
2.11.Time Kill.Time-killing experiments were achieved according to [18,19] with minor modifcations.Te peptide's concentration was 1 × MIC and 2 × MIC.At the time points of 15, 30, 45, and 60 min and after homogenization, suspensions were diluted in 10 mM sodium phosphate bufer (pH 6.8) and spread onto MH agar plates.After overnight incubation at room temperature, CFUs were counted.
2.12.In Vitro Peptide Stability.One mL of RPMI supplemented with 25% (v/v) rat serum was equilibrated at 37 °C for 15 min before adding the peptide (fnal concentration of 300 µM).At diferent time points, 300 µL of the reaction solution was removed and completed to 600 µL with 96% ethanol.Te sample was cooled at 4 °C for 15 min and then spun to precipitate serum proteins.Te supernatant was recovered and submitted to the viability assay as described in the cytotoxic assay section or analyzed by RP-HPLC on C18-300 100 × 4, 6 mm.A linear gradient from 100% bufer A (0.1% TFA in H 2 0) to 60% of bufer B (0.1% TFA in ACN) was used over 10 min with a fow rate of 1 mL/min.Te absorbance was detected at both 220 nm and 280 nm.

Apoptosis/Necrosis Assays. Induction of apoptosis or
necrosis by the peptides was tested by incubating 5.10 5 Jurkat E6-1 cells in 200 µl RPMI containing 10% FBS and 10 nM HEPES with the peptides at 5 µM for 2 hours at 37 °C, 5% CO 2 .A positive control was obtained by exposing the cells to UV light for 10 min (1 mJ/cm 2 , Stratagene UV Stratalinker 2400), before incubating them for 4 hours at 37 °C, 5% CO 2 , RPMI 10% FBS.Cells were then washed, centrifuged, resuspended, and incubated 30 min at 4 °C in the dark with Viability dye 780 (BioLegend).After washing the cells, Annexin-V-FITC in annexin-binding bufer was added, and the cells were incubated for 15 minutes at room temperature in the dark.Te cells were fxed with PFA at a fnal dilution of 2% before being analyzed by FACSCanto fow cytometry.

Peptide-Induced Permeabilization of the Inner Bacterial
Membrane [20].Permeabilization of the E. coli ML35p membrane by both peptides was assayed by measuring the β-galactosidase activity with the chromogenic substrate onitrophenyl-β-D-galactopyranoside (ONPG).Bacteria were grown in 10% Mueller-Hinton broth with 50 µg/mL ampicillin, washed twice with the test bufer (10 mM phosphate, 100 mM NaCl at pH 7.4 and 300 µg/mL Mueller-Hinton broth), and diluted in the test bufer to an absorbance of 0.6 at 630 nm (mid-log phase).50 µL aliquots of bacterial suspension were mixed with 25 µL pf the peptide at various concentrations and 25 µL ONPG (2 mM, i.e., 0.5 mM fnal) in a 96-well plate.Te hydrolysis of ONPG was monitored by measuring the absorbance of released o-nitrophenol at 412 nm with a Fluostar Galaxy microplate reader (BMG LabTech, France) [8].Data shown are the means of 3 independent experiments realized in duplicate.

4
International Journal of Infammation 2.16.Membrane Depolarization.E. coli cells were grown at 37 °C to the mid-logarithmic phase and were collected by centrifugation (4000 x g, 10 min), before being resuspended in MH with 0.1 mg/mL BSA and were diluted in the same bufer to an OD 620 of 0.6.Te bacterial suspension was divided into 125 µL aliquots in 96-well microplates.10 µL of DiSC 3 (5) in 1% DMSO was added in each well to a 10 µM fnal concentration.After 10 minutes, 10 µL of EDTA (15 mM fnal concentration) was added.After recording the baseline fuorescence, 5 µL of the aqueous peptide stock was added to the desired fnal concentration.Te increase in the fuorescence intensity registers dye release upon reduction of the transmembrane potential.Te fuorescence reading was monitored by using a microplate reader (BMG Fluostar) at an excitation wavelength of 620 nm and an emission wavelength of 670 nm.

ANTS/DPX Leakage Assay.
Te vesicles of POPE/ POPG (75/25) at 25 mM concentration were obtained as previously described and suspended in a solution of 12.5 mM ANTS and 45 mM DPX in 10 mM HEPES.After freeze-thaw and extrusion, LUVs were separated from external ANTS/DPX by ultracentrifugation (3 × 50 000 rpm, 15 min).Final lipid concentration was measured by the method of Rouser.

