Antimicrobial Peptides (AMPs) from Fish Epidermis: Perspectives for Investigative Dermatology

PERSPECTIVE

Antimicrobial Peptides (AMPs) from Fish Epidermis: Perspectives for Investigative Dermatology Sebastian Rakers1, Lars Niklasson2, Dieter Steinhagen3, Charli Kruse1, Ju¨rgen Schauber4, Kristina Sundell2 and Ralf Paus5,6 Mammalian and fish skin share protective activities against environments that are rich in infectious agents. Fish epidermis is endowed with an extrinsic barrier consisting of a mucus layer and antimicrobial peptides (AMPs). These operate together as a protective chemical shield. As these AMPs are evolutionarily well preserved and also found in higher vertebrate skin (including human epidermis), fish skin offers a unique opportunity to study the origins of innate antimicrobial defense systems. Furthermore, the broad spectrum of fish mucus antimicrobial activities renders piscine AMPs interesting to investigative dermatology, as these may become exploitable for various indications in clinical dermatology. Therefore, this article aims at casting light on fish mucus, the evolutionary relationship between human and fish AMPs, and the latter’s antibacterial, antifungal, and even antiviral activities. Moreover, we develop dermatological lessons from, and sketch potential future clinical applications of, fish mucus and piscine AMPs. Journal of Investigative Dermatology (2013) 133, 1140–1149; doi:10.1038/jid.2012.503; published online 14 February 2013

INTRODUCTION Human and fish skin are both continuously challenged by potentially infectious agents in their respective habitats (land/air vs. water). Although possessing distinct skin phenotypes reflecting life in different habitats, protective functions are shared between piscine and human skin, and anti-infection defense principles have been evolutionarily conserved (Rakers et al., 2010). Fish skin offers unique opportunities for studying the evolutionary origins of human innate antimicrobial defense systems, as many of these protective mechanisms, such as the piscine antimicrobial peptides (AMPs), have been preserved in higher vertebrate skin (including human epidermis). A careful investigation of fish skin defense systems also raises the possibility that some of the multiple fish mucus antimicrobial activities may be harnessed for selected indications in future clinical dermatology. After briefly synthesizing essential

differences and similarities between piscine and human skin, this Perspectives article invites mammalian skin biologists and dermatologists to more systematically explore the ‘‘piscine–human connection’’ in cutaneous anti-infection defense and to learn for potential future clinical applications in investigative dermatology. The piscine–human divide in skin biology: more common ground than may be apparent

Fish and human skin are structurally diverse (Figure 1). Yet, numerous essential functions are shared. These range from mechanical and chemical barrier formation via sensory functions and metabolism to the maintenance of osmotic homeostasis, and interindividual communication by cutaneous visual signals (e.g., pigments). Even the obvious differences between piscine and mammalian skin hide a shared basic design (Figure 1; Rakers et al.,

2010). If one focuses on the cutaneous production of glandular secretions (mucus vs. sebum) and of a large battery of protective molecules generated in response to biological, chemical, and physical stressors (Goldsmith, 1991; Woo, 2007; Proksch et al., 2008), the ‘‘piscine–human divide’’ in skin biology shrinks even further (Rakers et al., 2010). Evidently, different vertebrates have developed different anti-infection defense strategies that are best tailored to the specific pathogens that they encounter in their respective habitats. Fish are permanently surrounded by aquatic pathogens, whereas human skin is exposed to a variety of terrestrial and air-borne pathogens. Naturally, this requires specialized defense mechanisms. In spite of these differences, some central mechanisms to fight environmental pathogens have been well conserved, i.e., the first-line defense system

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Aquatic Cell Technology, Fraunhofer Research Institution for Marine Biotechnology, Luebeck, Germany; 2Department of Biological and Environmental Sciences, University of Gothenburg, Gothenburg, Sweden; 3University of Veterinary Medicine, Centre for Infection Medicine, Fish Disease Research Unit, Hanover, Germany; 4Department of Dermatology and Allergy, Ludwig Maximilian University, Munich, Germany; 5Department of Dermatology, University of Luebeck, Luebeck, Germany and 6School of Translational Medicine, University of Manchester, Manchester, UK

