Characterization of Two Antimicrobial Peptides from Antarctic Fishes (Notothenia coriiceps and Parachaenichthys charcoti)

We identified two antimicrobial peptides (AMPs) with similarity to moronecidin in Antarctic fishes. The characteristics of both AMPs were determined using moronecidin as a control. Moronecidin, which was first isolated from hybrid striped bass, is highly salt-resistant, and possesses broad-spectrum activity against various microbes. The moronecidin-like peptide from Notothenia coriiceps exhibited a narrower spectrum of activity and a higher salt sensitivity than moronecidin. The AMP from Parachaenichthys charcoti exhibited similar antimicrobial activity to moronecidin, and similar salt sensitivity. In an experiment to identify toxic effects, both of the moronecidin-like peptides from the Antarctic fishes exhibited lower hemolytic activity than moronecidin. In spite of its low toxicity, the AMP from N. coriiceps is unlikely to be considered as a candidate for antibiotic development, owing to its narrow spectrum of activity and high salt sensitivity. In contrast, the high salt resistance and broad-spectrum activity of the AMP from P. charcoti could be more advantageous for clinical use than moronecidin, since it could kill bacteria under physiological conditions with low toxicity. A further comparison of these two AMPs from Antarctic fishes with other AMPs could help to reduce the toxicity of AMPs for clinical use.


Introduction
Antimicrobial agents have defeated many infectious diseases and have improved public health significantly. However, many pathogenic microorganisms are becoming resistant to several antimicrobial agents/drugs, and demand for novel antibiotics continues to grow [1].
Antimicrobial peptides (AMPs) may be one of the new generation of antibiotics to meet this demand [2,3]. AMPs are crucial effector molecules of the innate immune response, present in most living organisms [4]. AMPs possess broad-spectrum antimicrobial activities against bacteria, fungi, and viruses [5]. Certain AMPs can kill pathogens that are resistant to almost all conventional antibiotics [6]. AMPs kill microorganisms using diverse mechanisms. AMPs can disrupt membrane structure by forming transmembrane pores, inhibiting cell-wall a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 synthesis, and by inhibiting cytoplasmic membrane septum formation. Certain AMPs can inhibit enzymes and can inhibit the synthesis of proteins and nucleic acids [3,7,8]. However, AMPs also have drawbacks; these include instability, hemolytic activity, salt sensitivity, toxicity toward eukaryotic cells, susceptibility to proteolysis, and a higher cost of production compared with conventional antibiotics [2,9,10].
In spite of their drawbacks, some AMPs from the pool of thousands of natural peptides have been developed and validated as therapeutic agents [9][10][11]. The costs could be decreased by commercial-scale production by the pharmaceutical industry [12][13][14]. Indeed, several AMPs have proceeded to clinical trials [9][10][11]. However, the Food and Drug Administration (FDA) of the United States of America has not yet granted approval for the clinical use of any AMP.
In this study, we discovered two moronecidin-like peptides in other Antarctic fishes (Notothenia coriiceps and Parachaenichthys charcoti). Using synthetic mature peptides, we investigated the spectrum of these AMPs, the effect of salt concentration on their activity, and their toxicity. To investigate whether these AMPs had distinct characteristics arising from their origin in fish that live in a cold environment, we tested the effect of temperature on their activity.

Molecular characterization of moronecidin-like peptides from Antarctic fishes
The amino acid sequence of a moronecidin-like peptide from N. coriiceps was obtained (NCBI reference sequence: XP_010768425.1). The cDNA sequence of a moronecidin-like peptide from P. charcoti was obtained from the assembled contigs, generated from mRNA-seq in the liver (GenBank accession number: KX344030). Theoretical isoelectric point (pI) values, net charges, and molecular weights (MW) were predicted using the Peptide Property Calculator from Innovagen (http://pepcalc.com/ppc.php). Schiffer-Edmundson wheel representations of AMPs were obtained using HeliQuest [28] from the ExPASy website (http://expasy.org/tools/). Homologous AMP sequences were obtained from the NCBI database and were aligned using ClustalW (http://www.genome.jp/tools/clustalw/). A phylogenetic tree was constructed by the neighbor-joining method using the Mega 5 program, on full-length amino acid sequences [29].

Microbial strains and culture conditions
The microbial strains used in this study are listed in Table 2. These include Gram-negative and Gram-positive bacteria, filamentous fungi, and yeast species. All microbial isolates were obtained from the Korean Collection for Type Cultures (KCTC), the American Type Culture Collection (ATCC), or the Polar and Alpine Microbial Collection (PAMC) of the Korea Polar Research Institute. For liquid cultures, tryptic soy broth (BD Diagnostic Systems, Sparks, MD, USA) and nutrient broth (BD Diagnostic Systems) were used. The culture medium and temperature used for each strain is listed in Table 2.