Intrinsic Tryptophan Fluorescence.
Te fuorescence spectra of DRS-DA2 peptides were recorded from 295 to 475 nm with excitation at 280 nm in a spectrofuorometer using a quartz cuvette (Jobin Yvon Horiba spectrometer H 10-61 UV, Kyoto, JAP; 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 was placed in a 10 × 2 mm cuvette (Hellman Analytics, Müllheim, GER), and increasing amounts of the lipid solution were added to obtain lipid concentrations of 10, 25, 50, 100, 200, 500, 1500, and 3000 μM.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.Te intensity of the emission was frst plotted against the wavelength of the emitted fuorescence of tryptophan.Taking into consideration that the peptide's concentration changes during the experiment because of the addition of amounts of lipidcontaining bufer, the data were formatted so that all information corresponds to the same peptide concentration.Te subsequent normalization of the graphs allows for an easy comparison of the results from diferent experiments.We chose to calculate Δλ max -hence the diference 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 ratio (concentration of lipid)/(concentration of peptide).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.

Isolation of a new peptide belonging to the dermaseptin family with immunosuppressive properties.
As previously reported [11], Pachymedusa dacnicolor skin exudate was extracted and purifed to homogeneity by a two-step protocol.In brief, 1.1 mL of skin exudate recovered by gently squeezing the skin parotid glands was frst fractionated on a Sephadex G-50 column (Fig S1 ), and the three fractions obtained were incubated with human leukocytes at 37 °C, 5% CO 2 .After 30 min of incubation with fraction I at a concentration of 100 µg mL −1 , 100% of the cells were dead (insert Fig S1).High molecular weight fraction I was then purifed by RP-HPLC (Figure 1(a)).Among all fractions incubated with leukocytes, the fraction with a retention time of 49 min was found to induce the death of leukocytes (data not shown).Tis RP-HPLC fraction was further analyzed by mass spectrometry for peptide identifcation and de novo sequencing.Te MALDI-TOF/TOF analysis of the RP-HPLC fraction revealed three peptides corresponding to monoprotonated ions [M + H] + with at m/z 3337.82,3209.77, and 3080.74 for the frst isotope (monoisotopic masses), respectively, and consistent with dermaseptin-like peptides (Figure 1(b)).Te three peptides, identifed with mass precision in the 10-20 ppm range, only difer from each other by the loss of one residue (Q) or two residues (EQ) at their C-terminus.Te nontruncated sequence, which corresponds to the sequence of DRS-International Journal of Infammation DA2NEQ, had been predicted from frog DNA in 1998 [7] but never confrmed until now, as the corresponding peptide was not isolated.Tese peptides were named DRS-DA2N, DRS-DA2NE, and DRS-DA2NEQ (Figure 1(b)).Synthetic peptides were used for their characterization.
DRS-DA2 peptides were then structurally characterized by circular dichroism (CD) and NMR.Antimicrobial peptides are generally unstructured in aqueous solution and tend to adopt a well-defned conformation in contact with membranes.CD spectra were recorded in the presence of Tris bufer (10 mM) or negatively charged LUVs composed of POPE and POPG phospholipids, with a 3 : 1 ratio that refects the PE/PG ratio in the inner membrane of E. coli.In the presence of negatively charged LUVs, both DRS-DA2N and DRS-DA2NEQ adopted dichroic spectrum characteristics of an α-helix conformation, like the one obtained for melittin, a well-known pore-forming peptide [21,22], as indicated by the two intense negative bands detected around 208 and 222 nm.However, a diference in the percentage of helicity was observed between the peptides, indicating that DRS-DA2N (91%) seemed to be better structured than DRS-DA2NEQ (62%) and melittin (77%).As expected, Scr (KGNVLVKAAMLTKVLAKNKGVQNANALTW), the random form of DRS-DA2N, remained unstructured (Figure 2(a)).On the contrary, in the presence of Tris bufer, all peptides remain unstructured (Fig. S2).Te conformation of DRS-DA2N was further characterized at the residue level by solution-state NMR in the presence of SDS micelles that mimic the amphipathic properties of the lipid bilayer.Analysis of NMR data indicated that the region from L2 to A26 adopted a well-defned helical structure, while the terminal residues A1 and N27-N29 are rather unstructured (Fig. S3).Te structure of DRS-DA2N exhibited a pronounced amphipathic character with the hydrophobic face predominantly formed by A, L, and V residues and the hydrophilic face containing 4 K residues (K8, K9, K12, and K16) (Figure 2(b)).Te interfacial position of residue K4 allowed the ammonium group of its long-side chain to potentially interact with the phospholipid head groups at the surface of the bacterial membrane or to make π-cation interactions with the aromatic group of W3 on the hydrophobic face of the helix.Despite its weak helical propensity, the presence of two G residues (G11 and G15) in a GXXXG motif did not interrupt the helical conformation in the central region of the peptide (Figures S3a and 3(b)).Altogether, these results demonstrate that DRS-DA2 is structured in an α-helix and that DRS-DA2N seems better structured than DRS-DA2NEQ.1), with DRS-DA2N being slightly more potent than DRS-DA2NEQ.As predicted, Scr did not induce the death of any of the human or mouse leukocyte populations.
To assess the kinetics of peptide-induced cell death, leukocytes were incubated with DRS-DA2N and DRS-DA2NEQ at a concentration of 10 µM for diferent times, up until two hours.As observed in Figure 3(b), around 50% of the leukocytes were killed after 15 min of incubation with both DRS-DA2N or DRS-DA2NEQ.However, DRS-DA2N seems to induce the death of the cells in a slightly faster way.
To fgure out if the induction of cell death by the peptides was restricted to leukocytes or afected other cell lineages, both peptides at diferent concentrations were incubated with human (Figure 4(a)) and mouse (Fig. S5) red blood cells, human (Figure 4(b)) and mouse (Fig. S5) primary epithelial cells, and the human tumoral epithelial cell line (A549) (Figure 4(c)).Interestingly, at the concentrations they induce the death of 100% of leukocytes (10 µM), none of the peptides induced the death of any of these cell types.
Tese results suggest that DRS-DA2N and DRS-DA2NEQ preferentially induce the death of leukocyte lineage cells but not that of other cells, such as epithelial cells or erythrocytes.