Correspondence: Sebastian Rakers, Aquatic Cell Technology, Fraunhofer Research Institution for Marine Biotechnology, Paul-Ehrlich-Strasse 1–3, Lu¨beck 23562, Germany. E-mail: [email protected] Abbreviation: AMP, antimicrobial peptide Received 18 April 2012; revised 19 November 2012; accepted 3 December 2012; published online 14 February 2013

1140 Journal of Investigative Dermatology (2013), Volume 133

& 2013 The Society for Investigative Dermatology

S Rakers et al. AMPs from Fish Epidermis

Fish

Human Inner root sheath Bulge— stem cells

Outer root sheath

Epidermis

Sebaceous gland

Dermal hair papilla

Dermis Hypodermis

Epithelial cell

Basal cell

Osteoblast

Corneocyte

Fibroblast

Mucous gland

Scale Melanocyte

Hair follicle Figure 1. General overview on skin structures. Simplified schematic drawing of (a) human and (b) fish skin. Main differences between teleost fish and human skin are the lack of a keratinized layer of corneocytes in most of the fish epidermis, the presence of bone tissue–related scales instead of hairs, including the osteoblasts being actively working with constant calcification, and the protection of the skin against pathogen entry by mucous glands (goblet cells) that secrete mucus to the outer surface. Several other cell types such as mast cells or merkel cells exist in fish and human skin, but are not explicitly named.

that is provided by AMPs. Fish and mammals possess similar innate immune mechanisms for dealing with pathogens, including AMPs generated by the epithelium and intramesenchymal mast cells, macrophages, and natural killer cells (for details, see Silphaduang and Noga, 2001; Wo¨lfle et al., 2009; Dzik, 2010). AMPs

AMPs were discovered about 30 years ago by Boman’s group (Hultmark et al., 1982), and the first AMP reports in teleosts date back to around the same time (Lazarovici et al., 1986; Thompson et al., 1986). AMPs have fascinated and attracted scientists since then (Zasloff, 1987; Agerberth et al., 1991; Schonwetter et al., 1995; Harder et al., 1997; Simmaco et al., 1998; FernandezLopez et al., 2001), and still AMPrelated research is a hot topic in life sciences (see, e.g., Peric et al., 2010; Schro¨der, 2010; Bernard and Gallo, 2011; Dombrowski et al., 2011; Jensen et al., 2011; Nakatsuji and Gallo, 2011; Schittek, 2011; Curtis and Radek, 2012). AMPs have been identified in plants and across a wide range of animal species from nonvertebrates to mammals, which underscores their evolutionary success (Zasloff, 2002; Sang and Blecha, 2008; Guanı´-Guerra et al., 2010; Steinstraesser et al., 2011). Nevertheless, the production and release of AMPs in fish and human skin

follows the same principle: wounding or contact with microorganisms or pathogens activates the synthesis and release of AMPs by certain cutaneous cells that destroy the pathogen by direct killing, membrane disruption, opsonization, or inhibition of DNA/RNA/ protein synthesis (Figure 2). This receptor-independent mechanism reduces the possibility for pathogens to develop resistance against the peptide (Sang and Blecha, 2008; Nakatsuji and Gallo, 2011). Special features of fish epidermis

Most fish lack a keratin-based epidermal barrier of dead cells characteristic of humans (Campinho et al., 2006), which may facilitate the entry of pathogens. Furthermore, the fish mucosal surface is an ideal (i.e., stable and nutrient-rich) habitat and colonization target for waterborne microorganisms (Smith and Fernandes, 2009). Control of microbial growth on exposed surfaces and within the tissues is important for the fitness, growth, and reproductive success of fish and mammals (Smith and Fernandes, 2009). Particularly after wounding, the skin is susceptible to proteolytic attack and needs to have some structural and immunological properties to prevent infections. To illustrate the immunological challenges faced by fish in human medical terms, it might be postulated that construction principles of the fish skin show similarities to the

human oral cavity. If this is really the case, a chief challenge of the piscine (teleost) organism might be to prevent ‘‘pan-body mucosal diseases.’’ Fish skin mucus

