<figure><img src="image_1.png" /></figure>

# Promising antibacterial efficacy of arenicin peptides against the emerging opportunistic pathogen *Mycobacterium abscessus*

Magali Casanova¹*†, Marc Maresca²†, Isabelle Poncin¹, Vanessa Point¹, Hamza Olleik², Céline Boidin-Wichlacz³, Aurélie Tasiemski³, Kamel Mabrouk⁴, Jean-François Cavalier¹* and Stéphane Canaan¹

**Abstract**

**Background** *Mycobacterium abscessus*, a fast-growing non-tuberculous mycobacterium, is an emerging opportunistic pathogen responsible for chronic bronchopulmonary infections in people with respiratory diseases such as cystic fibrosis (CF). Due to its intrinsic polyresistance to a wide range of antibiotics, most treatments for *M. abscessus* pulmonary infections are poorly effective. In this context, antimicrobial peptides (AMPs) active against bacterial strains and less prompt to cause resistance, represent a good alternative to conventional antibiotics. Herein, we evaluated the effect of three arenicin isoforms, possessing two or four Cysteines involved in one (Ar-1, Ar-2) or two disulfide bonds (Ar-3), on the in vitro growth of *M. abscessus*.

**Methods** The respective disulfide-free AMPs, were built by replacing the Cysteines with alpha-amino-*n*-butyric acid (Abu) residue. We evaluated the efficiency of the eight arenicin derivatives through their antimicrobial activity against *M. abscessus* strains, their cytotoxicity towards human cell lines, and their hemolytic activity on human erythrocytes. The mechanism of action of the Ar-1 peptide was further investigated through membrane permeabilization assay, electron microscopy, lipid insertion assay via surface pressure measurement, and the induction of resistance assay.

**Results** Our results demonstrated that **Ar-1** was the safest peptide with no toxicity towards human cells and no hemolytic activity, and the most active against *M. abscessus* growth. **Ar-1** acts by insertion into mycobacterial lipids, resulting in a rapid membranolytic effect that kills *M. abscessus* without induction of resistance.

**Conclusion** Overall, the present study emphasized **Ar-1** as a potential new alternative to conventional antibiotics in the treatment of CF-associated bacterial infection related to *M. abscessus*.

**Keywords** Mycobacterial infection, Cystic fibrosis, Hemolytic activity, Pore forming activity, Electron microscopy, Antibiotic resistance, Bactericidal activity, Antimicrobial peptide

<footnote>†Magali Casanova and Marc Maresca have contributed equally to this work.</footnote>

<footnote>*Correspondence:</footnote>

<footnote>Magali Casanova
magali.casanova@univ-amu.fr
Jean-François Cavalier
jfcavalier@imm.cnrs.fr
Full list of author information is available at the end of the article</footnote>
## Background

*Mycobacterium abscessus* is a non-tuberculous mycobacterium whose incidence is increasing worldwide, and which in some industrialized countries even exceeds that of *M. tuberculosis*, the etiological agent of tuberculosis [1]. *Mycobacterium abscessus* is responsible for severe lung infections and is associated with acute respiratory failure, notably in immunocompromised patients and/or patients with respiratory disorders such as cystic fibrosis (CF) [2, 3]. It is also responsible for cutaneous, bone and disseminated infections, and, in rare cases, for causing damage to the central nervous system [1]. Furthermore, the presence of *M. abscessus* may evolve into severe complications after lung transplantation, limiting the therapeutic arsenal offered to CF patients [4]. *Mycobacterium abscessus* exists as two phenotypically different morphotypes: a smooth (S) variant with a uniform, round and glossy appearance and that possesses glycopeptidolipids (GPL) at its surface; and an irregular and dry variant, called rough (R) variant, with no GPL at its surface thus resulting in cord formation [5, 6]. The S variant is associated with biofilm-like structures formation and should be responsible for the primo infection, whereas the R variant is usually found in severe and persistent forms of the clinical disease and represent the most virulent form of the bacteria [2, 7]. In addition, the naturally polyresistance of *M. abscessus*, not only to conventional anti-tuberculous drugs but also to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and β-lactams [1], leads to a treatment failure rate of nearly 60% [8]. Whether intrinsic, adaptive or acquired, *M. abscessus* resistance to many antibiotics involves several mechanisms: low permeability of the cell wall, induction of antibiotic efflux pumps, mutations inactivating antibiotic-activating enzymes or activation of enzymes that neutralize antibiotics or modify their targets [9, 10]. Consequently, in order to avoid bacterial resistance, the treatment of *M. abscessus* infections implements a multi-therapy possibly combined with surgery, resulting in an expensive and heavy treatment for the patient [11]. New drugs as well as new strategies are thus urgently needed to fight infections due to *M. abscessus*, ideally with a different mechanism of action than conventional antibiotics in order to minimize and prevent resistance.

In this context, some antimicrobial peptides (AMPs) have demonstrated particularly interesting antibacterial activities against *M. abscessus* [12–19]. AMPs are small-sized molecules (≤ 100 amino acids), with a cationic and a hydrophobic part [20]. They play function in the symmetry of organisms, from unicellular (bacteria, archaea) as anti-competitors, to pluricellular (plants, fungi, invertebrates and vertebrates) as key components of the innate

immune response [20, 21]. Often constituting the first line of defense, they possess a killing activity against a wide range of microorganisms from Gram-negative and Gram-positive bacteria, to fungi, enveloped viruses as well as parasites [22–24]. More precisely, through their cationic part, AMPs interact with the anionic components at the surface of microorganisms, facilitating the insertion of the hydrophobic domain into the phospholipid bilayer thus inducing membrane destabilization and permeabilization resulting in bacterial lysis [20, 21]. AMPs can also target key intracellular processes that lead to pathogenic microorganism death without necessarily disrupting the membrane. Such processes include the inhibition of cell wall components expression, DNA or protein synthesis, protein folding, and metabolic turnover [25]. Besides having a direct antimicrobial activity, several AMPs also possess the ability to modulate the host innate immune response and thereby indirectly to promote intracellular pathogen clearance [26]. Given such diverse modes of action, few pathogens have developed resistance to AMPs which are also found active against pathogens already resistant to conventional antibiotics [27–30].

Arenicins (*Ar*) are AMPs of 21 residues that have been biochemically isolated and identified from circulating blood cells of a marine annelid worm called *Arenicola marina*, inhabiting the intertidal zone of the European temperate shore [31]. Three isoforms (*Ar*-1, *Ar*-2 and *Ar*-3—Fig. 1) have been described, consisting in two antiparallel β-strands containing charged arginine residues as well as hydrophobic residues, explaining the amphipathic properties of these peptides [32–36].