DRS-DA2 Has a Broad-Spectrum Microbicidal Capacity.
Te antibacterial properties of DRS-DA2N, DRS-DA2NEQ, the control peptide Scr, and melittin as a reference poreforming peptide were tested on a series of Gram-positive and Gram-negative strains.As presented in Table 2, both peptides exhibited a wide spectrum of action, which was however greater on Gram-negative than on Gram-positive bacteria even if some MICs ≤ 6.3 µM were observed for S. aureus ATCC 6538 and ST 1065, S. epidermidis BM 3302, and Enterococcus faecalis CIP 103015.Interestingly, DRS-DA2N was generally four times more potent than DRS-DA2NEQ and also than melittin.Particularly, for the E. coli β-lactamaseresistant strain, the MIC observed for DRS-DA2N was 1.6 µM, whereas the one for melittin was only 6.25 µM.As predicted, Scr did not exhibit any bacterial activity.
As DRS-DA2N appeared to be more potent than DRS-DA2NEQ, its antibacterial activity was evaluated on various skin multiresistant strains and compared to reference antibiotics (Table 3).Interestingly, DRS-DA2N was active on multiresistant Gram-positive and Gram-negative strains and exhibited MICs lower than the reference antibiotics, even for Gram-positive strains.In the case of the clinical isolates of International Journal of Infammation P. acnes, responsible for acne vulgaris, DRS-DA2N was less efective than the reference antibiotic doxycycline, yet it exhibited good activity.
To further broaden the potential of DRS-DA2N and DRS-DA2NEQ to function as anti-microbial agents, we tested the antifungal activity of DRS-DA2N and DRS-DA2NEQ on C. glabrata CBS138 and C. albicans SC5314, two strains responsible for most bloodstream infections worldwide, associated with high mortality rates [23].C. glabrata CBS138 and C. albicans SC5314 were grown in the presence of DRS-DA2N and DRS-DA2NEQ at various concentrations.Although the peptides did not inhibit the growth of C. glabrata CBS138 and C. albicans SC5314, the lag phase was extended for both fungal pathogens, as compared to the no-treatment culture.In the absence of either peptide, C. albicans started growing at 125 min, but in the presence of DRS-DA2N (3 or 15 µM), the lag phase was extended to 250 min and that of C. glabrata up to 500 min (Figure 5(a)).In the presence of DRS-DA2NEQ, C. albicans also showed an extended lag phase and a reduced doubling time at 15 µM.At that same concentration of DRS-DA2NEQ, C. glabrata showed an extended lag phase with no efect in doubling time (Figure 5(b)).Tese data indicate that both peptides DRS-DA2N and DRS-DA2NEQ have also antifungal activities.
We next investigated if, like leukocytes, bacteria were killed rapidly by DRS-DA2N and DRS-DA2NEQ.Time-kill curves of DRS-DA2N and DRS-DA2NEQ against E. coli ATCC8739 were performed.Curves representing the amount of surviving bacteria (CFU/mL) in the absence or presence of DRS-DA2N, DRS-DA2NEQ, melittin, or omiganan, an antibiotic used in the treatment of skin infections at their MIC concentrations, are presented in Figure 6.Interestingly, the growth of E. coli ATCC8739 was stopped rapidly by DRS-DA2N, DRS-DA2NEQ, and melittin, as all the bacteria were killed after only 15 min of incubation with either peptide at 37 °C.In contrast, omiganan needed a much longer time to be active.
Altogether, these data show that DRS-DA2 peptides have rapid antibacterial and antifungal activities.