Fish epithelial cells act as a seal between the animal and its surroundings. They possess microridges, which are raised, actin-rich structures that serve to maintain and aid in the controlled release of mucus on the outer surface of the fish (Webb et al., 2008). They serve as microchannels and greatly increase the surface area (Figure 3). Beneath and in between this surface layer there are, among other differentiated cells (Figure 1), the mucous goblet cells of the stratum spinosum that supply the mucus (Figure 4). Goblet cells are structurally similar to its mammalian counterpart (Harris and Hunt, 1975), and their produced mucus contains neutral and acid (carboxylated and sulfated) glycoconjugates, disulfides, and other components, which are continuously accumulated in the direction of the external epidermal regions. They make the mucus a highly effective protection (Meyer et al., 2005). This protection shield includes most of the known piscine AMPs and is also made up of additional, AMP-independent anti-infection substances, including alkaline phosphatase, cathepsin B, complement, transferrin, lysozyme, and C-reactive protein (Subramanian et al., 2007; Zhao et al., 2008; see Table 1). Figure 3b shows the www.jidonline.org 1141

S Rakers et al. AMPs from Fish Epidermis

AMPs: H2A, H1, H6, HDL, peuricidin, pleurocidin, sal-2, defensins Direct killing Complement Inhibit DNA, RNA, protein synthesis

Peptides Carbohydrates Igs Lysozyme Lectins

Membrane disruption Bacteria/fungi/viruses

Protozoa Mucous goblet cells

Mucus

Cathelicidin Opsonization Piscidins

Mast cells β-Defensins wound healing?

Lymphocytes PMNs

IL-lβ, TNF-α2, hepcidin, apolipoproteins Inflammatory stimuli

Recruitment and activation of immune cells

Neutrophil β-Defensins

T cells

Figure 2. Schematic illustration of the antimicrobial defense mechanism in fish. When chemical or mechanical injury occurs, e.g., through bacteria, viruses, or other pathogens, epithelial cells start to release cytokines such as IL-1b, which is suggested to attract neutrophils and T cells to the superficial parts of the epidermis. At the same time, mucous goblet cells start to secrete their product, mucus, including antimicrobial peptides (AMPs). AMPs such as hepcidin are involved in the immune response to bacteria, whereas AMPs such as cathelicidins directly opsonize bacteria. PMNs, polymorphonuclear neutrophils. Modified after Rakers et al. (2010).

Sc

replenishment is costly, and mucus production itself may promote the growth of some bacteria and fungi that exploit it as a nutrient (Morgan and Adams, 2009). Fish mucus as a rich source of potent AMPs

Figure 3. Scale-derived epithelial cells of rainbow trout (Oncorhynchus mykiss) in culture. (a) Scanning electron micrograph of a scale-derived epithelial cell in culture. The typical microridges for the increase of the cell’s surface are highly visible. Bar ¼ 2 mm. (b) Scale-derived epithelial-like cells in culture (passage 0) stained with periodic acid–Schiff (PAS). Besides the unstained round epithelial cells, some polygonal cells stained positive for glycoconjugates indicating the presence of mucus (magenta/brownish, arrows). Cell nuclei in blue. Sc, scale. Scale bar ¼ 100 mm.

detection of glycoconjugates in some scale-derived epithelial cells. The substances lead to a specific glycocalyx composition, and their carbohydrate composition may change with stress, infection, or environmental conditions (Zaccone et al., 2001; Behrendt, 2005; Meyer et al., 2007; Neuhaus et al., 2007). When goblet cells of the fish skin release the mucus, they are not able to synthesize and discharge mucus a second time. Hence, there is a

continuous turnover of these cells in the outer layers of the epidermis. The physicochemical characteristics of fish skin mucus layer, as determined by the presence of bioactive substances and the epidermal migration of inflammatory cells and their secretions, may impair the establishment and proliferation of ectoparasitic bacteria, copepods, ciliates, or monogean helminths (Jones, 2001; van der Marel et al., 2010). Yet, continuous mucus production and

