Antimicrobial peptides
Antimicrobial peptides are increasingly recognized as a critical factor in host defence mechanism and found in organisms ranging from microbes, plants to animal species (Fernandes et al., 2004; Kennedy et al., 2009). It also plays an important role in fish when compared with mammals as fish rely more on their innate immune system (Hancock, 1997; Hancock and Scott, 2000).
Fish are a great source of these AMPs and they express all of the major classes of peptides like defensins, cathelicidins, hepcidins, histone-derived peptides, and a fish-specific class of the cecropin family, called piscidins (Valero et al., 2013). The fish peptides exhibit broadspectrum antimicrobial activity by killing both fish and human pathogens (Das et al., 2013).
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They can also be immunomodulatory and their genes are highly responsive to microbes and innate immuno-stimulatory molecules (Masso-Silva et al., 2014). Antimicrobial peptides are generally defined as broad spectrum cationic molecule having low molecular weight peptides (size <10 kDa; length 12-50 amino acids) with a net positive charge because of the presence of excess basic lysine and arginine residues over acidic residues.
They fold, owing to the presence of disulphide bridges or contact with membranes, into three dimensional amphiphilic structures. However, some anionic forms of AMPs have also been reported (Vizioli and Salzet, 2002). Antimicrobial peptides belong to a larger group of naturally occurring short polypeptides and share similar amphipathic α-helical structures that can interact strongly and permeate phospholipid membranes (Rakers et al., 2013).
The advantage of the AMPs is that they can function without either high specificity or memory. Also these are synthesized at low metabolic costs, capable of mass storage and readily available after infection. Such molecules are well suited for interacting with bacterial membranes having negatively charged and hydrophilic head groups and hydrophobic cores.
Antimicrobial peptides adopt several mechanisms for their activities: some appears to associate on the membrane surface and displace the outer bilayer (Shai, 2002) and others have shown to span the bilayer and associate to form the channel depolarizing the target cells. Few peptides have been proposed to cross the bilayer and interact with the microbial DNA similar to that of histones in eukaryotic DNA, disrupting the transcription and/or replication.
Antimicrobial peptides can interact with bacterial, fungal or protozoan cells and many recognize all three of them. There is also evidence regarding the role of peptides in stimulation of other immune processes and tissue repair through their action as signalling molecules in host response (Hancock and Scott, 2000; Shai, 2002). A few of the important AMPs found in fish are described below:
Pardaxin
Pardaxin is 33 residues in length and has a helixhinge-helix structure similar to cecropin and mellitin. This peptide shows characteristic pore forming properties in the metazoan membranes and also displays shark repellent properties. This AMP is highly effective against Gram +ve and Gram -ve bacteria. In fish, it was first reported from the skin mucus of Moses sole, Pardachirus marmoratus (Oren and Shai, 1986).
Pleurocidins and Moronecidins
Pluerocidin, a 25 residue cationic peptide was reported in winter flounder (Plueronectes americanus) and other white eyed flounders. Southern blot analysis of genomic DNA also revealed the presence of multigene families of pluerocidin related genes in Atlantic halibut (Hippoglossus hippoglossus), Yellow tail flounder (Pleuronectes ferruginea) and American plaice (Hippoglossoides platessoides) (Douglas et al., 2001). A 22 residue α-helix AMP, Monocidin is also found in skin and gill of Hybrid striped bass (Morone chrysops × Morone saxatilis). Like Paradaxin, monocidins showed wide spectrum activity against the Gram +ve and Gram - ve bacteria, fungi and yeast. Pluerocidins may provide pattern recognition with direct destruction and in many cases is likely to save the fish the cost of a more involved acute-phase response (Lauth et al., 2002).
Ribosomal peptides
A protein (6.7 kDa) isolated from the skin of Rainbow trout (Oncorhynchus mykiss) is effective against Gram +ve bacteria is found to be similar to the ribosomal protein S30 isolated from various species (Fernandes and Smith, 2002). Similarly, three 60S ribosomal proteins, L40, L36A, and L35 were identified from the skin of Atlantic cod which also reported to have antimicrobial activities (Bergsson et al., 2005).