DRS-DA2 Has a Short Half-Life in the Presence of Serum.
Before assessing the anti-infammatory activity of the peptides in vivo, the stability of DRS-DA2 to proteases was investigated.DRS-DA2N at a fnal concentration of     International Journal of Infammation 9 °C for diferent times (0, 0.5, 1, 2, 3, 4, and 24 h) before being recovered and analyzed by RP-HPLC.Peptide degradation was followed at 220 and 280 nm.Quantifcation of the remaining peptide DRS-DA2N over time was performed by comparing the areas of the intact peptide DRS-DA2N with the sum of the areas of all the peptides (intact + degraded) present in the rat serum (Figure 7(a)).Each fraction was then collected and characterized by MALDI TOF/TOF (Fig. S6).A 40% degradation of DRS-DA2N was observed after 30 min, and DRS-DA2N was totally degraded in the presence of serum for 24 h.MS/MS fragmentation of the degraded peptides, using MALDI-TOF/TOF, indicated a time-dependent Nterminal degradation (Fig. S6).Alternatively, DRS-DA2N (300 µM) fractions previously incubated with serum for 0 (t0), 15 min (t15), 30 min (t30), or 24 h (t24 h) were recovered and incubated with cells from the mouse bone marrow for two hours at a concentration of 5 µM.Cell death was evaluated by fow cytometry.As observed in Figure 7(b), no signifcant diferences in the percentage of dead cells could be seen in the presence of DRS-DA2N t0 or Both experiments indicated that DRS-DA2 degraded rapidly in the presence of proteases contained in the serum, as a consequence of which the peptide lost its immunosuppressive function.

DRS-DA2N Decreases Locally the Infammation in a Mouse Model of Noninfectious Peritonitis.
As DRS-DA2 induces the death of immune cells and bacteria rapidly, in less than 30 min, without afecting the viability of erythrocytes or epithelial cells, and since it also degrades rapidly in the presence of serum, we considered the possibility of using DRS-DA2 in infammatory pathologies that could beneft of a local treatment, which would have the advantage to decrease the risks of adverse efects.
To validate the possibility of using the peptides in vivo as a local anti-infammatory treatment, we used a mouse model of noninfectious peritonitis and injected the peptides directly in the peritoneal cavity.Basically, peritonitis was induced by injecting thioglycolate 3% in the peritoneal cavity.DRS-DA2N, DRS-DA2NEQ, Scr, or the vehicle (NaCl) were intraperitoneally (i.p.) injected 14, 19, and 24 h later.A group of mice injected with NaCl only was used as a control of the induction of peritonitis.Mice were sacrifced 12 h after the last injection of the peptides or vehicle.Te efect of the peptides on the number of leukocytes in the peritoneal cavity, blood, and bone marrow was analyzed by fow cytometry, with monocytes, macrophages, and neutrophils being defned on the expression of CD11b, Ly6G, and F4/80 cell surface markers.
Te injection of thioglycolate 3% and the peptide vehicle (NaCl) increased the level of monocytes, macrophages, and neutrophils in the peritoneal cavity, compared to the control group (NaCl only).Tree i.p. injections of DRS-DA2 after the thioglycolate 3% injection resulted in a signifcant decrease in the number of monocytes, macrophages, and neutrophils in the peritoneal cavity of the mice compared to the group of mice injected with thioglycolate 3% and vehicle (NaCl) or Scr (Figure 8(a)).
To investigate whether the lesser number of leukocytes observed in the peritoneal cavity after treatment with DRS-DA2N and DRS-DA2NEQ was restricted to a local environment (peritoneal cavity) or was systemic, we evaluated the number of infammatory cells in blood and in the bone marrow.As expected, induction of peritonitis led to an egress of monocytes and neutrophils from the bone marrow (Figure 8(b)) and an increase in blood (Figure 8(c)).Interestingly, this egress of infammatory cells from the bone marrow and this increase in blood observed after induction of peritonitis and treatment with DRS-DA2N or Scr were conserved, whereas the egress from the bone marrow and the increase of monocytes in the blood were reduced after induction of peritonitis and treatment with DRS-DA2NEQ (Figures 8(b) and 8(c)).Tus, these results indicate that the i.p. treatment with both peptides reversed the increase of infammatory cells in the peritoneal cavity after induction of peritonitis.However, whereas the efect of DRS-DA2N was restricted to the peritoneal cavity, DRS-DA2NEQ seemed to have a more systemic efect, as there is a signifcant diminution of the egress of monocytes in the bone marrow and of the increase of monocytes in blood.