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Owing to their antibacterial and immunomodulatory activity, fish AMPs are of interest in veterinary medicine, namely in the context of fish aquaculture (one of the fastest growing industries in the past decades). During the past few years, a number of studies on piscine skin AMPs (Subramanian et al., 2008a, b; Fernandes et al., 2010; Ruangsri et al., 2010; Wei et al., 2010; Table 2) have raised the possibility that fish mucus extract may be an excellent antimicrobial agent for treating human pathogens as well. Piscine skin generates a large variety of AMPs such as hepcidin (Cuesta et al., 2008), defensin-like peptides (Zou et al., 2007; van der Marel et al., 2012), cathelicidins (Maier et al., 2008; Scocchi et al., 2009), certain

S Rakers et al. AMPs from Fish Epidermis

apolipoproteins (Villarroel et al., 2007), piscidin (Silphaduang et al., 2006), and pleurocidin (Birkemo et al., 2003; Patrzykat et al., 2003), often with selective properties against pathogenic bacteria, fungi, algae, viruses, or parasites (Komatsu et al., 2009; Urquhart et al., 2009; Table 2). Fish AMP research could also increase its impact on research of innate immunity in human skin (see, e.g., Harder et al., 2007; Lai and Gallo, 2009; Peric et al., 2009; Reithmayer et al., 2009; Nakatsuji and Gallo, 2011; Emelianov et al., 2011; Schittek, 2011; Jensen et al., 2011). On one hand, understanding the mechanisms of AMP regulation in fish could lead to a better understanding of human AMP regulation. Unique evolutionary links such as those discovered for B cells (Li et al., 2006) show that there is evidence for a real connection between the most primitive forms of immunological defense, which

is still present in fish, and a more specialized, adaptive immune response seen in humans and other mammals. To examine AMP regulation in fish that is similar to humans may be far better than to jump ahead to clinical trials. Some AMP regulation pathways in fish may of course differ from human AMP regulation, and some pathways simply have been further developed in humans. Interspecies comparisons may help to find explanations as to why one regulation has coevolved and the other differs in fish and humans. Importantly, fish AMPs might be a source of novel anti-infection agents (Najafian and Babji, 2012). For example, AMPs identified in fish (e.g., piscidins), which are not found in humans, could be developed as novel drugs for human skin diseases with impaired innate skin defense such as atopic dermatitis (Noga et al., 2009). Current and future

Epi

Der

Sc

Sc

Epi

Sc Hyp

Sc

Figure 4. Fish skin histology. (a) Aldehydfuchsin-Goldner staining of rainbow trout skin. Huge cells in the epidermis (Epi) are mucous goblet cells (arrowheads), secreting the mucus to the surface (arrow). Green part in the dermis is collagen-rich connective tissue. Nuclei in blue. Der, dermis; Hyp, hypodermis. Scale bar ¼ 50 mm. (b) Detailed picture of periodic acid–Schiff (PAS) staining. Mucous goblet cells (arrowheads) are strongly stained positive and secrete mucus to the outer epithelial surface (arrows). Epi, epidermis. Bar ¼ 20 mm.

research, thus, may well identify several AMPs in fish mucus that are worth developing as antibiotics for human disease. For example, piscidin-2 of hybrid striped bass was shown to exhibit potent antifungal activity against human pathogenic fungi (Sung et al., 2008). Some fish AMPs (epinecidin-1) also act as potential antitumor agents against human fibrosarcoma cells: they inhibited cell proliferation and reduced the migration of tumor cells (Chen et al., 2009a,b; Lin et al., 2009). Evolutionary conservation: human skin defense and AMPs from fish

