Antimicrobial Spectrum of Activity and Mechanism of Action of Linear Alpha-Helical Peptides Inspired by Shrimp Anti-Lipopolysaccharide Factors

Shrimp antilipopolysaccharide factors (ALFs) form a multifunctional and diverse family of antimicrobial host defense peptides (AMPs) composed of seven members (groups A to G), which differ in terms of their primary structure and biochemical properties. They are amphipathic peptides with two conserved cysteine residues stabilizing a central β-hairpin that is understood to be the core region for their biological activities. In this study, we synthetized three linear (cysteine-free) peptides based on the amino acid sequence of the central β-hairpin of the newly identified shrimp (Litopenaeus vannamei) ALFs from groups E to G. Unlike whole mature ALFs, the ALF-derived peptides exhibited an α-helix secondary structure. In vitro assays revealed that the synthetic peptides display a broad spectrum of activity against both Gram-positive and Gram-negative bacteria and fungi but not against the protozoan parasites Trypanosoma cruzi and Leishmania (L.) infantum. Remarkably, they displayed synergistic effects and showed the ability to permeabilize bacterial membranes, a mechanism of action of classical AMPs. Having shown low cytotoxicity to THP-1 human cells and being active against clinical multiresistant bacterial isolates, these nature-inspired peptides represent an interesting class of bioactive molecules with biotechnological potential for the development of novel therapeutics in medical sciences.


Introduction
The selection of microorganisms with resistance against multiple conventionally used antibiotics represents one of the leading health problems worldwide [1]. The study of antimicrobial host defense peptides (AMPs) has recently expanded and emerged as an
Crude peptide (50 mg) was first reduced with 10% β-mercaptoethanol (95 • C for 5 min) then dissolved in 50% (v/v) AcOH/H 2 O and later diluted in 32 mL of oxidation buffer (2 mM guanidinium chloride, 10% isopropyl alcohol, and 10% dimethyl sulfoxide). The pH was adjusted to 5.8 with ammonium hydroxide. The peptide solution was subjected to air oxidation at room temperature for 18 h. The peptide solution was then acidified to pH 2.5 and purified using a SPE C18 (Waters Corp., Milford, MA, USA). The peptides were eluted with 5%, 20%, 40%, 60%, and 80% acetonitrile in 0.05% TFA ultrapure water at a flow rate of 1 mL/min. The fractions were collected, and the acetonitrile was evaporated on a Savant SPD 1010 SpeedVac Concentrator (Thermo Scientific, Asheville, NC, USA). The fractions were analyzed with MALDI-TOF mass spectrometry.

Circular Dichroism (CD) Measurement
Circular dichroism (CD) spectroscopy was carried out on a JASCO J-815 CD Spectrometer coupled to a Peltier JASCO CDF-426S/15 system for temperature control (JASCO Corp., Tokyo, Japan) in the far ultraviolet range (λ = 190-250 nm), using quartz cuvettes with a 0.1 cm path length and 1 nm bandwidth at 0.1 nm resolution. Each spectrum was recorded as an average of four scan repetitions in continuous scanning mode with 50 nm/min scanning speed and a response time of 1 s. The solvent contribution blank was subtracted from each sample spectrum. Molar ellipticity was calculated for each synthetic peptide. CD spectra of the peptides were recorded in ultrapure water and trifluoroethanol (TFE, 30% v/v in ultrapure water). The spectra were recorded at 20, 30, and 37 • C.

Antibacterial Assays
The antibacterial activity of the synthetic ALF-derived peptides was assayed against reference, clinical, and environmental strains of Gram-positive and Gram-negative bacteria (phyla Actinobacteria, Firmicutes, and Proteobacteria). Minimum inhibitory concentrations (MICs) were determined in duplicate with the liquid growth inhibition assay in 96-well microtiter plates, as previously described [21]. In brief, 10 µL of peptides (final concentration, range from 40 to 1.25 µM) was incubated with 90 µL of bacterial suspension brought to the exponential growth phase and adjusted to A 600nm = 0.001 in its respective media and culture conditions under shaking (Table S1). In negative controls, peptides were substituted with sterile ultrapure water. Growth was monitored spectrophotometrically (λ = 600 nm) on a Tecan Infinite M200 spectrophotometer (Tecan, Männedorf, Switzerland) at 24 h. MIC values are expressed as the lowest concentration tested (in µM) that caused 100% growth inhibition. The minimum bactericidal concentration (MBC) was determined by plating 100 µL of overnight cultures onto nutrient agar plates from each of the wells from the MIC test that showed no turbidity. The plates were incubated for 48 h. MBC values were expressed as the lowest concentration tested (in µM) for which no bacterial growth was observed on the plates.