DRS-DA2 Afects the Integrity of the Membrane of Immune Cells and Bacteria.
Te rapid killing action of the peptides (under 30 min) of both leukocytes and bacteria supports evidence that the peptides induced the death of cells and bacteria in a like manner, i.e., by a nonreceptormediated mechanism.
As a frst step, we investigated if the peptides induced the death of immune cells by directly disrupting the cytoplasmic membrane (necrosis) or by the more regulated process of apoptosis, where membrane integrity is maintained.First, to assess that DRS-DA2 induced the death of Jurkat E 6-1 cells similarly to primary leukocytes, Jurkat E 6-1 cells were incubated with DRS-DA2N or Scr at various concentrations, and viability was measured.Similarly, to what was observed on primary leukocytes, DRS-DA2N International Journal of Infammation induced the death of Jurkat cells after 2 hours with an LC 50 of 4 µM (Figure S7).Ten, to diferentiate the two death pathways, Jurkat E 6-1 cells were incubated for 2 hours with DRS-DA2N or Scr at a concentration of 5 µM or with the vehicle (PBS) as a negative control, and the cells were stained with a viability dye (eFluor 780, V) and annexin V (A) (Figure 9(a)).A positive control of apoptosis was generated by exposing the cells to UV (1 mJ/cm 2 ) for 10 min and incubating them for 4 hours at 37 °C, 5% CO 2 prior to staining for annexin binding and viability.After incubation with DRS-DA2N, around 70% of the remaining cells were annexin and eFluor 780 positive (A + V + ), indicating they died of necrosis (Figure 9(a)).As expected, around 40% of the UV-irradiated cells were in the apoptotic cells' quadrant (A + V − ).Like untreated cells (negative control), hardly any cell death (<4%) was observed after the treatment with Scr, as more than 96% of cells were alive (A − V − ) (Figure 9(b)).Tese results suggest that DRS-DA2N could induce the death of immune cells by permeabilizing the membrane leading to the loss of membrane integrity.We next investigated if DRS-DA2N and DRS-DA2NEQ also permeabilized the membrane of the bacteria.Te extent of membrane permeabilization due to the peptides' activity was evaluated by using the ONPG test on the E. coli ML35p strain.Tis mutant is constitutive for cytoplasmic β-galactosidase, lacks lac permease, and expresses a plasmidencoded periplasmic β-lactamase.Since there is no lactose permease in this mutant, ONPG cannot cross its inner membrane to be cleaved by cytoplasmic β-galactosidase to ONP unless permeabilization of the inner membrane occurs.ONPG cleavage produces a color change that can be measured spectrophotometrically at 420 nm [24].DRS-DA2N and DRS-DA2NEQ, as well as melittin, induced inner membrane permeabilization, whereas, even after 60 min, omiganan did not reach the plateau (Figure 10(a)), indicating that omiganan seems to adopt another mode of action.Te slight diferences observed between the DRS-DA2 peptides and melittin were not signifcant because they probably were due to minor diferences in concentrations, taking account of the very small concentrations used in this test.
Te cytoplasmic membrane depolarization activity of the peptides was determined by using the membrane potentialsensitive fuorescent dye diSC 3 -5 on E. coli membranes [25].If bacteria permeabilization and disruption of the cytoplasmic membrane occur following contact with the peptides, an enhancement in fuorescence will be observed as a consequence of the dispersion of the fuorescent probe in the medium.Our frst results on E. coli showed that DRS-DA2N and DRS-DA2NEQ, as well as melittin, induced rapid depolarization within 10 min (Figure 10(b)).
To go further in the study of DRS-DA2 peptide interaction with lipids, we used liposomes as a reliable model to characterize AMP action on a membrane [26].Te permeabilization of LUV membranes induced by the peptides was assessed by using the ANTS/DPX assay that can be used to measure the leakage of small molecule probes from lipid vesicles [27].ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt) is a highly negatively charged dye used together with the fuorescent quencher DPX (N,N′p-xylene-bis (pyridinium bromide)) for the studies of membrane fusion or permeability.Te mixture of ANTS-DPX is minimally fuorescent initially but becomes increasingly fuorescent upon dilution by leakage into the bufer.To mimic E. coli membranes, a mixture of POPE and POPG (75 : 25) LUVs was used.In this model system, DRS-DA2N and DRS-DA2NEQ were shown to act similarly as melittin, allowing a rapid increase in fuorescence when enhancing the peptide's concentration (Figure 10   International Journal of Infammation whereas melittin was slightly less active (Table 4).As previously reported by Melo et al. [28], omiganan did not induce any leakage.
Te tryptophan fuorescence spectrum depends on the tryptophan environment, giving information on aqueous or lipid surroundings of the indole nucleus.As the DRS-DA2 peptides contain tryptophan in position 3, the study of the tryptophan fuorescence emission spectra could give indications of the peptides' ability to insert in membranes that would be characterized by a blue shift of λ max .Te tryptophan fuorescence spectra of DRS-DA2N and DRS-DA2NEQ in aqueous bufer had a maximum emission circa 355 nm.Addition of increasing concentration of LUVs made with lipids extracted directly from E. coli ML35p showed a large blue shift (30-35 nm) in the emission maxima of the DRS-DA2N and DRS-DA2NEQ together with an enhancement of emission intensity maxima, indicating the incorporation of the tryptophan aromatic ring into the hydrophobic medium of lipids (Figure 10(d)).
Altogether, these results indicate that similarly to what is observed for immune cells, the induction of bacterial death by DRS-DA2 involves a strong disturbance of the membrane.