Arenicins exhibit antifungal and antibacterial activities against Gram-negative and Gram-positive bacteria including drug-resistant clinical isolates [33–35]. They induce cell death in fungi as well as in bacteria via membrane permeabilization and disruption by formation of toroidal pores [33]. It has been recently demonstrated that *Ar*-1 is also able to induce an apoptosis-like response in *Escherichia coli*-treated cells through accumulation of reactive oxygen species, imbalance of intracellular calcium concentrations, disruption of membrane potential, caspase-like activation, and DNA damage [37]. Another important issue for the therapeutic potential of AMPs is related to their anti-inflammatory activity. Their potential role in inflammation may indeed result from the regulation of various processes such as cytotoxicity towards eukaryotic cells, complement activation, steroid synthesis, mast cell activation, monocyte chemo-attraction, and enhancement of cytokine expression [38]. In the case of *Ar*-1, this peptide has been found to modulate complement activity in vitro depending on its concentration: stimulation at low concentrations (3.5–14.5 µM)
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** 3-dimensional structures of arenicin peptides. Secondary structure of **Ar-1** (left, PDB id: 2JSB [32]), **Ar-2** (center, PDB id: 2JNI [36]) and **Ar-3** (right, PDB id: 5VOY [35]) represented as cartoons displaying anti-parallel β-sheets stabilized by disulfide bond(s). Below are reported their respective amino acid sequences showing cysteines highlighted in yellow and disulfide bond connectivity; as well as the disulfide-free derivatives **Ar-1-Abu**, **Ar-2-Abu**, **Ar-3-Abu** in which all cysteines were replaced by alpha-amino-*n*-butyric acid (Abu) residues, and **Ar-3(3–20)** and **Ar-3(7–16)** in which cysteines 1–16 and cysteines 3–20 were replaced by Abu residues, respectively</figcaption></figure>

and acting as a complement inhibitor at higher concentrations (29–58 µM) [39].

Finally, in contrast to many other antimicrobial peptides, such as β-defensins [40, 41] and polymyxin B [32], arenicins are very stable AMPs that retain their antibacterial activity at high salt concentrations [42] and under high temperature conditions. A property probably due to the presence of one or two disulfide bonds along with several hydrogen bonds, allowing the formation of a ring that contributes to the conformational stability of the molecule (Fig. 1) [33, 43]. Considering that salt-induced inactivation of host defense peptides is an important issue in CF patients [44, 45], the rapid and efficient killing of pathogenic bacteria in the presence of NaCl concentrations makes arenicin peptides particularly interesting for lead structures for drug development.

From such physicochemical properties, together with their promising antibacterial activities against four pathogens commonly isolated from CF patients, namely

*Pseudomonas. aeruginosa*, *Staphylococcus aureus*, *Bacillus subtilis*, and the yeast *C. albicans* [33]; we hypothesized that these three arenicin isoforms might be active against *M. abscessus*, the most drug-resistant mycobacterial species.

Hence, in this study we first compared the antimycobacterial activity, cytotoxicity and hemolytic activity of **Ar-1**, **Ar-2** and **Ar-3**, as well as their respective disulfide-free derivatives (Fig. 1), with the aim of understanding the role of disulfide bridges in peptide function. To achieve this goal, the selective substitution of Cysteine residues by alpha-amino-*n*-butyric acid (Abu) was performed. Although the effects of replacing half-cystine residues in peptides and proteins with non-bridging residues such as alanine, valine, serine or threonine have been extensively studied, Abu has proved to be a more attractive substitutable residue because it is isosteric to half-cystine and has a similar polarity [46]. As a result, such a Cys to Abu replacement has become the classic strategy used
for decades to prevent the formation of disulfide link-
age while leaving the peptide sequence globally unaltered
[47–49]. We further evaluated the potential of **Ar-1** to
permeabilize bacterial cytoplasmic membranes upon
interaction with *M. abscessus* cells, and also investigated
its binding affinity with monolayers of total lipids from
*M. abscessus*.

**Methods**

Mycobacterium abscessus strains, growth conditions
and arenicin peptides

*Mycobacterium abscessus* S and R strains (CIP 104536 T) were grown in Middlebrook 7H9 medium supplemented with 0.05% (v/v) Tween 80, 0.2% (v/v) glycerol and 10% (v/v) oleic acid–albumin–dextrose–catalase (OADC enrichment; BD Difco) (7H9-S<sup>OADC</sup>) at 37 °C under stirring (200 rpm). We used **Ar-1** (RWC<sup>3</sup>VYAYVRVRGV-LVRYRRC<sup>20</sup>W) and **Ar-1-Abu**, in which the cysteines in position 3 and 20 were replaced by alpha-amino-*n*-butyric acid (Abu) residues. We also tested **Ar-2** (RWC<sup>3</sup>VYAY-VRIRGVLVRYRRC<sup>20</sup>W), **Ar-2-Abu** (cysteines 3 and 20 replaced), **Ar-3** (GFC<sup>3</sup>WYVC<sup>7</sup>VYRNGVRVC<sup>16</sup>YRRC<sup>20</sup>N), **Ar-3-Abu** (all four cysteines were replaced by Abu), **Ar-3(3–20)** (cysteines 7 and 16 replaced), and **Ar-3(7–16)** (cysteines 3 and 20 replaced). All arenicins and arenicin-Abu derivatives were obtained from GenScript (Piscataway, NJ, USA) with purity higher than 95%.

Minimal inhibitory concentration (MIC) determination
via the resazurin microtiter assay

For determining the antimicrobial activity of the differ-
ent peptides, the Middlebrook 7H9 broth microdilution
method was used in sterile 96-well flat-bottom Greiner
Bio-One polypropylene microplates with lid (Thermo
Fisher Scientific) using the resazurin microtiter assay
(REMA) [50–52]. Briefly, *Mycobacterium abscessus* was
cultured up to mid log-phase (OD<sub>600</sub> ~ 1.5–2) and diluted
to a cell density of 5 × 10<sup>6</sup> cells/mL in 7H9-S<sup>OADC</sup>. Then,
100 µL of this above inoculum was distributed to each
well containing serial two-fold dilutions of arenicin,
arenicin derivative or controls to a final volume of 200
µL. Growth controls containing no peptide (i.e., bacteria
only = B), inhibition controls containing 50 µg/mL kana-
mycin (Euromedex, Souffelweyersheim, France) and ste-
rility controls (i.e., medium only) without bacteria were
included. After 5 days incubation at 37° C in a humidity
chamber to prevent evaporation, 20 µL of a 0.025% (w/v)
resazurin solution was added to each well. The microplate
was re-incubated until appearance of a color change from
blue to pink in the control well (i.e., bacteria without anti-
biotics). Fluorescence of the metabolite resorufin (λ<sub>ex</sub>/
λ<sub>em</sub> = 530/590 nm) was measured using a Tecan Spark
10 M <sup>TM</sup> multimode microplate reader (Tecan Group

Ltd, France). Relative fluorescence units (RFU) were
defined as: RFU% = (test well FU/mean FU of control B
wells) × 100. MIC₅₀ and MIC₉₀ values were determined
by fitting the RFU% sigmoidal dose-response curves in
Kaleidagraph 4.2 software (Synergy Software, Reading,
PA). The concentration of peptide leading to 50% and
90% inhibition of *M. abscessus* growth was defined as
the MIC₅₀ and MIC₉₀, respectively. All experiments were
performed independently at least three times.