In recent years, a wide array of cutaneous peptides with in vitro antimicrobial activity have been identified in mammals (Harder et al., 1997, 2007; Braff et al., 2005). Although proteins from the fish skin could be suspected to be involved in antibacterial defense processes in fish (Lemaıˆtre et al., 1996), the AMPs detected in fish skin until recently have been scarce (Narvaez et al., 2010, Cuesta et al., 2011; Ruangsri et al., 2012). Fish AMPs are— in line with mammalian AMPs— classified either through their conserved secondary structure or their evolutionary relationship (Gennaro and Zanetti, 2000). One class of well-studied AMPs are the cathelicidins; these peptides have an overall cationic and often amphipathic character (Tomasinsig and Zanetti, 2005; Chang et al., 2006). The highly conserved nature of the cathelin domain has been used to

Table 1. Composition and functions of fish mucus Mucus component

Antiviral

Antiparasitic, e.g., antiprotozoan

Reference

Complement factors

|

|

(Lim, 2008)

Hydrolytic enzymes (e.g., lysozyme, cathepsin B, proteases)

|

|

(Ross et al., 2000; Subramanian et al., 2007)

| IgM, IgT

(Ebran et al., 2000; Hansen et al., 2005; Danilova et al., 2005)

| C-reactive proteins | Histone-like proteins

(Woo, 2007)

|

(Suzuki et al., 2003; Tsutsui et al., 2005)

Igs

Lectins Mucus extract1

|

(Raj et al., 2011)

IFN

|

(Caipang et al., 2011; Cui et al., 2011; Lu¨ et al., 2012)

1

As described by (Hellio et al. (2002).

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Table 2. Antimicrobial peptides of fish skin Known human1 Peptide (type)

Expression site

H2A

homologs

Spectrum Species

Gram þ

Gram 

Fungi

Reference

Mucous goblet cells

Rainbow trout

Yes

Yes

Yes

(Fernandes et al., 2002)

H1 (oncorhyncin II)

Mucous goblet cells

Rainbow trout

Yes

Yes

Yes

(Noga et al., 2001; Fernandes et al., 2004)

H6 (oncorhyncin III)

Mucous goblet cells

Rainbow trout

Yes

Yes

Yes

(Fernandes et al., 2003)

HDL (high-density lipoprotein)

Mucous goblet cells

Carp, rainbow trout

Pleurocidin

Mucous goblet cells, skin epithelium

Winter flounder, American plaice, American halibut, mud dab

Yes

Yes

Yes

Sal-2

Gill, skin epithelium

Atlantic salmon

No

Yes

Yes

Defensins (e.g., b-defensins, in some fish a primitive form of b-defensins)

Pituitary, testis, skin epithelium

(Concha et al., 2003; Villarroel et al., 2007)

ND hBD-1, hBD-2, Zebrafish, hBD-3 pufferfish, rainbow trout, common carp

(Cole et al., 1997; Douglas et al., 2003; Patrzykat et al., 2003; Brocal et al., 2006)

(Zou et al., 2007; Casadei et al., 2009; Jin et al., 2010; Cuesta et al., 2011; van der Marel et al., 2012)

Mucous goblet cells

Carp

ND

(van der Marel et al., 2012)

Hepcidin

Peritoneal leukocytes, head, kidney, liver, and skin epithelium

Gilthead seabream, tilapia

Yes

Cathelicidins (e.g., rtCath1, rtCath2)

Mast cells, skin epithelium Cathelicidins (hCAP-18/ LL-37)

Various, e.g., rainbow trout, Atlantic salmon, Atlantic cod

ND

(Maier et al., 2008; Scocchi et al., 2009)

Piscidin

Mast cells in the skin, gill, and gastrointestinal tract

Various

ND

(Noga and Silphaduang, 2003; Silphaduang et al., 2006)

Yes

(Cuesta et al., 2008; Chen et al., 2009a, Chia et al., 2010)

Abbreviations: hBD, human b-defensin; ND, not determined; rtCath1, rainbow trout cathelicidin 1; rtCath2, rainbow trout cathelicidin 2. According to Schro¨der (2002) and Wiesner and Vilcinskas (2010), these antimicrobial peptides (AMPs) are also prominently expressed in the human skin.