Discussion
In this study, a new member of the demarseptin family, DRS-DA2, exhibiting in vitro antimicrobial and immunosuppressive activities at a micromolar range and in vivo antiinfammatory activity; both within 30 min; was isolated from the endemic Mexican tree frog Pachymedusa dacnicolor.Among the recorded 3569 AMPs (APD3, https://aps.unmc.edu/) [29], there are only 32 peptides reported with both antibacterial and anti-infammatory activities.Most of these peptides exert their anti-infammatory activity by inhibiting the production of infammatory cytokines and chemokines, such as TNF-alpha, IL-6, IL-1-beta, or CCL2, or increasing the production of anti-infammatory cytokines such as IL-10 [30], by inhibiting interaction of LPS to toll-like receptors [31] or the production of nitric oxide [32].To our knowledge, DRS-DA2 is the frst AMP to induce specifcally the death of immune cells and thus regulating locally the pool of leukocytes at the site of infammation in an in vivo infammatory model.
Interestingly, three diferent forms of this peptide, DRS-DA2NEQ, DRS-DA2NE, and DRS-DA2N, appear to coexist naturally.Te existence of such truncated peptides has already been reported in the skin secretions of Phasmahyla jandaia where bradykinin appears as a full (RPPGFSPFR) or a C-terminal truncated form (RPPGFSPF) [33], though this does not completely refute a possible degradation of the sample, leading to the loss of residues in the C-terminal part of the peptide DRS-DA2.Although this is unlikely, since all the peptides present in the other fractions are intact.Te full-length peptide was predicted by Wechselberger in 1998 but had never been isolated until now [7].DRS-DA2NEQ and DRS-DA2NE present a net charge of +4, while DRS-DA2N has a net charge of +5 and is thus slightly more charged than most of the recorded AMPs having a net charge between +2 and +4.Like numerous AMPs, and particularly those of the dermaseptin family, DRS-DA2 is structured in the alpha-helix in contact with micelles that mimic amphipathic properties of the membranes lipid bilayer, with the C-terminal region of the peptide rather unstructured, and exhibits a pronounced amphipathic character (Figures 2(a) and 2(b)).Interestingly, the short form of the peptide DRS-DA2N presents a higher percentage of helicity, indicating that it seems to be better structured.Tese diferences in terms of the net charge and structural conformation seem to play a role in the activity of the peptide as DRS-DA2N exhibits slightly better antimicrobial and immunosuppressive activities than DRS-DA2NEQ (Tables 1 and 2).Tese observations are consistent with the literature as the antibacterial mechanism of action of 14 International Journal of Infammation these kinds of peptides depends on the positive net charge and alpha-helix conformation of the peptide [34].Te detailed mechanism by which cationic AMPs inhibit bacterial growth leading to their death has not yet been totally described.However, it is now known that most of these peptides interact through their N-terminal region with the membrane before disrupting and permeabilizing it, whereby several biophysical properties of the peptide (amphipathicity, net charge, hydrophobicity, structural conformation, or fexibility) and also of the membrane (charge, conformation, curvature, fexibility, or composition) are implicated [34,35].In the present study, the possible mechanism of bacterial death induced by DRS-DA2 was evaluated by various biophysical techniques and showed that the peptide disrupted the bacterial membrane within 30 minutes.As the immunosuppressive efect was also observed within 30 minutes after incubating leukocytes with DRS-DA2, we hypothesized that the peptide would induce the death of the cells by necrosis, which is a mechanism of death very similar to the bacterial membrane disruption and which occurs rapidly.Indeed, using necrotic and apoptotic markers, we showed that the peptide induced necrosis rather than apoptosis.Cancer cell membranes difer from primary cells by exhibiting more negatively charged components, greater fuidity, and cell surface area [36].Tese properties play an important role in the induction of cancer cell death by enabling cationic peptides to interact better with the membrane of cancer cells than with that of primary cells.However, we could not note signifcant diferences in the induction of death of primary versus cancer cells (Figures 3(a), S7, 4(b) and 4(c)), indicating that the biophysical properties mentioned above are not sufcient for DRS-DA2 to induce cell death.Moreover, as far as we are aware, this is the frst report documenting a peptide that induces specifcally the death of one type of cells that are not tumor cells, without afecting other types of eukaryotic cells at this range of concentration.Te exact molecular mechanisms by which DRS-DA2 induces bacteria and leukocyte death, as well as the diference in sensitivity of DRS-DA2 on leukocytes and other eukaryotic cells, are for the moment, not fully understood.To clarify these points, further investigations will be needed.