Cytotoxic assay on human cells

The toxicity of each peptide was evaluated on human cell
lines using a resazurin assay as previously described [53,
54]. Human lung epithelial cells BEAS-2B (ATCC CRL-
9609), liver cells HepG2 (ATCC HB-8065), and kidney
cells A498 (ATCC HTB-44) were used. All cells were cul-
tured in Dulbecco's modified essential medium (DMEM)
supplemented with 10% heat-inactivated fetal calf serum
(FBS, Thermo Fisher Scientific), and 1% L-glutamine
(Thermo Fisher Scientific) (DMEM<sup>FBS</sup>) at 37 °C in a 5%
CO<sub>2</sub> incubator to subconfluent concentrations. Cells
were detached with trypsin-EDTA solution (Thermo
Fisher Scientific), and around 1×10<sup>4</sup> cells/well were
seeded in 96-well flat-bottom microplates (Greiner Bio-
One) with a lid, and cultured for additional 48-72 h at
37 °C and 5% CO<sub>2</sub> until cell confluence. The medium was
removed, and 100 µL of serial two-fold dilutions of the
peptides in DMEM<sup>FBS</sup> were added to each well. Following
a 48 h-incubation period at 37 °C and 5% CO<sub>2</sub>, cell viabil-
ity was evaluated using the resazurin assay as described
above. Fluorescence of the resazurin metabolite resorufin
(λ<sub>ex</sub>/λ<sub>em</sub> = 530/590 nm) was measured after 2 h incuba-
tion at 37 °C and 5% CO<sub>2</sub> in the dark, with a microplate
reader (Biotek, Synergy Mx, Colmar, France). The cyto-
toxic concentration of peptide leading to 50% cell death
as compared to the control was defined as the CC<sub>50</sub>, and
was determined by fitting the RFU% sigmoidal dose-
response curves in Kaleidagraph 4.2 software (Synergy
Software). All experiments were conducted in triplicate.

Hemolytic activity assay

The hemolytic activity of each peptide was determined
as previously described [55]. Briefly, human erythro-
cytes (red blood cells, hRBC; Divbioscience, Ulvenhout,
Netherlands) were washed 2-times with sterile phos-
phate buffered saline (PBS; Thermo Fisher Scientific)
and pelleted by centrifugation at 800 rpm for 5 min;
then resuspended in DMEM<sup>FBS</sup> to a final erythrocyte
concentration of 8% (v/v). Then, 100 µL of this hRBC
solution was distributed to each well of sterile 96-well
flat-bottom microplates (Greiner Bio-One) containing
serial two-fold dilutions of arenicin, arenicin-Abu deriva-
tive or controls in DMEM<sup>FBS</sup> to a final volume of 200 µL.
After 1 h incubation at 37 °C, microplates were centrifuged at 800 rpm for 5 min. Then, 100 µL of supernatant were carefully transferred to a new 96-well microplate, and OD₄₀₅ corresponding to the release of hemoglobin was measured using a microplate reader (Biotek, Synergy Mx, Colmar, France). DMEM<sup>FBS</sup> and Triton-X100 at 0.1% (v/v) were used as negative (i.e., 0% hemolysis) and positive (i.e., 100% hemolysis) controls, respectively. The concentration of peptide leading to 10% and 50% of hemolysis compared to the positive control was defined as the HC₁₀ and HC₅₀, respectively, and was determined by fitting the dose-response curves in Kaleidagraph 4.2 software (Synergy Software). All experiments were done in triplicate.

## Membrane permeabilization assay

For evaluating whether **Ar-1** induces *M. abscessus* membrane permeabilization, bacterial cells in exponential growth phase were resuspended at 10⁹ cells/ mL in 7H9-S<sup>OADC</sup>, before addition of 1 mg/mL propidium iodide (Sigma Aldrich, Lyon, France), a DNA/ RNA probe that is cell-impermeable. Then, 100 µL of this mix were distributed in the wells of a 96-well flat-bottom Greiner Bio-One polypropylene microplate already containing **Ar-1** at 4×MIC₉₀, 150 µM cetyltrimethylammonium bromide (CTAB) as positive control, or 7H9-S<sup>OADC</sup> for the negative control [56]. Fluorescence (λ<sub>ex</sub>/λ<sub>em</sub> = 530/590 nm) was recorded at 5, 15, 30, 60 and 120 min with a Tecan Spark 10 M™ multimode microplate reader (Tecan Group Ltd, France). Results were expressed in percentage of permeabilization compared to the maximum fluorescence obtained with CTAB, after removing the fluorescence of the negative control. All experiments were done independently at least three times.

## Lipid insertion assay through surface pressure measurement

The insertion of **Ar-1** into total lipids from *M. abscessus* was measured using reconstituted lipid monolayer at the air-water interface as previously described [56]. Briefly, total lipids from *M. abscessus* S or R dry extracts were incubated at room temperature and under shaking with CHCl₃-MeOH (1:2; v/v) for 16 h. Re-extraction of the residual lipids was done first with CHCl₃-MeOH (1:1; v/v), and then with CHCl₃-MeOH (2:1; v/v) during 16 h. The organic phases were pooled, concentrated under reduced pressure, re-suspended in CHCl₃-MeOH (3:1; v/v), and then washed with 0.3% (w/v) NaCl solution. Centrifugation at 5000 rpm for 15 min allowed separation of the organic and aqueous phases. Then, the organic phase was dried over MgSO₄. Extracted total lipids were

dried, solubilized in CHCl₃-MeOH (2:1, v/v), and stored at -20 °C under nitrogen.

Surface pressure measurement was performed in a controlled atmosphere at 20 ± 1 °C using a fully automated microtensiometer (µTROUGH SX, Kibron Inc., Helsinki, Finland). This apparatus allowed the recording of the adsorption kinetics of a ligand onto a monomolecular film using a set of specially designed Teflon troughs. A few microliters of each total lipid extracts were spread using a 50 µL Hamilton's syringe at the surface of a sterile ultra-pure water (volume 800 µL) until the desired initial surface pressure (Πᵢ) was reached, thus generating a stable lipid monolayer at the air-water interface. The waiting time for the spreading solvent evaporation and for the lipid monolayer to reach equilibrium varied from 5 to 10 min depending on the spreading volume and the initial surface pressure. **Ar-1** was further injected into the ultra-pure water sub-phase below the stable monolayer, with a 10 µL Hamilton's syringe. The surface pressure increase (ΔΠ<sub>max</sub>) due to the insertion of the peptide into the monolayer of total lipids was then continuously recorded until the equilibrium surface pressure was reached. The data were analyzed using the Filmware 2.5 program (Kibron Inc., Helsinki, Finland).

## Electron microscopy

Cultures of *M. abscessus* S (10⁹ cells/mL) were incubated with **Ar-1** at 4×MIC₉₀ for 120 min, then fixed with a solution of 2% glutaraldehyde in 200 mM HEPES (Thermo Fisher Scientific) (pH 7) directly in the culture medium (v/v) for 20 min at room temperature. After centrifugation and removal of the supernatant, the pellet was immediately covered with a second solution of 1% glutaraldehyde for 1 h at 4 °C. After four rinses of 5 min each with the HEPES buffer, the samples were post-fixed with 1% OsO₄ (Electron Microscopy Sciences, Hatfield, PA, USA) in H₂O for 1 h at 4 °C. Then, four rinses of 5 min each were done with water. At this stage, the samples were gradually dehydrated using acetone-water mixtures of 50:50, 70:30, 90:10, and 96:4 (v/v), respecting a 5 min pause between each bath. Subsequently, the samples were transferred to 100% acetone and placed on a circular coverslip to complete the dehydration process. The coverslip was glued onto a nail-shaped support. Following carbonization, the samples were observed using the scanning electron microscope Tescan VEGA3 SBH (30 kV; Tescan, Brno—Kohoutovice, Czech Republic) and Jeol JSM-7900F (5 kV, 6 mm, LED detector) for higher magnification (Jeol, Croissy-sur-Seine, France).