Antimicrobial activity assay
The minimal inhibitory concentration (MIC) was determined as described previously [30]. To determine the MIC, a microbial culture was incubated with an AMP for 18 h in 96-well plate (Bioneer, Republic of Korea). Each well contained 90 μL of a microbial cell suspension at 1 × 10 5 cfu/mL, and 10 μL of a particular AMP that had been serially diluted in growth medium. MICs were defined as the lowest peptide concentrations that inhibited microbial growth completely. To evaluate the effect of temperature on the MICs, microbial cultures in

Hemolytic activity assay
The hemolytic activity of AMPs was determined against sheep blood and horse blood (Oxoid Ltd., London, United Kingdom) [24]. Freshly packed sheep and horse erythrocytes (1 mL) were washed with phosphate-buffered saline (pH 7.4). AMPs were added to 90 μL of a 1% erythrocyte suspension (1:10 dilution of washed erythrocytes) in microcentrifuge tubes. The samples were incubated for 30 min at 37˚C, and then centrifuged for 10 min at 4000 rpm at room temperature. The supernatants were transferred carefully to a 96-well plate, and the optical density was determined at 405 nm. The percentage of hemolysis was defined relative to the hemolysis obtained by treating the erythrocyte suspension with 0.1% SDS (100% hemolysis).

Genes encoding moronecidin-like peptides in antarctic fishes
Piscidin, which is a cationic peptide comprising 22-amino acids, has broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria [24]. It is also known to induce apoptosis in cancer cells [31]. To detect piscidin homologs in Antarctic fishes, we investigated the genome of N. coriiceps using the BlastP tool, and we identified a gene encoding a moronecidin-like peptide with NCBI accession number XP_010768425.1. The gene encoding a moronecidin-like peptide in N. coriiceps consists of 4 exons, which encode 77 amino acids.
Since we had assembled contigs using RNA-seq data generated from the liver tissue of P. charcoti, we were also able to identify the cDNA sequence encoding a 79 amino acid moronecidinlike peptide among those contigs.

Phylogeny and molecular evolution of piscidins
The amino acid sequence of the newly identified AMP from P. charcoti shares 64% identity with that from hybrid striped bass, and 78% identity with that from Chionodraco hamatus. For N. coriiceps, the amino acid sequence of its AMP has comparatively low identity (43%) with moronecidin from hybrid striped bass. The AMP from P. charcoti has 43% identity with the AMP from N. coriiceps. An alignment with homologous AMPs shows that both the signal peptide and mature peptide are well conserved in the two novel moronecidin-like peptides (Fig 1). A phylogenetic analysis was also constructed, based on the amino acid sequences of other known piscine AMPs. The AMP from P. charcoti is closely related to the AMP from C. hamatus. In contrast, the AMP from N. coriiceps is more closely related to piscidin-4 and piscidin-5 from hybrid striped bass (Fig 2). All three AMPs from Antarctic fishes are located in single clade, together with piscidin-4 and -5 from hybrid striped bass.

Secondary structures of the AMPs
Schiffer-Edmundson helical wheel modeling of mature peptides from Antarctic fishes shows amphipathic alpha-helix conformations, in which hydrophobic and hydrophilic residues are on opposite sides of the alpha-helix (Fig 3). The mature moronecidin (moro) from hybrid striped bass and the mature peptide from P. charcoti (moroPC) each have a higher hydrophobicity score (0.627 and 0.617, respectively) than the mature peptide from N. coriiceps (mor-oNC; hydrophobicity score: 0.449). The hydrophobic moment, which is a measure of the amphiphilicity of a helix, was represented using HeliQuest [28]. The hydrophobic moments of moro, moroPC, and moroNC are 0.556, 0.559, and 0.365, respectively. Of the three peptides, moroNC has the lowest amphiphilicity.

Antimicrobial activity
Moronecidin from hybrid striped bass displayed broad-spectrum antibacterial activity. Consequently, we determined the antimicrobial activity using synthetic, amidated AMPs (moro-NH 2 , moroNC-NH 2 , and moroPC-NH 2 ) bearing the amino acid sequences found in Antarctic fishes ( Table 3). The AMPs from Antarctic fishes showed strong activity against Shigella sonnei, Psychrobacter sp., and Escherichia coli DH5α (MIC < 12.5 μM), but did not exhibit antibacterial activity (up to 50 μM) against the Gram-negative bacteria Pseudomonas aeruginosa or Burkholderia cepacia (Table 3). Only moro-NH 2 showed activity against Pseudomonas aeruginosa at 50 μM. Synthetic AMPs from hybrid striped bass and P. charcoti exhibited antibacterial activity against Enterobacter cloacae above 25 μM. In the case of Gram-positive bacteria, Enterococcus faecalis, Streptococcus pyogenes, Staphylococcus aureus and Listeria monocytogenes were sensitive to both AMPs from the Antarctic fishes below 25 μM, with the exception of moroNC-NH 2 against E. faecalis. All three of the AMPs had antimicrobial activity against Candida tropicalis. A similar spectrum of activity and MIC against bacteria was seen for mor-oPC-NH 2 and moronecidin from hybrid striped bass. In contrast, moroNC-NH 2 exhibited weaker or non-existent antimicrobial activity against certain species. A relatively low hydrophobicity and pI value for moroNC-NH 2 might account for its narrower spectrum and lower    antibacterial activity. Nonetheless, for particular bacterial strains, moroNC-NH 2 has similar antibacterial activity as the other two peptides, in spite of these differences in its physicochemical properties. Since Antarctic fishes live in a cold environment, below 2˚C [32], and since AMPs from icefish have been shown to kill bacteria in a temperature-dependent manner [27], we evaluated the effect of temperature on the activities of moro-NH 2 , moroNC-NH 2 , and moroPC-NH 2 against certain bacteria. Psychrobacter sp. PAMC 25501, isolated from Ny-Ålesund in Svalbard, Norway, was used to test activity up to~25˚C. E. coli DH5α was used to test activity from 15˚C to 37˚C (Tables 4 and 5). However, the activities of AMPs from hybrid striped bass and Antarctic fishes were unaltered by these temperature changes. To assess whether the AMPs from Antarctic fishes could be effective as innate immunity molecules in a cold environment, we also measured their antibacterial activity against an additional cold-loving bacterium ( Table 6). Lacinutrix algicola AKS293 T isolated from marine sediment in the Southern Ocean [33], and Flavobacteria sp. PAMC 22217 isolated from the Arctic Ocean, were selected, along with Psychrobacter sp. PAMC 21119, which was isolated from the Antarctic permafrost [34]. Although moro-NH 2 exhibited a narrow spectrum of activity in Table 3, both AMPs from Antarctic fishes were active enough to kill these additional cold-loving bacteria.