Antifungal Assays
The antifungal activity of the synthetic ALF-derived peptides was assayed against reference and environmental strains of filamentous fungi and yeasts (phyla Ascomycota, Basidiomycota, and Mucoromycota). The MIC for filamentous fungi was determined in duplicate with the liquid growth inhibition assay using spores, as previously described [12]. In brief, pure cultures were grown in potato dextrose agar (Table S1) at 28 • C until abundant sporulation. Spores were harvested with 0.1% Tween-20 (v/v in sterile ultrapure water), filtered through lint, and centrifuged (3000× g for 15 min). Spore pellets were washed twice with 0.1% Tween-20, and their concentration was adjusted in a Neubauer chamber. For the tests, 90 µL of fungal spores (final concentration, 10 4 spores/mL) suspended in potato dextrose broth at half-strength ( 1 /2 PDB, Table S1) were added to 10 µL of peptides (final concentration, range from 40 to 1.25 µM) in 96-well microtiter plates. In negative controls, peptides were substituted with sterile ultrapure water. Spore germination was observed under an inverted microscope after 48 h of incubation with shaking in a humidity chamber at 28 • C in the dark. The minimum fungicidal concentration (MFC) was determined by plating 100 µL of fungal cultures onto PDA plates from each of the wells from the MIC test that showed no spore germination. The plates were incubated for 48 h at 28 • C. MFC values were expressed as the lowest concentration tested (in µM) for which no growth was observed on PDA plates.
The MIC values for the antiyeast assays were determined as described for the antibacterial tests. In brief, 10 µL of peptides (final concentration, range from 40 to 1.25 µM) were incubated with 90 µL of yeast suspension brought to the exponential growth phase and adjusted to A 600nm = 0.001 in Sabouraud medium (Table S1) at 28 • C under shaking conditions. In controls, peptides were substituted by sterile ultrapure water. Growth was monitored spectrophotometrically (λ = 600 nm) on a Tecan Infinite M200 spectrophotometer (Tecan, Männedorf, Switzerland) at 48 h. MIC values are expressed as the lowest concentration tested (in µM) that caused 100% yeast growth inhibition. The MFC was determined by plating 100 µL of yeast cultures onto Sabouraud agar plates from each of the wells from the MIC test that showed no growth. Cultures were incubated for 48 h at 28 • C. MFC values were expressed as the lowest concentration tested (in µM) for which no growth was observed on the plates.

Determination of Fractional Inhibitory Concentrations (FICs)
The synergic effect of the synthetic ALF-derived peptides in inhibiting the growth of the bacterial strains M. maritypicum CIP 105733 and E. coli SBS 363 and the shrimp midgut-associated yeast Rhodotorula sp. LIAA-UFSC was evaluated using the titration test [22] with some modifications. To assess the reduction in the inhibitory concentration of each peptide, serial dilutions of one peptide (peptide "A") were combined with half the MIC of the other peptide (peptide "B") and vice versa. The results obtained were expressed using the fractional inhibitory concentration (

Assays for Bacterial Membrane Permeability
The effect of the synthetic ALF-derived peptides on the integrity of bacterial membranes was assessed with the Sytox Green uptake assay. Exponential phase cultures of E. coli SBS 363 were diluted at A 600nm = 0.2 in 10 mM phosphate-buffered saline (PBS) supplemented with 138 mM NaCl and 2.7 mM KCl (pH 7.4). Then, 45 µL of bacterial solution containing 1 µM Sytox Green (Invitrogen, Carlsbad, CA, USA) were dispensed into 0.1 mL MicroAmp Fast 96-well reaction plates (Thermo Scientific, Asheville, NC, USA) containing 5 µL of each peptide in triplicate (final concentration, 5 µM). In positive controls, peptides were substituted with 1.25 µM of an amidated analog of magainin, a recognized pore-forming AMP from the African clawed frog Xenopus laevis (amino acid sequence = GIGKFLKKAKKFGKAFVKMKK-NH2, molecular weight = 2495.97 Da, pI = 10.9 [23]). In negative controls, peptides were substituted with sterile ultrapure water. Sytox Green uptake was measured every 30 s over 1 h (λ excitation = 480 nm; λ emission = 550 nm) at 37 • C using a StepOnePlus Real-time PCR System (Thermo Scientific, Asheville, NC, USA).