DRS-DA2 also displays a diferent sensitivity on Gram-negative and Gram-positive bacteria by exhibiting an enhanced efect on Gram-negative bacteria, with a MIC and an MBC signifcantly lower.Tese fndings are quite unusual, as observed by Malanovic [34].Indeed, it has been demonstrated that the peptidoglycan composition of the outer membrane wall of Gram-positive bacteria would 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.
Nevertheless, we showed that DRS-DA2N inhibited the growth of the skin multiresistant Gram-positive strain S. aureus ATCC 700699 (MecA +), frequently responsible for nosocomial infections, and is though better than the reference antibiotic (10 times better).DRS-DA2N also inhibited the growth of the other skin Gram-positive bacteria Propionibacterium acnes, responsible for acne vulgaris, which is one of the most common skin diseases afecting more than 630 million people in the word [37].However, even if DRS-DA2 exhibited an MIC higher than the reference antibiotic doxycycline, it still presented a good activity.It should be noted that doxycycline is used as oral treatment and can result in severe aplastic anemia [38], whereas DRS-DA2 could be used topically and locally.DRS-DA2N also inhibited the growth of two Gramnegative skin bacteria responsible for nosocomial infections, S. epidermidis ST20140436 (MecA +) and E. coli OXA-48 (carbapenemase) with MICs lower than the reference antibiotic.DRS-DA2 was also efcient against two candida species, Candida glabrata CBS138, which present an innate resistance to azole antimycotic therapy that is very efective in treating infections caused by other Candida species, and Candida albicans.Most Candida infections are benign, though they can lead to death in immunocompromised hosts.Until recently, C. glabrata was considered a relatively nonpathogenic fungal organism.However, with the increased use of immunosuppressive agents, mucosal and systemic infections caused by C. glabrata have raised signifcantly [39].
Most treatments used in infammatory diseases are small drugs that interact with various pharmacological targets that exhibit poor selectivity and high toxicity that are associated with numerous side efects.Most of them present systemic efects, which in long-term therapies usually weaken the ability of the immune system to combat infections [40].Tus, the use of peptides, and in particular, peptides exhibiting dual action, i.e., anti-infammatory and antibacterial, could be an alternative treatment to small molecules.Compared to small molecules, peptides present high potency and selectivity, low toxicity, low risk of drug interaction, and low accumulation in tissue.Nevertheless, poor metabolic stability and rapid clearance of bioactive peptides are usually seen as a restriction in their use [40,41].As expected for a peptide, DRS-DA2 demonstrated poor metabolic stability as it started to degrade within one hour.However, we also showed that it induced the death of bacteria and immune cells in less than 30 min.Tus, this rapid clearance should not afect its antibacterial and immunosuppressive activities in vivo after a local application.Furthermore, it would have the advantage of minimizing the dissemination of the peptide in the body, thus decreasing the risks of side efects.Moreover, the fact that DRS-DA2 exhibits a rapid action is very promising as less peptide would be necessary to kill bacteria in comparison with slow-acting peptides or small molecules (time-kill greater than 2 h) International Journal of Infammation since bacteria are able to double in the number every 20 min [42].We showed that the local application (i.p. injection) of DRS-DA2 after induction of noninfectious peritonitis reversed the number of leukocytes in the peritoneal cavity.Additional in vivo experiments would be necessary to confrm if the decrease observed is due to the death of leukocytes as hypothesized or to another efect on leukocytes, such as inhibition of migration.Notably, the short form DRS-DA2N exhibited a local efect, as no modulation of the number of leukocytes was observed in the bone marrow or in blood after peritonitis induction.Furthermore, as the in vivo efect of the peptide was validated in a short-term infammatory model, it would be of great interest to investigate its efect on a more relevant infammatory model, such as a chronic skin infammatory disease, to ensure that the death induction of leukocytes does not trigger proinfammatory signals, such as the production of infammatory cytokines, angiogenesis, or vasodilation.