## Induction of resistance assay

Induction of bacterial resistance by **Ar-1** was achieved by exposure of *M. abscessus* S and R to **Ar-1** or to the
control antibiotic clarithromycin (CLR) for 42 consecutive days, and with repeated broth microdilution susceptibility testing and subsequent MIC determination. After each MIC determination, the well containing the highest concentration of **Ar-1** or CLR that allows bacterial growth was diluted 1:1000 in 7H9-S<sup>OADC</sup> and used as inoculum for the next MIC assay with fresh amounts of **Ar-1** or CLR.

## Statistical analysis

Graphpad Prism 8 (GraphPad Inc.) was used to perform all statistical analyses, which details are given in

<table><caption>**Table 1** Antibacterial activities (µM) of arenicin and arenicin-Abu derivatives against *M. abscessus* S and R compared to standard drug Amikacin<sup>a</sup></caption><thead><tr><th rowspan="2"></th><th colspan="2">MIC<sub>50</sub>/MIC<sub>90</sub> (µM)</th></tr><tr><th>M. abscessus CIP 104536<sup>T</sup></th><th></th></tr><tr><th></th><th>S variant</th><th>R variant</th></tr></thead><tbody><tr><td>Ar-1</td><td>11.6 ± 1.0/20.2 ± 1.6</td><td>11.4 ± 0.5/17.5 ± 0.8</td></tr><tr><td>Ar-1-Abu</td><td>> 100</td><td>> 100</td></tr><tr><td>Ar-2</td><td>19.8 ± 1.3/29.3 ± 0.6</td><td>53.1 ± 0.3/ > 100</td></tr><tr><td>Ar-2-Abu</td><td>89.3 ± 9.0/ > 100</td><td>> 100</td></tr><tr><td>Ar-3</td><td>5.3 ± 0.3/12.2 ± 0.1</td><td>44.7 ± 3.0/ > 100</td></tr><tr><td>Ar-3-Abu</td><td>> 100</td><td>> 100</td></tr><tr><td>Ar-3(3-20)</td><td>48.3 ± 1.4/ > 100</td><td>77.8 ± 10.1/ > 100</td></tr><tr><td>Ar-3(7-16)</td><td>17.2 ± 2.7/21.3 ± 3.6</td><td>24.6 ± 0.5/ > 100</td></tr><tr><td>AMK</td><td>3.9 ± 0.19 / 5.8 ± 0.20</td><td>7.4 ± 0.26/10.1 ± 0.45</td></tr></tbody></table>

All values are expressed as mean ± SD (n ≥ 3). AMK: amikacin

each figure legend. Differences were considered significant when the calculated *p*-values were smaller than 0.05.

## Results

### Antimicrobial activity and cytotoxicity

Drug susceptibility testing of the arenicin and arenicin-Abu derivatives was assessed against both S and R variants of *M. abscessus*, with amikacin (AMK) as standard drug. The corresponding MIC<sub>50</sub>/MIC<sub>90</sub> values for each peptide, as determined by the REMA assay [51], are reported in Table 1. The three arenicin peptides displayed comparable antibacterial activity against *M. abscessus* S growth, with MIC<sub>50</sub>/MIC<sub>90</sub> values in the range 5.3–19.8 µM and 12.2–29.3 µM, respectively (Table 1 and Fig. 2). Considering the MIC values reached on *M. abscessus* R, they are 2.5- to eightfold greater for **Ar-2** and **Ar-3**, respectively, than those obtained on the S variant (Table 1). In contrast, only **Ar-1** was found to exhibit similar highly potent MIC<sub>50/90</sub> values against the two morphotypes (mean MIC<sub>50</sub>=11.5 ± 1.0 µM/mean MIC<sub>90</sub>=18.8 ± 0.9 µM). Furthermore, among the three native arenicin peptides tested, the fact that **Ar-1** also displayed MIC<sub>90</sub> values comparable to those of the reference drug amikacin (AMK), made it the best growth peptide inhibitor of the two variants S and R of *M. abscessus*.

Replacement of all cysteines by Abu residues in the **Ar-1-Abu**, **Ar-2-Abu** and **Ar-3-Abu** resulted in a total loss of their antibacterial activity against the two *M. abscessus* variants (Table 1 and Fig. 2). Interestingly, the selective replacement of only two cysteines by Abu residues in **Ar-3**, likely to remove only one disulfide bond out of two, led to two different antibacterial behaviors. While **Ar-3(7-16)** exhibited MIC<sub>50/90</sub> values in the same order

<figure><img src="image_3.png" /><figcaption>**Fig. 2** Dose-response activity of the different arenicin and arenicin-Abu peptides against **A. M. abscessus** S and **B. M. abscessus** R strains replicating in broth medium, expressed as normalized relative fluorescence units (RFU%). Results are expressed as mean ± SD of at least three independent assays (n ≥ 3)</figcaption></figure>

<footnote><sup>a</sup> MIC<sub>50</sub>/MIC<sub>90</sub>: peptide minimal concentration leading to 50% and 90% inhibition of in vitro growth, respectively, as determined by the REMA assay</footnote>
of magnitude as **Ar-3** against both *M. abscessus* morphotypes, a significant increase of around 10 times was observed for **Ar-3(3-20)** MIC values (MIC<sub>50</sub>=48.3 µM/MIC<sub>90</sub> > 100 µM vs. MIC<sub>50</sub>=5.3 µM/MIC<sub>90</sub>=12.2 µM, for **Ar-3(3-20)** and **Ar-3**, respectively) against *M. abscessus* S, with no real change in its antibacterial activity against the R variant compared to **Ar-3** (Table 1 and Fig. 2). These results highlight the importance of the disulfide bonds (one for **Ar-1**/*Ar-2* and two for **Ar-3**) which in such small peptides enables the integrity of the 3D structure to be maintained, and suggest that their presence is crucial for the antimycobacterial activity of each peptide against *M. abscessus*.

