## 1 Introduction  
Liver-expressed antimicrobial peptides (LEAPs) are cysteine-containing cationic peptides with antimicrobial activity, which were first discovered in human blood ultrafiltrate and urine samples [1]. In humans, LEAPs with eight and four cysteine residues are termed as hepcidin [1] or LEAP-1 [2] and LEAP-2 [3], respectively. The human LEAP-1 (hLEAP-1) is a cysteine-containing cationic peptide comprising about 20-25 amino acid residues [2]. It has a characteristic \(\beta\)-sheet structure, which is stabilized by eight cysteine residues [2]. These residues form four disulfide bonds, one of which is an unusual vicinal bond between adjacent cysteines at the hairpin turn with an amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif [4]. The N-terminal region of human LEAP-1 (hepcidin-25) has a metal binding site specific for the coordination of Cu(II) and Ni(II) known as ATCUN motif [5]. The histidine residue at the amino-terminal (XXH) of ATCUN motif in teleosts and mammals are highly conserved, which has been reported to form a putative metal binding motif. It was found that Ni(II) and Cu(II) interaction with the peptide, results in formation of metal and peptide complex which leads to the oxidative damage of DNA [6]. The trout LEAP with ATCUN motif in the presence of Cu(II) and ascorbate was able to hydrolyze a DNA plasmid. On the contrary, the trout LEAP without ATCUN motif was unable to hydrolyze the plasmid DNA [5]. Thus, ATCUN motif is essential for antimicrobial activity via DNA damage [7]. LEAPs have been identified in many other vertebrates such as fish, chickens, pigs, and mice [8; 9; 10; 11]. LEAPs play a significant role in the fish innate immune system [8; 12]. The hybrid striped bass was the first fish from which LEAPs were isolated [8]. Since then, LEAPs have been identified in at least 30 fish species [13]. Similar structure of LEAPs are identified in human and fish with respect to their distorted \(\beta\)-sheet shape with a hairpin loop, position of cysteines, and ATCUN motif [14; 15; 16]. Nevertheless, sequence analysis revealed the presence of four, six, and seven cysteines in mature LEAPs from fish [13]. There are many differences between the fish and mammalian LEAPs, including the presence of several LEAP variants within a single fish species, such as the seven variants identified in _Acanthopagrus schlegelii_[17]. Moreover, the expression of these isoforms is observed not only in hepatocytes, but also in kidney and hematopoietic tissues [18], contrary to the mammalian LEAPs. The typical LEAPs with eight cysteine residues show prominent antibacterial activity against bacterial pathogens, fungi, viruses, and some neoplasms [19]. For a potent response against pathogens, fish extensively depend on the production of antimicrobial peptides, which is a part of their first line of defense [20]. The Antarctic notothenioid and Antarctic eelout were the first Antarctic fish in which four-cysteine LEAP was reported [18]. Recently, synthetic LEAPs with four cysteine residues from _Epinephilus coioides_ were reported, which possess antimicrobial and antiviral activities [21]. The existence of several LEAP isoforms in the same organism highlights the need for multiple functions in a complex aquatic environment [22; 23]. To date, no studies have addressed whether LEAPs plays a vital role in the innate immunity of Antarctic notothenioid (_Dissostichus mawsoni_) and Antarctic eelout (_Lycodichthys debarborni_). To determine the potential role of novel four- and eight-cysteine LEAPs in the innate immunity of Antarctic fish, there is a need to evaluate their antibacterial activity. In this study, we attempted to evaluate the antibacterial activity and mechanism-of-action of novel LEAPs with four (4 cys) and eight cysteine (8 cys) residues from _Dissostichus mawsoni_ and _Lycodichthys debarborni_. In the Antarctic Ocean, the temperature stays below 2 \({}^{\circ}\)C. Therefore, it is important for Antarctic antimicrobial peptides to remain active against cold-loving bacteria at low temperatures.