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search for cathelicidins in species other than humans. In fish, several cathelicidins have been identified (Uzzell et al., 2003; Chang et al., 2005, 2006; Maier et al., 2008). Fish cathelicidins are processed and activated by the protease elastase, which is the major cleaving enzyme in mammals, together with the kallikreins (Yamasaki et al., 2006; Morizane et al., 2010). However, phylogenetic analysis has demonstrated that the amphibian cathelicidin predates reptilia but postdates fish cathelicidin, suggesting a convergent evolution (Hao et al., 2011). In fish, there are several cathelicidin genes, whereas in humans only one gene exists. As discussed before, the regulation of cathelicidins has been further developed during evolution. Epithelial fish cathelicidins are regulated through alarm signals, such as DNA- or

pathogen-associated molecular patterns; human cathelicidin is also regulated by vitamin D (Park et al., 2011). Even if the cathelin-like domain in the middle of the molecule is highly conserved, the C-terminal antimicrobial domain is less conserved. The C-terminal antimicrobial cathelicidins are markedly variable in structure, and this highly functional sequence is cleaved off by appropriate proteases (Yang et al., 2001). In humans, there exists only the a-helical structure, which is cleaved by neutrophil elastase (Yang et al., 2001), whereas, e.g., in cod the predicted mature peptides are unusual peptides made mainly of arginine, glycine, and serine (RGS) residues, which form a novel class of AMPs (Maier et al., 2008). Thus, the less conserved C-terminal antimicrobial domain seems to be the one that is

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responsible for the different functions of the cathelicidin molecule. The expression pattern in trout could suggest different functions for the cathelicidins and derived peptides in innate immunity of fish: rainbow trout cathelicidin 1 (rtCath1) was found to be expressed only after infection, whereas rtCath2 was constitutively expressed in many tissues and further upregulated after bacterial challenge (Chang et al., 2006). Cathelicidins, together with b-defensins, are constitutively expressed in the skin of arctic char (Salvelinus alpinus) (Maier et al., 2008), zebrafish (Danio rerio) (Zou et al., 2007), and carp (Cyprinus carpio) (van der Marel et al., 2012), and upregulated 24 hours after bacterial (Maier et al., 2008) or viral (M Adamek and D Steinhagen, unpublished) challenge. Several other AMPs,

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including b-defensins and pleurocidin, have been immunolocalized in the mucus layer covering the epithelium (Cole et al., 1997; Cuesta et al., 2011; Ruangsri et al., 2012). Further AMPs in skin and epithelial tissues of perciform fishes, called piscidins, are produced in mast cells, a population of tissue granulocytes also found in other vertebrates (Silphaduang and Noga, 2001; Silphaduang et al., 2006). As discussed for cathelicidin, other conserved sequences of mature fish AMPs might emerge in the mammalian repertoire. To address this subject, there is a need to evaluate human homology with ancestral fish–derived immune effectors and mechanisms and evaluate the effect it may exert on our epithelium. From fish, chemically synthesized peptides have been tested for their cognate activities, e.g., by predictions of active peptide sequences in flatfish, and a highly active peptide had inhibitory activity against a test panel of pathogens including antibiotic-resistant bacteria and fungi (Patrzykat et al., 2003). Interestingly, other flatfish AMPs, such as members of the pleurocidin family that originally derived from the winter flounder, had an effect on human breast cancer cells (Hilchie et al., 2011). Without doubt, it deserves further studies on fish-specific peptides to explore a clear clinical relevance of homologous or nonhomologous fish AMPs. The evolutionary pattern suggests that invertebrates tend to have a less amount of specialized immune cell types compared with vertebrates, and lymphocytes have its closest, now living, holdings in fish, although some scientists argue that fish lack certain immune cell types compared with mammals (Rowley, 1996; Ottaviani and Franceschi, 1998). Fish rely on their innate immune response to a large extent. As in mammals, the cytokine IL-1b seems to act as an important activator, and IFN-g may act as a switch from the innate to a more specific response (Lindenstrm et al., 2003; Zou et al., 2005; Niklasson et al., 2011). This response in fish is dependent upon secreted products, i.e., cytokines and AMPs (Figure 2), and includes the activity of epithelial cells, skin-associated