In parallel, the cytotoxic activity of the different peptides towards nontumorigenic human lung epithelial cells (BEAS-2B cells), human cells from a hepatocellular carcinoma (HepG2), and human kidney cells from a kidney cancer patient (A498) was investigated using a classical dose-response assay [57]. The calculated response parameter was CC<sub>50</sub>, which corresponds to the concentration required to elicit a 50% decrease in cell viability in vitro, compared to the control. As seen in Table 2 and Fig. 3A, B, no toxicity towards BEAS-2B and HepG2 cells was observed, as demonstrated by CC<sub>50</sub> values > 100 µM. Regarding kidney A498 cells, although **Ar-1** and **Ar-1-Abu**, and to a lesser extend **Ar-2-Abu** (CC<sub>50</sub>=92.1 µM), were nontoxic, all other peptides displayed high toxicity with CC<sub>50</sub> values ranging from 36.6 to 46.7 µM (Table 2 and Fig. 3C). Such toxicity towards A498 cells was best characterized by calculating the respective therapeutic index [58] (TI<sub>A498</sub>); i.e., the ratio between CC<sub>50</sub> on A498 cells and MIC<sub>50</sub> on *M. abscessus* strains, for each peptide (Table 2). Accordingly, and

except for **Ar-1** (TI<sub>A498</sub>>8.6 for both variants) and **Ar-3** (TIA<sub>498</sub>=8.8 on *M. abscessus* S), all tested arenicin and arenicin-Abu peptides displayed TI<sub>A498</sub> values < 2.2 synonymous with a very narrow window between efficacy and toxicity.

To better characterize the innocuity of the eight peptides, their hemolytic activity on human erythrocytes was further assessed through determination of their respective HC<sub>50</sub> and HC<sub>10</sub> values and calculation of their corresponding therapeutic indexes, TI<sub>H50</sub> and TI<sub>H10</sub> (Table 3 and Fig. 2D). First, in our experimental conditions, only **Ar-2** was found to exert a strong hemolytic activity against red blood cells with a HC<sub>50</sub> of 29.2 µM resulting in TI<sub>H50</sub> of 1.5 and 0.5 with regards to *M. abscessus* S and R variant, respectively (Table 3 and Fig. 2D). Considering their HC<sub>10</sub> values, except **Ar-1-Abu** and **Ar-3(3, 20)** totally inactive, all other arenicin and arenicin-Abu peptides tested showed variable hemolytic activities on human erythrocytes, resulting in predominantly low (<0.4) and up to moderate (1-2.5) therapeutic indexes; with **Ar-1** displaying the most promising TI<sub>H10</sub> values (Table 3).

Overall, all these results clearly emphasized the efficacy of **Ar-1** against *M. abscessus* S and R. Indeed, **Ar-1** exhibited the most potent MIC values against both variants of *M. abscessus* (mean MIC<sub>90</sub>~19 µM—Table 1), was nontoxic to host cells (CC<sub>50</sub>>100 µM—Table 2), and showed only very weak hemolytic activity thus exhibiting the best therapeutic indexes related to hemolysis of human erythrocytes (TI<sub>H50</sub>>~8.7/TI<sub>H10</sub>~1.1—Table 3). Given all these findings, amongst the 8 tested peptides, **Ar-1** was therefore found to display the best combination of antibacterial and cytotoxic properties and was then selected for further investigations.

<table><caption>**Table 2** Evaluation of the cytotoxicity of the arenicin and arenicin-Abu peptides</caption><thead><tr><th rowspan="2"></th><th colspan="3">CC<sub>50</sub> (µM)<sup>a</sup></th><th colspan="2">TI<sub>A498</sub><sup>b</sup></th></tr><tr><th>BEAS-2B</th><th>HepG2</th><th>A498</th><th>S variant</th><th>R variant</th></tr></thead><tbody><tr><td><b>Ar-1</b></td><td>&gt; 100</td><td>&gt; 100</td><td>&gt; 100</td><td>&gt; 8.6</td><td>&gt; 8.8</td></tr><tr><td><b>Ar-1-Abu</b></td><td>&gt; 100</td><td>&gt; 100</td><td>&gt; 100</td><td>-</td><td>-</td></tr><tr><td><b>Ar-2</b></td><td>&gt; 100</td><td>&gt; 100</td><td>37.7 ± 1.1</td><td>1.9</td><td>0.28</td></tr><tr><td><b>Ar-2-Abu</b></td><td>&gt; 100</td><td>&gt; 100</td><td>92.1 ± 2.3</td><td>1.0</td><td>&lt; 1.0</td></tr><tr><td><b>Ar-3</b></td><td>&gt; 100</td><td>&gt; 100</td><td>46.7 ± 1.7</td><td>8.8</td><td>1.0</td></tr><tr><td><b>Ar-3-Abu</b></td><td>&gt; 100</td><td>&gt; 100</td><td>36.6 ± 0.9</td><td>&lt; 0.4</td><td>&lt; 0.4</td></tr><tr><td><b>Ar-3(3-20)</b></td><td>&gt; 100</td><td>&gt; 100</td><td>42.0 ± 1.3</td><td>0.87</td><td>0.54</td></tr><tr><td><b>Ar-3(7-16)</b></td><td>&gt; 100</td><td>&gt; 100</td><td>37.7 ± 1.2</td><td>2.2</td><td>1.5</td></tr></tbody></table>

All values are expressed as mean ± SD (n = 3)

## Mechanism of action

The fact that **Ar-1** is known to induce bacterial death through a pore forming mechanism in Gram-negative and -positive bacteria [33, 42, 43, 59–63] prompted us to evaluate its effect on membrane integrity of *M. abscessus* S and R using a propidium iodide assay (Fig. 4). Indeed, propidium iodide is a cell permeable dye which can enter bacteria only if the membrane is damaged, thus forming a highly fluorescent complex with DNA and RNA. As a consequence, the monitoring of propidium iodide fluorescence allows evaluation of membrane integrity. The membranolytic detergent cetyltrimethylammonium bromide (CTAB) was used as a positive control, corresponding to 100% permeabilization [15].

With the two S & R variants of *M. abscessus*, **Ar-1** induced comparable permeabilization pattern over time (Fig. 4). As shown in Fig. 4, a plateau value corresponding to a non-significantly different membrane

<footnote><sup>a</sup> CC<sub>50</sub>: Cytotoxic concentration of peptide leading to 50% cell toxicity in vitro, compared to the control</footnote>

<footnote><sup>b</sup> Therapeutic Index (TI) calculated by dividing the CC<sub>50</sub> value on A498 cell lines by the MIC<sub>50</sub> reached for each *M. abscessus* morphotype</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 3** Cytotoxic and hemolytic activity of the arenicin and arenicin-Abu peptides. **A–C** Cytotoxicity dose-response curves following 48 h-exposure to increasing concentrations of each peptide on human **A** BEAS-2B cells, **B** HepG2 cells, and **C** A498 cells. **D** Dose-response curves for human erythrocyte hemolysis after exposure to increasing concentrations of each peptide. Results are expressed as the mean ± SD of three independent experiments</figcaption></figure>