Salt sensitivity
AMPs are initially attracted to microbial membranes by electrostatic interactions, prior to forming pores [35]. These electrostatic interactions can be disrupted by salts, inhibiting Table 3. Antimicrobial spectrum of synthetic, amidated moronecidin-like peptides from Antarctic fishes.

Hemolytic activity
Since some types of AMP can lyse mammalian erythrocytes, hemolytic activity was tested as a therapeutic index. It is important to establish a low hemolytic activity for clinical use [2]. To investigate the possibility of using the AMPs from Antarctic fishes in a clinical setting, we used both sheep and horse erythrocytes to evaluate their hemolytic effects (Fig 4). Twelve concentrations were used for each AMP, and moro-NH 2 was used as a control. Both AMPs from the Antarctic fishes caused a relatively lower percentage of hemolysis than moro-NH 2 . The hemolytic activity of moroNC-NH 2 did not reach 10% with the highest concentration tested (50 μM). With 25 μM of an AMP from either of the Antarctic fishes, the hemolytic activity is below 10%. In contrast, moro-NH 2 lysed up to 25% and 62% of the sheep and horse erythrocytes, respectively. moroNC-NH 2 had a hemolytic activity below 1% at 25 μM, in both sheep and horse erythrocytes.

Conclusions
The moronecidin-like peptide from N. coriiceps shows distinctive features for an AMP. The amino acid sequence has very low similarity with other AMPs. The most similar amino acid sequence is that of the piscidin-like antimicrobial peptide from the icefish, C. hamatus, with which it shares 55% identity. It shares 43% identity with moronecidin from hybrid striped bass. However, we could not find any advantages conferred by this distinctive amino acid sequence. In spite of its low toxicity, moroNC-NH 2 has a narrow spectrum of antibacterial activity, and high salt sensitivity. These characteristics make it difficult to consider mor-oNC-NH 2 for clinical use.  The moronecidin-like peptide from the Antarctic dragonfish, P. charcoti, is 88% identical to a piscidin-like antimicrobial peptide from C. hamatus, and has 64% identity with an AMP from hybrid striped bass. However, moroPC-NH 2 produced similar results to moro-NH 2 in experiments testing its salt sensitivity and its spectrum of activity against microbes. Furthermore, its toxicity was lower than that moro-NH 2 . At 12.5 μM of the AMPs tested, almost none of the sheep or horse erythrocytes were lysed. A characterized AMP from C. hamatus also has low hemolytic activity and broad-spectrum antimicrobial activity [27]. Although we could not test all of the AMPs from Antarctic fishes, AMPs from the species (C. hamatus, N. coriiceps, and P. charcoti AMPs) exhibit lower toxicity than moronecidin.
Currently, no AMPs have been approved as therapeutic agents by the FDA. Nonetheless, several cationic antimicrobial peptides (Pexiganan, Omiganan, Iseganan, and others) have entered into Phase III trials and have had their use clinically validated [9][10][11]. Moronecidin, which is a piscidin homolog, is also cationic antimicrobial peptide [36]. Therefore, in this study we investigated the characteristics of two moronecidin-like peptides from Antarctic fishes and tested whether those AMPs are suited for use as therapeutic agents. The AMP from P. charcoti exhibited high salt resistance, low toxicity, and broad-spectrum activity; these characteristics suggest that this AMP could be considered for inclusion in future clinical trials. We could not identify any temperature dependency for the activity of AMPs from N. coriiceps or P. charcoti; in contrast, the activity of the AMP from C. hamatus is known to be temperaturedependent [27]. We established that low toxicity appears to be a distinctive feature of AMPs from the Antarctic fishes studied to date. A further comparison of other AMPs, and AMPs from other Antarctic fishes, might facilitate the development of AMPs with lower toxicity [37].