Antiparasitic Activity
Cultures of Trypanosoma cruzi (Tulahuen strain) epimastigotes and Leishmania (L.) infantum (MHOM/BR/74/PP75 strain) promastigotes were grown in liver infusion tryptose (LIT) and M199 supplemented with 10% fetal bovine serum (FBS), with the latter also supplemented with 5% human urine, at 26.5 • C with 5% CO 2 in an incubator. For the tests, 90 µL of parasite suspension (5.4 × 10 5 parasites/well) was added to each well of 96-well microtiter plates, followed by 10 µL of peptides (final concentration, range from 20 to 1.25 µM). In negative controls, peptides were substituted with sterile ultrapure water, whereas 20 µM benznidazole (Sigma-Aldrich, St. Louis, MO, USA) and 2 µM amphotericin B (Bristol-Myers, Squibb, Woerden, Netherlands) were used as positive controls for trypanocidal and leishmanicidal activities, respectively. A sample control was included for each peptide dilution containing only the peptide and the respective parasite medium. The 96-well microtiter plates were incubated for 72 h at 26.5 • C. The antiparasitic activity was determined in triplicate with a quantitative colorimetric assay using the oxidation-reduction (blue-pink) indicator resazurin as an indicator for metabolic function. T. cruzi and L. (L.) infantum parasites were incubated for 5 h and 1 h 30 min, respectively, with 3 mM resazurin solution at 26.5 • C in the dark. Parasite viability was monitored using fluorescence quantification (λ excitation = 560 nm; λ emission = 590 nm) on a Tecan Infinite M200 spectrophotometer (Tecan, Männedorf, Switzerland). Relative viability was calculated for each sample by averaging fluorescence readings across the triplicates, subtracting the average for the sample control, and then dividing by the value obtained for the negative control.

Cytotoxicity Assays
The human leukemia monocytic cell line THP-1 (ATCC TIB202), obtained from the cryobank of the Laboratory of Protozoology at the Federal University of Santa Catarina (Florianópolis, Brazil), was cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate and grown at 37 • C with 5% CO 2 in an incubator. For the tests, in a 96-well microtiter plate, 100 µL of THP-1 cell suspension (2.2 × 10 5 cells/mL) was differentiated into macrophages using 100 ng of phorbol-12myristate-13-acetate (PMA) and incubated for 72 h at 37 • C with 5% CO 2 . Then, the medium was replaced with 90 µL of RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, and 1 mM sodium pyruvate followed by 10 µL of peptides (final concentration, range from 80 to 1.25 µM), and the 96-well microtiter plate was incubated for 72 h at 37 • C. DMSO 50% and RPMI-1640 were used as positive and negative controls, respectively. Cytotoxicity was determined in triplicate with a quantitative colorimetric assay using the oxidation-reduction (blue-pink) indicator resazurin as an indicator for metabolic function. Fluorescence was measured after 24 h incubation with 3 mM resazurin solution using fluorescence quantification (λ excitation = 560 nm; λ emission = 590 nm) on a Tecan Infinite M200 spectrophotometer (Tecan, Männedorf, Switzerland). Cell viability was calculated by averaging fluorescence readings across the triplicates, subtracting the average for the sample control, and then dividing by the value obtained for the negative control.
ture-based design of novel therapeutics, we synthesized short, linear, cysteine-free peptides (20 amino acid residues) based on the central β-hairpin (the functional domain) of three ALF members (Litvan ALF-E to -G) recently characterized in the penaeid shrimp L. vannamei [11]. This region was selected for peptide synthesis due to the presence of hydrophobic and cationic residues, resulting in high amphipathicity ( Figure 1A). Remarkably, while the whole mature peptides Litvan ALF-E (GenBank: FE069658) and Litvan ALF-G (GenBank: GETZ01049665) exhibited anionic properties (pI 6.11 and 5.02, respectively) [11], their central β-hairpins (Litvan ALF-E33-52 and Litvan ALF-G35-54) were highly cationic (pI 9.70 and 11.12, respectively) ( Figure 1A). An increase in the pI was also observed for Litvan ALF-F31-50 ( Figure 1A) when compared to the mature Litvan ALF-F [11]. In all mature ALFs, the two conserved cysteine residues delimit a central β-hairpin structure that is considered essential for their biological activities [9]. The circular dichroism (CD) spectra of the linear, cysteine-free peptides were recorded in an aqueous solution added to TFE 30% at room temperature (20 °C) and at microorganism growth temperatures (30 and 37 °C) ( Figure 1B). A standard α-helix signal The circular dichroism (CD) spectra of the linear, cysteine-free peptides were recorded in an aqueous solution added to TFE 30% at room temperature (20 • C) and at microorganism growth temperatures (30 and 37 • C) ( Figure 1B). A standard α-helix signal with characteristic peaks at 195, 208, and 222 nm was detected for the three synthetic peptides in all temperatures ( Figure 1B). These results indicate that the synthetic ALF-derived peptides have an α-helical structure similar to classical cationic antimicrobial peptides (cAMPs), which is distinct from that observed for the cysteine-stabilized β-hairpin of ALFs [14].