Conclusions
In conclusion, to our knowledge, DRS-DA2 is the frst AMP to exhibit antimicrobial activity combined with the capacity to regulate the pool of infammatory cells by inducing specifcally the death of leukocytes at a concentration within the micromolar range without being toxic to other eukaryotic cells such as erythrocytes or primary and transformed epithelial cells at that concentration.As the short form DRS-DA2N is slightly more potent at inducing the death of bacteria and leukocytes, as well as presenting a capacity to regulate the pool of infammatory cells locally in the infammatory model of peritonitis, it could be a promising therapeutic agent in the treatment of infammatory diseases, and particularly, in skin infammatory diseases.

Figure 1 :
Figure 1: Isolation and purifcation of a new immunosuppressor peptide, dermaseptin DRS-DA2.(a) Fraction I that was shown to induce the death of leukocytes was purifed by RP-HPLC.Among all the fractions tested for an immunosuppressive efect, the one with a retention time of 49 min (arrow) induced the death of leukocytes.(b) De novo characterization by MALDI-TOF/TOF mass spectrometry identifed three peptides in fraction 49: DRS-DA2NEQ, DRS-DA2NE, and DRS-DA2N.Tose peptides were all carboxylated at their C-terminus.Isotopic masses are given for the protonated molecules, and mass errors are given in ppm.Te sequence of the nontruncated peptide corresponds to PD-2-2.

Figure 2 :
Figure 2: Structural properties of DRS-DA2N and DRS-DA2NEQ.(a) Far-UV CD spectra of DRS-DA2N, DRS-DA2NEQ, melittin, and Scr in POPE: POPG (75 : 25) LUVs.Data shown are representative of at least three diferent experiments.(b) NMR structure of DRS-DA2N bound to SDS micelles (ribbon representation of the lowest-energy conformer).Side chains of residues forming the polar and hydrophobic faces are represented by sticks.

Figure 3 :
Figure 3: DRS-DA2N and DRS-DA2NEQ induce the death of human leukocytes.(a) Diferent populations of human leukocytes (PMN, Mo, NK, and T cells) were incubated for two hours at 37 °C, 5% CO 2 in the presence of various concentrations of DRS-DA2N, DRS-DA2NEQ, or Scr.Te percentage of live cells was determined by fow cytometry.(b) Total human leukocytes were incubated in the presence of DRS-DA2N, DRS-DA2NEQ, and Scr at a concentration of 10 µM for diferent time periods until 2 hours, and the percentage of live cells was determined by fow cytometry.Data represent three independent experiments run in triplicate.

Figure 4 :
Figure 4: DRS-DA2N and DRS-DA2NEQ are not toxic for erythrocytes or epithelial cells.(a) Human erythrocytes in PBS were incubated with various concentrations of DRS-DA2N and DRS-DA2NEQ for one hour.Triton 1% was used as a positive control.Hemoglobin release was monitored by measuring the absorbance at 550 nm (mean of 3 independent experiments run in triplicate).(b) Human primary epithelial cells were incubated with DRS-DA2N and DRS-DA2NEQ at the indicated concentrations for 2 hours at 37 °C, 5% CO 2 .Te percentage of live cells was determined by fow cytometry.(c) A549 cells were incubated for 1 hour at 37 °C, 5% CO 2 in the presence of increasing concentrations of the peptides.Te percentage of live cells was determined by the CCK8 assay.Experiments were run in duplicate.

Figure 5 :Figure 6 :
Figure 5: DRS-DA2N and DRS-DA2NEQ present antifungal activities.C. albicans and C. glabrata cells were grown for 48 h at 30 °C and diluted into fresh YPD media containing DRS-DA2N (a) and DRS-DA2NEQ (b) at 1.5, 3, or 15 µM.OD 600 was determined every 15 min.Te control corresponds to fungi without any peptide.

Figure 7 :
Figure 7: DRS-DA2N and DRS-DA2NEQ are resistant to enzymatic degradation for 15 minutes.(a) Quantifcation of the remaining peptide based on relative UV absorption at 220 nm of DRS-DA2N upon incubation with rat serum for diferent incubation time points (n � 3 independent experiments).(b) Recovered DRS-DA2N previously submitted to serum for diferent time points was incubated with mouse bone marrow cells for 2 hours, and cell viability was determined.Tree independent experiments were run in triplicate.

Figure 9 :
Figure9: DRS-DA2N induces the death of immune cells by membrane disruption.Jurkat E6-1 cells were incubated for two hours with DRS-DA2N, Scr, or PBS or exposed to UV for 10 min and put to rest for 4 hours, 5% CO 2 at 37 °C, before being stained with annexin V and the viability dye eFluor 780.(a) Representative fow cytometry analysis of Jurkat E6-1 cells double stained with annexin V (A) and the viability dye eFluor 780 (V).(b) Percentage of cells A − V − , A + V − , A − V + , and A + V + after the diferent treatments.Two experiments were run in triplicate.

Table 2 :
Antimicrobial activities of DRS-DA2N and DRS-DA2NEQ on Gram-negative and Gram-positive strains.

Table 3 :
Antibacterial activities of DRS-DA2N on some skin multiresistant bacteria in comparison with reference antibiotics.

Table 4 :
Membrane perturbation studied by the ANTS/DPX assay from POPE/POPG LUVs.