<table><caption>**Table 3** Evaluation of the hemolytic activity of the arenicin and arenicin-Abu peptides against Human erythrocytes</caption><thead><tr><th rowspan="2"></th><th rowspan="2">HC<sub>50</sub> (µM)<sup>a</sup></th><th colspan="2">TI<sub>H50</sub><sup>b</sup></th><th rowspan="2">HC<sub>10</sub> (µM)<sup>a</sup></th><th colspan="2">TI<sub>H10</sub><sup>c</sup></th></tr><tr><th>S variant</th><th>R variant</th><th>S variant</th><th>R variant</th></tr></thead><tbody><tr><td>Ar-1</td><td>&gt; 100</td><td>&gt; 8.6</td><td>&gt; 8.8</td><td>20.2 ± 1.8</td><td>1.0</td><td>1.2</td></tr><tr><td>Ar-1-Abu</td><td>&gt; 100</td><td>-</td><td>-</td><td>&gt; 100</td><td>-</td><td>-</td></tr><tr><td>Ar-2</td><td>29.2 ± 2.4</td><td>1.5</td><td>0.5</td><td>4.9 ± 0.7</td><td>0.2</td><td>-</td></tr><tr><td>Ar-2-Abu</td><td>&gt; 100</td><td>&gt; 1.1</td><td>-</td><td>15.4 ± 1.0</td><td>&lt; 0.2</td><td>&lt; 0.2</td></tr><tr><td>Ar-3</td><td>&gt; 100</td><td>&gt; 18.9</td><td>&gt; 2.2</td><td>30.3 ± 2.5</td><td>&gt; 2.5</td><td>&lt; 0.3</td></tr><tr><td>Ar-3-Abu</td><td>&gt; 100</td><td>-</td><td>-</td><td>39.3 ± 3.2</td><td>&lt; 0.4</td><td>&lt; 0.4</td></tr><tr><td>Ar-3(3–20)</td><td>&gt; 100</td><td>&gt; 2.1</td><td>&gt; 1.3</td><td>&gt; 100</td><td>-</td><td>-</td></tr><tr><td>Ar-3(7–16)</td><td>&gt; 100</td><td>&gt; 5.8</td><td>&gt; 4.1</td><td>41.1 ± 4.3</td><td>1.9</td><td>&lt; 0.4</td></tr></tbody></table>

All values are expressed as mean ± SD (n = 3)

<footnote><sup>a</sup> HC10 and HC<sub>50</sub>: hemolytic concentration of peptide leading to 10% and 50% hemolysis in vitro, compared to the control</footnote>

<footnote><sup>b</sup> TI<sub>50</sub>: Therapeutic Index calculated by dividing the HC<sub>50</sub> value by the MIC<sub>50</sub> reached for each *M. abscessus* morphotype</footnote>

<footnote><sup>c</sup> TI<sub>10</sub>: Therapeutic Index calculated by dividing the HC<sub>10</sub> value by the MIC<sub>90</sub> reached for each *M. abscessus* morphotype</footnote>
<figure><img src="image_5.png" /><figcaption>**Fig. 4** Effect of Ar-1 on *M. abscessus* membrane integrity. *M. abscessus* S (blue circles) or R (green squares) variant was exposed to **Ar-1** at 4×MIC~90~ concentration. The effect of Ar-1 on the integrity of the mycobacterial membrane was followed over 120 min using propidium iodide and compared to the maximum fluorescence obtained with 150 µM CTAB (100% permeabilization). Results are expressed as mean ± SD of three independent experiments. Statistical analysis was done using a non-parametric Mann-Whitney test using Prism 8.0 (Graphpad Inc.): ns, not significant (*p* > 0.05)</figcaption></figure>

permeabilization of 36.0 ± 3.0% and 47.4 ± 16.0% (*p* > 0.05) was reached between 60- and 120-min incubation for *M. abscessus* S and R, respectively. Such a result is in line with the similar MIC~50/90~ values obtained by **Ar-1** against the two morphotypes.

### Ar-1 interaction with monolayers of total lipids from *M. abscessus*

Given the ability of **Ar-1** to cause membrane permeabilization in *M. abscessus*, its ability to insert into monolayers of total lipids from this bacterium [56] was further assessed (Fig. 5). In a first set of experiments, the dose-dependent insertion of **Ar-1** into total lipid monolayers was investigated (Fig. 5A). Total lipids were first spread until an initial surface pressure $Π_i = 30 \pm 0.5$ mN/m, corresponding to a lipid packing density theoretically equivalent to that of the outer leaflet of a cell membrane [64]. Increasing concentrations of **Ar-1** were then injected into the aqueous phase below the lipid monolayer, and the variation in surface pressure due to the insertion of the peptide into the total lipid monolayer was continuously recorded until the new equilibrium surface pressure ($ΔΠ_{max}$) was reached (around 90 min—Fig. 5A). In agreement with propidium iodide assay results (Fig. 4), data showed that **Ar-1** was able to insert into total lipid monolayers from both *M. abscessus* S and R with a maximal insertion reached at a concentration of 1.25 µM (i.e., ~0.11×MIC~50), above which a plateau value in terms of $ΔΠ_{max}$ was obtained (Fig. 5A). Moreover, **Ar-1** insertion resulted in a significantly higher equilibrium surface pressure, in the 1.25–10.0 µM concentration range, with total lipid monolayers from *M. abscessus* S ($ΔΠ_{max} = 9.3 \pm 0.69$ mN/m) vs. *M. abscessus* R ($ΔΠ_{max} = 7.5 \pm 0.65$ mN/m; *p*-value < 0.001) (Fig. 5A).

In a second series of experiments, the influence of $Π_i$ [65] on the insertion process of **Ar-1** into total lipid monolayers from *M. abscessus* was studied. The optimal final

<figure><img src="image_6.png" /><figcaption>**Fig. 5** Insertion of Ar-1 into monolayers of total lipids from *M. abscessus*. A Variations of the surface pressure increase ($ΔΠ_{max}$) as a function of the peptide concentration upon **Ar-1** insertion into total lipid monolayers from *M. abscessus* S and R. The increase in surface pressure due to the insertion of **Ar-1** into the monolayers of total lipids, spread at an initial surface pressure $Π_i = 30$ mN/m, was continuously monitored. Data are mean values ± SD of three independent experiments. Statistical analysis was done using a non-parametric Mann-Whitney test with Prism 8.0 (Graphpad Inc): ***$p$ < 0.001. B Binding parameters of **Ar-1** into a monomolecular film of total lipids from *M. abscessus* S and R. Variation of surface pressure ($ΔΠ_{max}$) after **Ar-1** injection (1.25 µM final concentration) is represented as a function of the initial surface pressure, $Π_i = 10–30$ mN/m</figcaption></figure>
peptide concentration of 1.25 µM, determined above by performing ΔΠ measurements at various **Ar-1** concentrations, was used in these experiments. The plot ΔΠ<sub>max</sub>=f(Π<sub>i</sub>) depicted in Fig. 5B, was first used to evaluate the binding parameters of **Ar-1**. Linear extrapolation to no increase in the surface pressure (ΔΠ<sub>max</sub>=0) allowed to determine the critical surface pressure (Π<sub>c</sub>), also named “maximum insertion pressure” [66], as being equal to 47.2 and 43.7 mN/m for *M. abscessus* S and R, respectively. Above this Π<sub>c</sub> value, which is specifically related to the peptide and the lipid forming the monolayer, no increase in the surface pressure will then occur. In addition, the synergy factor (slope of the linear regression +1) which has been demonstrated to modulate protein/peptide insertion, as well as ΔΠ<sub>0</sub> (y-intercept of the curve corresponding to Π<sub>i</sub>=0) [65–67] can also be deduced from the same plot. Indeed, and as defined by Salesse group [66, 67], the observed positive synergy values (0.42–0.43) and the occurrence of a ΔΠ<sub>0</sub> value (25.3–26.9 mN/m) lower than the related Π<sub>c</sub> (43–47 mN/m) are consistent with an ‘insertion’ surface pressure and favorable interactions between the peptide and the monolayer of total lipids from *M. abscessus*.