Shrimp ALF-Derived Peptides Permeabilize Bacterial Membranes
To gain insight into the mechanism of action of the synthetic peptides, we evaluated their capacity to permeabilize the membrane of the Gram-negative E. coli SBS 363 by using the Sytox Green uptake assay. The increase in fluorescence was observed at the beginning of incubation with Litvan ALF-G 35-54 and after less than 10 min with Litvan ALF-F 31-50 ( Figure 2). No significant increase in fluorescence was observed in wells incubated with sterile ultrapure water (negative control) or with Litvan ALF-E 33-52 , which was not active against E. coli SBS 363 ( Figure 2; Table 1). These results indicate that these synthetic α-helical peptides are membrane-disrupting molecules with the ability to permeabilize bacterial membranes as typically observed for cAMPs. for distinct combinations of synthetic peptides using the checkerboard assay. Results showed that Litvan ALF-F31-50 and Litvan ALF-G35-54 acted synergistically against bacteria (FIC = 0.5-1) but not against the yeast Rhodotorula sp. (FIC = 2) ( Table 3). In contrast, Litvan ALF-E33-52 and Litvan ALF-F31-50 acted synergistically against the yeast but not against the bacteria (Table 3). Finally, the combination of Litvan ALF-E33-52 and Litvan ALF-G35-54 (FIC = 0.63) led to an eightfold reduction in the concentration of Litvan ALF-E33-52 required to inhibit the growth of M. maritypicum CIP 105733 (Tables 1 and 3).

Shrimp ALF-Derived Peptides Permeabilize Bacterial Membranes
To gain insight into the mechanism of action of the synthetic peptides, we evaluated their capacity to permeabilize the membrane of the Gram-negative E. coli SBS 363 by using the Sytox Green uptake assay. The increase in fluorescence was observed at the beginning of incubation with Litvan ALF-G35-54 and after less than 10 min with Litvan ALF-F31-50 ( Figure 2). No significant increase in fluorescence was observed in wells incubated with sterile ultrapure water (negative control) or with Litvan ALF-E33-52, which was not active against E. coli SBS 363 ( Figure 2; Table 1). These results indicate that these synthetic α-helical peptides are membrane-disrupting molecules with the ability to permeabilize bacterial membranes as typically observed for cAMPs. Green uptake assay. Bacterial cells were exposed to 5 μM of each ALF-derived peptide or an equal volume of sterile ultrapure water (negative control). In positive controls, peptides were substituted with 1.25 μM of magainin, a recognized pore-forming antimicrobial peptide [23]. measured with the Sytox Green uptake assay. Bacterial cells were exposed to 5 µM of each ALF-derived peptide or an equal volume of sterile ultrapure water (negative control). In positive controls, peptides were substituted with 1.25 µM of magainin, a recognized pore-forming antimicrobial peptide [23].