Derivatives of histones
Certain AMPs in fishes are believed to be derived from various histones. Hipposin of Atlantic halibut (H. hippoglossus) and parasin I of Amur catfish (Parasilurus asotus), are identified to be the N-terminal fragments of H2A (Cho et al., 2002b). Hipposin, a 51 residue long cationic peptide effective against a large number of Gram-positive as well as negative bacteria is found in the skin mucus of the healthy halibut (Birkemo et al., 2003). A 19 residue AMP, parasin I was discovered in the mucus of the injured fish. It was found to be quite similar to Buforin I, a H2A derived peptide in toads (Park et al., 1996). It was active against various Gram +ve and Gram -ve bacteria and fungi as well. Cleavage of large sized protein like cathepsin-D is believed to give rise to these AMPs. This protease is activated by matrix metalloprotease-2 whose production is increased during the immune response or tissue repair (Cho et al., 2002a). A 30-residue N-terminally acetylated peptide derived from the N-terminal part of histone H1 in skin mucus of Atlantic salmon (S. salar) (Luders et al., 2005) is termed as salmon antimicrobial peptide [SAMP H1] and found to be active against both Gram-negative and Grampositive bacteria. Amongst the cysteine-rich AMPs, cathelicidins, defensins, and liver-expressed antimicrobial peptides (LEAPs) are mostly found in teleosts (Silphaduang and Noga, 2001). Nevertheless, putative cathelicidins have also been reported in fish like Rainbow trout (Smith and Fernandes, 2009), Atlantic salmon (Chang et al., 2006), Arctic char (Salvelinus alpines), Atlantic cod and brook trout (Salvelinus fontinalis) (Maier et al., 2008). Furthermore, genes coding for cathelicidin has also been reported for jawless fish, like Atlantic hagfish (Myxine glutinosa) (Uzzell et al., 2003). Defensins from teleost were identified by different molecular methodologies (Matsuzaki et al., 1999; Uzzell et al., 2003; Roussel and Delmotte, 2004; Zou et al., 2007; Maier et al., 2008; Casadei, 2009). The genomic data on the defensin indicate resemblance with the βdefensins of birds and mammals (Zhao et al., 2009). Falco et al. (2008) demonstrated antiviral activity against viral haemorrhagic septicaemia rhabdovirus (VHSV). Recently, three novel β-defensins from Rainbow trout were cloned that constitutively expressed that further increased during bacterial and simulated viral challenges (Zou et al., 2007). In another study, a β-defensin-like gene from the olive flounder has been identified and expressed in larval fish, one day after hatching (Nam et al., 2010). Other cysteine-rich AMPs found in fish are the LEAPs (Park et al., 2001). The LEAP family include different peptides such as hepcidins isolated from winter flounder, turbot, and red sea bream, Sal-1 and Sal-2 from Atlantic salmon, JF-1 and JF-2 from Japanese flounder, and LEAP-2 from catfish and trout (Smith and Fernandes, 2009). Antimicrobial peptides isolated from fish skin mucus with their structure and specificity have been summarized in Table 3.
Lysozyme
Lysozyme is an important component of the innate immune system and mediates protection against pathogenic invasion (Dash et al., 2011). It is a mucolytic enzyme of leucocytic origin. The common feature of lysozyme is their ability to hydrolyse β-(1,4)-glycosidic bonds between the alternating N-acetylmuramic acid (NAM) and N-acetyl glucosamine (NAG) residues of peptidoglycan of bacterial cell wall resulting in rapid cell lysis in a hypo-osmotic environment. In addition to bacteria, lysozyme has also been reported to inhibit viruses (Lee-Huang et al., 1999), parasites (LeonSicairos et al., 2006) and fungi (Wu et al., 1999) despite the absence of typical peptidoglycan in their envelopes. The enzyme also attacks structures containing muramic acid, hydrolyses glycol chitin and has a restricted degrading effect on chitin, which is a major component of the cell walls of fungi and the exoskeletons of certain invertebrates (Wu et al., 1999). Lysozyme also promotes phagocytosis directly by activating polymorphonuclear leucocytes and macrophages or indirectly by an opsoninic effect. Broadly, lysozymes are classified into five major types such as (i) chicken-type lysozyme (c-type) that includes stomach lysozyme and Ca+2 binding lysozyme, (ii) goose-type lysozyme (g-type), (iii) plant-type lysozyme, (iv) bacterial lysozyme, and (v) T4 phage lysozyme (phage-type). However, in fishes only c- and g-type lysozyme have been reported (Beintema and Terwisscha van Scheltinga, 1996; Fastrez, 1996; Irwin et al., 1996; Prager and Jolles, 1996; Qasba and Kumar, 1997). The lysozyme response has been found to be variable in its potency depending on the species and the tissue location (Qasba and Kumar, 1997). It was reported that increase in mucus lysozyme activity in Rainbow trout and Atlantic salmon when infected with sea lice in the earlier days of infection that decreased further (Fast et al., 2002). However, lysozyme activities did not increase in coho salmon in earlier days, but increased significantly in the later stage of post infection. It appears that the lysozyme response in fish may be induced very rapidly and is not only related to bacterial presence but also to other alarm situations such as stress. Thus, lysozyme in fish would be involved in the overall alarm response, acting as an acute-phase protein. The estimation of lysozyme may be of diagnostic value to determine the disease status of fish.