Microscopic observations

*Mycobacterium abscessus* S cultures were treated with **Ar-1** at 4×MIC<sub>90</sub> for 120 min, then fixed and processed for scanning electron microscopy experiments. As can be seen in Fig. 6, different modifications in the cell shape could be observed on **Ar-1**-treated *M. abscessus* S cells (Fig. 6B, D, F, and H) as compared to untreated one (Fig. 6A, C, E, and G). Indeed, bacteria incubated with **Ar-1**, especially when they are in clusters, seem to be covered by a sticky substance (Fig. 6B, D vs. A, C). Furthermore, clear alteration of cell wall morphology can be seen on single isolated bacteria treated with **Ar-1** which seem to be empty with edges appearing blurred (see blue arrows in Fig. 6F and H). It should be noted that some cell fragments are also visible (see yellow arrowhead in Fig. 6H), thus strengthening the appearance of empty cells. In contrast, untreated control cells show a smooth and intact bacterial membrane (Fig. 6E and G). All these data clearly support disruption of the bacterial membrane through the pore forming activity of **Ar-1** on *M. abscessus* S cells.

Induction of resistance assay

Regarding **Ar-1**, all results obtained above suggest that this peptide may target the bacterial membrane of *M. abscessus*. Given such membranolytic activity, **Ar-1** should be less prompt at selecting resistant bacteria compared to conventional antibiotics targeting enzymes/proteins for which mutations may induce resistance. This

is especially true with *M. abscessus*, which is naturally resistant to many antibiotics and possesses the ability to acquire reversible and irreversible resistance to various antibiotics after a long period of treatment. In this context, we evaluated whether this mycobacterium could develop resistance to **Ar-1** (Fig. 7).

As depicted in Fig. 7, a 42-days continuous exposure to **Ar-1** induced no significant change in the MIC<sub>90</sub> value of this peptide against *M. abscessus* S and R variants, contrary to the conventional antibiotic clarithromycin (CLR) for which a 32-fold increase in MIC<sub>90</sub> values was observed after 20–30 days of exposure.

Discussion

Infections due to non-tuberculous mycobacteria represent a major public health problem worldwide, including developed countries where their incidence can even be higher than that of tuberculosis [68–70]. Among non-tuberculous mycobacteria, *M. abscessus* is a problem, causing skin infections as well as infections involving soft tissues [71, 72], being increasingly prevalent during the last decade in CF patients [73, 74]. Yet, the treatment usually lasts 18 to 24 months with a cocktail of antibiotics, inducing severe adverse events in patients and also being a high cost for society [70, 75–77]. Furthermore, the treatment outcomes are often disappointing with a cure rate of patients with a *M. abscessus* pulmonary infection of only 25–58% [76, 78, 79]. This can be explained by several characteristics of *M. abscessus*, notably biofilm-like aggregation and intracellular survival, limiting antibiotic efficiency, as well as *M. abscessus* high resistance to many antibiotics [69, 80]. This resistance, whether intrinsic, adaptive or acquired, is such that *M. abscessus* is referred to as the “antibiotic nightmare” [10]. As a consequence, new therapeutic tools, efficient against both *M. abscessus* S and R morphotypes, generating no resistance, are needed.

With no novel antibiotics entering the market in the past twenty years and the emergence of multidrug resistance (MDR) in many pathogenic bacteria, AMPs are making a comeback as the tool of choice against such pathogens. Indeed, thirty years after their discovery, a better understanding of their mode of action, their modification feasibilities and their synthesis at lower cost are reigniting the commercial development of AMPs [20, 81]. Among AMPs, arenicins are β-hairpin peptides stabilized by disulfide bonds which have been evidenced to exert antibiotic activities in vivo in mouse models of urinary tract infection, peritonitis and endotoxemia [82]. Arenicin analogues are currently undergoing preclinical studies against infections caused by Gram-negative and Gram-positive MDR bacteria [35, 83], thus highlighting
<figure><img src="image_7.png" /><figcaption>**Fig. 6** Scanning electron microscopy of untreated control cells (left panels **A**, **C**, **E**, and **G**) and cells treated with **Ar-1** at 4×MIC~90~ concentration for 120 min (right panels **B**, **D**, **F**, and **H**). Control cells appear well distinguishable, with clear cell contour. Cells treated with **Ar-1**, when in clusters, seem to be covered in a sticky substance (**B** and **D**). When isolated, cells treated with **Ar-1**, contrary to control cells, have blurred edges (blue arrow in **F** and **H**). Some cell pieces can also be seen in the treated sample (yellow arrowhead in **H**), contrary to the control one</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 7** *Mycobacterium abscessus* is not able to develop resistance mechanisms to **Ar-1**. The ability of *M. abscessus* A S or B R to develop resistance to **Ar-1** was assessed by repeated MIC determination over 42 consecutive days exposure to this peptide. Clarithromycin (CLR) prompted to cause resistance was used as a positive control</figcaption></figure>

the relevance of this almost unexplored family of AMPs
in comparison with conventional antibiotics.

In this context, we have evaluated the antimycobacte-
rial activity of the three arenicin isoforms; **Ar-1**, **Ar-2**
and **Ar-3**; as well as their respective disulfide-free Abu-
derivatives; **Ar-1-Abu**, **Ar-2-Abu**, **Ar-3-Abu**, **Ar-3(3-
20)** and **Ar-3(7-16)**; on the growth of the S & R variants
of *M. abscessus*.

Among these arenicin and arenicin-Abu peptides,
we showed that **Ar-1** displayed the best combination of
antimycobacterial activity against both morphotypes
(MIC<sub>90</sub>~19 µM), cytotoxicity on eukaryotic cells and
hemolytic activity towards human erythrocytes, resulting
in relatively good therapeutic indexes [58] (Figs. 2, 3 and
Table 1, 2). Concerning **Ar-2** and **Ar-3**, they displayed
good antibacterial activity against *M. abscessus* S-variant
only, but also showed potent cytotoxicity against A498
cells and hemolytic activity (for **Ar-2**), which consider-
ably limits their use in humans. Interestingly, the replace-
ment of the two bridged Cys<sup>3</sup> & Cys<sup>20</sup> residues by Abu led
to totally inactive and nontoxic peptides in the case of the
**Ar-1-Abu** and **Ar-2-Abu**. Similar results were reported
by Lee et al. [60] who found that a linear derivative of
**Ar-1**, generated by substituting Cys<sup>3</sup> & Cys<sup>20</sup> with serine
residues, was fourfold less active against Gram-negative
bacterial cells than native **Ar-1**. In contrast, when the two
cysteines were replaced by alanine residues, Krenev et al.
showed that the resulting Ar-1(C/A) peptide was still able
to form a twisted β-hairpin structure despite the absence
of its disulfide bond, and also to retain both antibacte-
rial and hemolytic activities as compared to natural pep-
tide [84]. From these biological data, it can be concluded
that the hydrophobic-hydrophilic balance, the disulfide

bridge, and the amphipathic β-sheet structure of the
peptide play important roles in the biological activities
of arenicins. Regarding **Ar-3**, neither the selective nor
the complete replacement of the four cysteine residues
improved the toxicity of the **Ar-3(3–20)**, **Ar-3(7–16)**
and **Ar-3-Abu** resulting peptides (Fig. 3 and Table 2). In
contrast, the fact that **Ar-3(7–16)** retained similar anti-
bacterial activity to **Ar-3**, whereas **Ar-3-Abu** was inac-
tive and **Ar-3(3–20)** weakly active thus highlighted the
importance of Cys<sup>7</sup>-Cys<sup>16</sup> bridge, which appears to drive
the global antibacterial activity of all **Ar-3** peptides prob-
ably by maintaining a correct folding of their respective
3D structure.