Shrimp ALF-Derived Peptides Display Low Cytotoxicity to Human THP-1 Cells
The cytotoxic effect of the synthetic peptides was evaluated against the human leukemia monocytic cell line THP-1. No cytotoxic effect was observed at any of the tested concentrations (80 to 1.25 µM; cell viability > 94%) ( Table 4). Due to the low influence on cell viability at the tested concentrations, it was not possible to calculate the half-maximal cytotoxic concentration (CC50) of these peptides. These results suggest that the synthetic peptides show low cytotoxicity to human cells at antibacterial and antifungal concentrations.  Table 4).

Discussion
Results showed that 20 residue linear peptides based on the central β-hairpin of the newly described L. vannamei ALFs from groups E to G [11] adopt an α-helix secondary structure and display a broad spectrum of activity against clinically relevant microorganisms, including multiresistant strains. Remarkably, these modified α-helical cysteine-free peptides exhibited a broader and stronger spectrum of antimicrobial activity than their correspondent forms adopting a β-hairpin structure stabilized by two cysteines [11]. Different studies have shown that the cysteine-stabilized β-hairpin is the functional domain of ALFs, which displays the ability to mimic the biological activity of the whole molecule [12,16,17]. ALFs have high binding properties for microbial components (e.g., LPS, LTA, and β-glucans) and their affinity for these components has been described as essential for their antimicrobial activities [12]. Microbial-binding involves seven charged residues located in the cysteine-stabilized β-hairpin [15], and it has been proposed that the amino acid diversity found among ALF groups in this functional domain severely impacts ALF antimicrobial activity and mechanism of action [11,12,24]. Based on the results of membrane permeabilization assays, it is most likely that the α-helical cysteine-free ALF-derived peptides have a mechanism of action similar to that of classical cationic AMPs (cAMPs) and different from whole mature ALFs. Indeed, cAMPs act on the membranes of microorganisms through electrostatic interactions with anionic phospholipids and hydrophobic interactions with the lipid bilayer, leading to their destabilization and permeabilization [25]. Thus, alterations in the primary amino acid structure represent an alternative for producing ALF-derived bioactive molecules with distinct properties and mechanisms of action.
AMPs have been considered one of the most promising classes of potential drug candidates for combatting multidrug resistance [1,29,30]. AMPs show a broad spectrum of biological activities, including antiviral, antifungal, antimitogenic, anticancer, and antiinflammatory properties. Here, we investigated the activity of linear α-helical peptides against clinically relevant bacteria and yeast, including different strains of MRSA and Candida spp. Remarkably, Litvan ALF-G 35-54 was active against two MRSA isolates: 16003 (resistant to cefoxitin) and 17022 (resistant to cefoxitin, ciprofloxacin, clindamycin, erythromycin, gentamicin, rifampicin, and trimethoprim-sulfamethoxazole). S. aureus is the most frequently isolated pathogen from human skin and wound infections and the emergence of MRSA strains exhibiting resistance to conventional drugs is a significant public health challenge that requires novel therapeutic alternatives [29]. In addition, Litvan ALF-G 35-54 was active against the yeasts C. krusei and C. parapsilosis, while all synthetic peptides showed activity against C. tropicalis. Invasive candidiasis is an important fungal disease among hospitalized patients associated with significant mortality and excessive medical costs [31]. Candida spp. have been reported to be significant clinical pathogens that can persist in hospital environments and are able to form biofilms on central venous catheters and other medically implanted devices [32]. Although C. albicans still accounts for most invasive candidiasis overall, infections caused by non-C. albicans species have been widely reported (reviewed in [33]). In fact, C. parapsilosis is often the second most commonly isolated Candida spp. from blood cultures and can overcome C. albicans in some locations [32,33]. The activity of Litvan ALF-G 35-54 against these relevant clinical pathogens reinforces the potential of AMPs to replace or to be implemented together with conventional drugs to combat the issue of multidrug resistance.