macrophages, or mast cells, and on a broader range of complement factors and Toll-like receptors as compared with mammals (Rebl et al., 2010; Nakao et al., 2011). With regard to the immune repertoire, in terms of immunoglobulins and T-cell receptors, fish seems to lack the great diversity of immunoglobulins that mammals possess, but they still have the ability to mount a specific response (Amemiya and Litman, 1990; Boudinot et al., 2001). V(D)J recombination of DNA segments, responsible for the variability of antibodies, is seen for the first time in evolution in teleosts, and IgM and IgD have been proposed as the humoral immunoglobulins in fish (Hordvik et al., 1999). The slow, less-pronounced and less-diversified antibody production in fish compared with mammals is mainly explained by fish being ectothermic, and is thereby affected by seasonal and environmental variations. However, there might also be an underestimation of the complexity of the acquired immune response due to previous failure to successfully immunize fish in aquaculture as a result of the their inability to mount an efficient response upon re-exposure (Killie and Jrgensen, 1995). The available diversity in the fish antibody repertoire could therefore be much greater than the expressed diversity (Castro et al., 2011). IgM has for a long time been accounted as the mucus-secreted antibody of fish, as an equivalent antibody to IgA in mammals seems to be lacking. However, a new IgH-derived antibody was recently characterized, the IgT/IgZ (Danilova et al., 2005; Hansen et al., 2005). Further investigations led to the conclusion that this antibody is secreted into the mucus, and it seems to have a higher local defensive affect than IgM under specific conditions (Zhang et al., 2010). These results show that although counterparts for a significant amount of mammalian antibodies have not been found in fish, there is still a need for further research in this respect. Cationic peptides produced in the epithelia of fish after exposure to bacteria often have homologs in other organism groups; e.g., pleurocidin, found in winter flounder, is related to

AMPs found in both frog and fruitfly (Cole et al., 1997). Furthermore, the activation mechanism is likely to involve the same basic properties with Toll-like receptors and the highly conserved transcription factor NF-kB as important hallmarks (Rebl et al., 2009). Although antibiotics and bacterial activities induce cytokine expression and inflammatory responses, the AMPs have the benefit that they may act to inhibit cytokine expression and still exert an effect on the pathogen. In fish, cathelicidins have been shown to be expressed and to modulate the immune response to different pathogens (Bridle et al., 2011; Broekman et al., 2011). Interestingly, mammalian cathelicidins are important for cutaneous wound healing and barrier formation (Ramos et al., 2011). Alibardi et al. (2012) found an upregulation of b-defensins in lizard tails after wounding. It would thus not be surprising if cathelicidins or b-defensins had an important role in wound healing in fish as well. Fish mucus and AMPs with their broad spectrum might therefore be good candidates for dermatological therapy. Key open questions and research challenges

In the future, mucus from selected fish species may be a source of novel antimicrobial agents for human health– related applications, besides its importance in aquaculture health and welfare. However, the variation in habitat and life history of fish and mammals may complicate the issue. In mammals, different glands have been formed— sebaceous, mammary, or sweat glands. Do fish have a similar ‘‘overall strategy’’ of diverse mucogenic cell types? It is known that different types of glands have evolved, depending on the fish species and its habitat, e.g., the tetrodotoxin-secreting gland in the skin of pufferfish (Kodama et al., 1986) or serotonin-secreting club cells in the epidermis of the sea eel and other species (Zaccone et al., 1989). Some studies show that parts of the fish mucus support human wound healing, e.g., fatty and amino acids (Jais et al., 1994, 1998; Zakaria et al., 2007) or Sebastes schlegeli antibacterial protein (L-amino acid oxidase) (Kitani et al., 2007). The www.jidonline.org 1145