Such differences, however, between **Ar-1** and **Ar-2**,
were surprising as the two isoforms share 95% sequence
identity and differ in only one residue: Val¹⁰ in **Ar-1** and
Ile¹⁰ in **Ar-2** (Fig. 1). Such a single Val to Ile mutation
has however been reported in the case of transthyretin
transport protein, and has been shown to be responsi-
ble for a cardiomyopathy characterized by accelerated
cardiac amyloid deposition [85]. In a recent study on
the link between arenicins' structure and antimicrobial
and hemolytic activities [84], the authors suggested that
the overall 3D conformation of **Ar-1** could be described
as more compact compared with the more elongated
**Ar-2** conformation. From our data, the specific posi-
tion of the Val¹⁰ and Ile¹⁰ residues at the extremity of
one β-sheet of **Ar-1** and **Ar-2**, respectively, proved to
be crucial for their inhibitory effect as well as for their
cytotoxic and hemolytic activities. This is particularly
true for the *M. abscessus* R variant, for which the MIC
values of **Ar-2** are ≥5 times higher than those of **Ar-1**
(Table 1). On the other hand, the effects of **Ar-3** may be
explained by its amino acid composition, which differs mainly from **Ar-1** and **Ar-2** with only 47% sequence identity.

Given their very limited application in humans due to lack of significant antibacterial activity combined with high toxicity to A498 cells, the use of Abu derivatives should be limited to comparative mechanistic studies with arenicin peptides. However, it might be interesting to test them on cancer cell lines other than A498 kidney cells to check whether these Abu-peptides have the same effect, which could pave the way for the possibility of a novel treatment for cancer cells.

We further focused on **Ar-1** to better characterize its mechanism of action against *M. abscessus* S and R. Our data showed that this peptide provokes a ~42% mean permeabilization of *M. abscessus* membrane regardless of the S or R variant tested (Fig. 4). Moreover, **Ar-1** was also able to efficiently interact and insert into monolayers of total lipids from *M. abscessus* S and R at high surface pressures: it was not excluded from the monolayer, and it exhibited a slightly higher affinity for lipid extracts from the S morphotype (Fig. 5). A recent study has underlined the importance of the surface-exposed glycopeptidolipids (GPL) present only in the S variant in *M. abscessus* hydrophobicity [86]. By using quantitative imaging atomic force microscopy, the authors demonstrated that the transition from a smooth to a rough colony morphology, caused by the loss of GPL, led to a dramatic change in surface hydrophobicity, smooth bacteria displaying unusual nanodomains with varying degrees of hydrophobicity. In the case of **Ar-1**, the presence of GPL in *M. abscessus* S may alter cell-wall fluidity/permeability thus slightly favoring the insertion of this peptide at the origin of membrane permeabilization, cytoplasm leakage and bacterial cell lysis as depicted in Fig. 6.

As previously observed with another class of synthetic copolymers [56], such a pore-forming mode of action is likely to prevent the bacteria from developing any kind of resistance mechanism. This was clearly confirmed by the absence of appearance of **Ar-1** resistance in *M. abscessus* S and R variants after a 42 days continuous exposure to this peptide (Fig. 7).

Overall, the fact that **Ar-1** possesses good bactericidal activity against the S and R morphotypes of *M. abscessus*, without significant toxicity or hemolytic activity and without inducing resistance, is therefore very promising. Furthermore, since CF patients are initially infected with the S variant of *M. abscessus*, which can evolve into the R variant capable of forming cords and leading to severe and almost incurable lung infection, **Ar-1** could represent a valuable alternative strategy to kill *M. abscessus* from the early stage of infection to the chronic lung infections.

## Conclusion

In this article we have confirmed the great interest of **Ar-1**, a 21-residue antimicrobial peptide, in the fight against *M. abscessus* infections. Moreover, the fact that **Ar-1** also exhibits a broad spectrum of activities against Gram-positive and Gram-negative bacterial as well as fungal pathogens [33], such as *Pseudomonas aeruginosa*, *Staphylococcus aureus*, and *Candida albicans*, respectively, either alone or in combination with classical antibiotics as its synergistic activity has also been demonstrated [87], opens new perspectives for CF patients. Future studies should however be conducted, notably in vivo studies, to confirm the interest of this peptide in the therapeutic arsenal against bacterial infections in CF patients. In particular, to circumvent the described low hemolytic activity, encapsulation experiments might be done in order to use this peptide as an aerosol in human therapy, similar to amikacin liposome inhalation suspension [88], rather than injected into patients or administered orally.

### Abbreviations

Abu Alpha-amino-n-butyric acid residue

AMK Amikacin

AMPs Antimicrobial peptides

Ar Arenicin

CC₅₀ Cytotoxic concentration leading to 50% cell toxicity in vitro

CF Cystic fibrosis

CLR Clarithromycin

CTAB Cetyltrimethylammonium bromide

DMEM Dulbecco's modified essential medium

FBS Fetal calf serum

GPL Glycopeptidolipids

MIC Minimal inhibitory concentration

REMA Resazurin microtiter assay

TI Therapeutic index

### Acknowledgements

We thank Hugo Le Guenno from the microscopy facilities of the "Institut de Microbiologie de la Méditerranée" (IMM FR3479), and Damien Chaudanson from the electronic microscopy facilities of the "Centre Interdisciplinaire de Nanoscience de Marseille" (CINaM).

### Author contributions

Conceptualization: MC, MM and J-FC; methodology: MC, MM and J-FC.

Validation: MC, MM and J-FC. Formal analysis: MC, MM, IP, VP, HO and J-FC.

Investigation: MC, MM and J-FC. Formal analysis: MC, MM, IP, VP, HO, CB-W, AT, KM and J-FC. Visualization: J-FC. Writing—review and editing: MC, MM, CB-W, AT, KM, J-FC and SC. Supervision: MC, MM, J-FC and SC. Project administration: J-FC and SC.

### Funding

Not applicable.

### Availability of data and materials

All data generated and analyzed during this study are included in this article.

### Declarations

#### Ethics approval and consent to participate

Not applicable.
**Consent for publication**

Not applicable.

**Competing interests**

The authors declare that they have no competing interests.

**Author details**

Received: 19 December 2023 Accepted: 22 January 2024

Published online: 29 January 2024

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