In addition to their application in the clinic, recombinant or synthetic AMPs can be safely used as therapeutics in aquaculture, food additives for livestock, or food preservatives [34]. Many AMPs have been observed, when applied as food additives, to improve production performance and livestock immunity and promote intestinal health in aquaculture, as well as in poultry, swine, and ruminants breeding [34]. Our results showed that Litvan ALF-F 31-50 and Litvan ALF-G 35-54 could inhibit the growth of microorganisms responsible for significant loss in aquaculture, such as Gram-negative bacteria of the genus Vibrio [35] and the filamentous fungus F. oxysporum [9]. In fish farming, it has been shown that the injection of the AMPs epinecidin-1 and pleurocidin resulted in lower cumulative mortality against Vibrio vulnificus and V. anguillarum infections, respectively [36,37]. In addition, oral administration of synthetic FSB-AMP was shown to have the potential to protect L. vannamei shrimp against V. parahaemolyticus infections [38]. Altogether, these results support the applicability of shrimp ALF-derived peptides as therapeutic agents for the health of both humans and cultivated species, and future studies evaluating the effects of these peptides as therapeutics or food additives in aquaculture should be performed.
Antimicrobials are, as a class of drugs, particularly troublesome regarding cytotoxicity for hosts, since their role is to ultimately achieve microbial cell death. cAMPs have been shown to exert their antimicrobial effect by selective permeabilization of predominantly negatively charged bacterial membranes. However, their detergent-like effect can sometimes compromise both microbial and mammalian cell membranes. Our results showed that the shrimp ALF-derived peptides synthesized in this study were not active against the protozoan parasites T. cruzi and L. (L.) infantum; however, they showed low cytotoxicity to the human leukemia monocytic cell line THP-1, even at high concentrations. Peptide net charge determines the extent of the initial electrostatic interactions with both prokaryotic and eukaryotic membranes, with a more significant cationic charge favoring antimicrobial action [28]. Tailoring of these properties is likely to be the key to successfully transferring ALF-derived peptides from laboratory experiments into clinical practice as safe pharmaceutical formulations. For cAMPs, there is a correlation between a larger ratio of aromatic residues-especially tryptophan-to cationic residues and a high degree of hemolysis and cell cytotoxicity [28,39]. Hence, the absence of these residues in Litvan ALF-E 33-52 and Litvan ALF-F 31-50 and the low prevalence in Litvan ALF-G [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53][54] can contribute to the low cytotoxicity observed for these peptides.
In an era critically lacking in new antibiotics, manipulating AMPs for therapeutic application emerges as a pivotal strategy. Microorganisms' resistance to AMPs can still remain problematic, since both Gram-positive and Gram-negative bacteria have evolved strategies to neutralize the net negative charge of their cell surfaces to avoid electrostatic interactions with cationic AMPs [40]. In addition, when activated by misfolding of outer membrane proteins, bacteria can produce factors that help to preserve and/or restore cell envelope integrity [40]. Finally, bacteria have also evolved a series of efflux pumps to transport AMPs out of their cytoplasmic space in case they have breached the bacterial membrane barriers [40]. To deal with this issue, the use of AMPs in conjunction with other antimicrobials has been considered a promising approach [3]. In this scenario, synergistic antimicrobial combinations are promising candidates that reduce potential bacterial resistance, overcome preexisting resistance to current antibiotics, prevent host toxicity, and increase antimicrobial efficacy [3]. To address this question, we evaluated the synergistic activity of the linear α-helical peptides synthesized in this study. Results showed that combinations of Litvan ALF-E [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52] or Litvan ALF-F 31-50 with Litvan ALF-G 35-54 acted synergistically against bacteria, while Litvan ALF-E 33-52 and Litvan ALF-F 31-50 acted in synergism against yeast cells. These results reveal that distinct aims can be achieved based on combinations of ALFs to improve their antimicrobial activities or to target specific microorganisms. In addition, introducing antibiotics inside bacteria has often been a challenge. We showed that ALF-derived α-helical peptides can address this challenge by disrupting bacterial membranes that might facilitate the entry of antibiotics into the cytoplasm. Therefore, combining shrimp ALF-derived peptides with other therapeutics can be an effective strategy to limit bacterial resistance through the use of different mechanisms of action.

Conclusions
We demonstrated that linear α-helical peptides inspired by the central β-hairpin of L. vannamei ALFs from groups E to G display a broad and interesting range of antimicrobial activities. Shrimp ALF-derived peptides were shown to be active against different bacterial and fungal strains, including human pathogens. In addition, these amphipathic AMPs were able to act synergistically to improve their antimicrobial properties and their ability to disrupt bacterial membranes. Finally, shrimp ALF-derived peptides showed low cytotoxicity to the human cell line THP-1 and were active against clinical MRSA isolates. Altogether, our results emphasize the biotechnological potential of this diverse crustacean AMP family for the development of novel therapeutic agents.