S Rakers et al. AMPs from Fish Epidermis

‘‘Antimicrobial peptide design’’ Fish

Amphibian

Conserved protein regions

Mammals

Modification *

Modified synthethic AMP molecule Mature peptide

Figure 5. Using evolution to design synthetic antimicrobial peptide (AMP) molecules. A synthetic AMP would include common structures of AMPs found throughout the animal kingdom, such as a mature peptide, consisting of amphiphatic and cationic amino acids. Recently, sophisticated approaches include modifications such as hybrid AMPs, AMP congeners, cyclotides, and stabilized AMPs, AMP conjugates, and immobilized AMPs (Brogden and Brogden, 2011). In the manner described, would it be possible to create synthetic AMPs that work both in human skin and fish (e.g., in fish aquaculture)? *Modifications as described by Brogden and Brogden (2011).

Box 1. Why fish AMPs matter General properties of fish antimicrobial peptides (AMPs) and some interesting features of selected fish AMPs: | Almost all fish AMPs have antibacterial or bacteriostatic functions against several Gram-negative and Gram-positive strains (Najafian and Babji, 2012). | Fish rely more heavily on their innate immune defenses than mammals, and might therefore constitute a potential rich source of antiviral compounds for fighting against mammalian viral infections (Esteban, 2012). | Piscidin-2 of hybrid striped bass was shown to exhibit potent antifungal activity against human pathogenic fungi (Sung et al., 2008). | Epinecidin-1 acts as a potential antitumor agent against human fibrosarcoma cells (Lin et al., 2009, Chen et al., 2009a, b). | The diversity of genomic sequences encoding fish AMPs can serve as a source for synthetic (novel) recombinant AMPs for use in medicine. Aquatic sources contain thousands of fish species, and each secretes AMPs with structural differences.

great diversity in mucus ingredients might similarly be the key for broad treatment possibilities of skin diseases. As mucus IgM, and may be IgT as well, are important in the mucus-associated immune system of fish skin (Shepard, 1994; Lim, 2008; Zhang et al., 2010), the anti-infection potential of fish mucus might be of interest for the development of topical dermatological therapy (Baie, 1999; Rakers et al., 2010). For dermatological applications, high amounts of pure peptides have to be provided. To this end, genetic technologies present are at hand to isolate peptide genes from nature without having to go through a protein purification process (Patrzykat et al., 2003).

Unfortunately, often neither the sequence nor the structure is conserved among gene families. A reasonable hypothesis is that small peptides with antimicrobial abilities have evolved multiple times (Tennessen, 2005). The most prominent family of peptides exhibiting a common genetic organization and a common N-terminal flanking sequence are the cathelicidins. Even so, mature cathelicidins display a range of amino acid compositions, charges, lengths, and structures, which make it difficult to decide the most important features. A design of a synthetic AMP, however, would include common structures of AMPs found throughout the animal kingdom, starting

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with the mature peptide, consisting of amphiphatic and cationic amino acids (Figure 5). Interspecies comparisons can be made to identify specific residues or conserved structures in the mature antimicrobial peptide to design the most active or specifically active peptide. Czihal et al. (2012) demonstrated that the antibacterial peptide apidaecin 1b could be the origin for a lead compound, in this case named ‘‘apa88,’’ and the sequence has then been optimized to treat systemic infections. To conclude, several hurdles have to be overcome, such as the amount of AMPs compared with other antibiotics (Marr et al., 2006). With more highly conserved sequences discovered, as for cathelecidins and defensins (Zasloff, 2002; Patrzykat et al., 2003; Dean et al., 2011; Lu et al., 2011), and possible modifications of AMPs with functionalized biomaterials (Brogden and Brogden, 2011; Veerapandian and Yun, 2011), the creation of synthetic ‘‘super-AMP’’ molecules (Figure 5) with widespread applications from agriculture and aquaculture to clinical dermatology can be envisioned. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank Paul M Maderson for critical comments on the fish skin structures and Dr Marina Gebert for substantial comments on the manuscript. The writing of this review was made possible in part by grants from the European Fond for Regional Development (EFRE) to CK and from Manchester NIHRS Biomedical Research Center to RP. This work has been supported by the EU project Lifecycle (FP7-222719) to LN and KS.

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