Antimicrobial Peptides in Echinoderms ISJ 7: 132-140, 2010 ISSN 1824-307X MINIREVIEW Antimicrobial peptides in Echinoderms C Li, T Haug, K Stensvåg Norwegian College of Fishery Science, Faculty of Bioscience, Fisheries and Economics, University of Tromsø, Breivika, N-9037 Tromsø, Norway Accepted May 5, 2010 Abstract Antimicrobial peptides (AMPs) are important immune effector molecules for invertebrates, including echinoderms, which lack a vertebrate-type adaptive immune system. Here we summarize the knowledge of such peptides in echinoderms. Strongylocins are a novel family of cysteine-rich AMPs, recently identified in the sea urchins, Strongylocentrotus droebachiensis and S. purpuratus. Although these molecules present diverse amino acid sequences, they share an identical cysteine arrangement pattern, dissimilar to other known AMPs. A family of heterodimeric AMPs, named centrocins, are also present in S. droebachiensis. Lysozymes and fragments of larger proteins, such as beta-thymocins, actin, histone 2A and filamin A have also been shown to display antimicrobial activities in echinoderms. Future studies on AMPs should be aimed in revealing how echinoderms use these AMPs in the immune response against microbial pathogens. Key Words: sea urchin; host defence peptides; celomocyte; innate immunity Introduction Antimicrobial peptides (AMPs) are evolutionarily conserved small molecular weight proteins of the innate immune response, with a broad spectrum of antimicrobial activities against bacteria, viruses, and fungi (reviewed by Mookherjee and Hancock, 2007). AMPs appear naturally throughout all three domains of life from unicellular to multicellular organisms (Zasloff, 2002; Riley and Chavan, 2007; Wang et al., 2009). By February 2010, more than 1,500 AMPs have been identified (http://aps.unmc.edu/AP/main.php). AMPs are characterized as having less than 100 amino acids and are usually cationic in nature. Nearly all antimicrobial peptides form amphipathic structures which reflect the relative abundance and polarization of hydrophobic and hydrophilic domains within the peptides. The hydrophobicity enables the water-soluble antimicrobial peptides to interact with the hydrophobic lipid bilayer of the microbial membranes. AMPs are likely to be attracted by and attach to the negatively charged bacterial surfaces because of their cationic nature. The mechanism by which AMPs interact with the cell wall structures of Gram- ___________________________________________________________________________ Corresponding author: Klara Stensvåg Norwegian College of Fishery Science Faculty of Bioscience, Fisheries and Economics University of Tromsø, Breivika, N-9037 Tromsø, Norway E-mail: Klara.Stensvag@uit.no negative and Gram-positive bacteria has rarely been addressed and is therefore not yet understood (Brogden, 2005). However, once the peptides come into contact with the outer leaflet of the cell membrane and the peptide/lipid ratio increases, the peptides start forming multimers or self-associating on top of the membrane (Yang et al., 2001). At sufficiently high concentrations of the peptides orientate perpendicularly and insert into the bilayer, thereby interfering with membrane integrity. However, new paradigms imply that pore-forming is not the only mechanism of antimicrobial activity. Some peptides are also able to interact with intracellular targets without disrupting the membrane integrity. Intracellular targets of antimicrobial peptides vary from nucleic acids to enzymatic proteins (Brogden, 2005). AMPs are crucial immune effector molecules for invertebrates which lack a vertebrate-type adaptive immune system. They have been identified both in plasma and in various blood cells and their distribution in the host can be site-specific or systemic. They are either expressed constitutively or the expression is induced by exposure to pathogens. They do not only inactivate bacteria in vitro and in vivo , thereby protecting host organisms against a variety of infections, but they also modulate immunity ( Hancock and Diamond, 2000; Zasloff, 2002; Hancock et al., 2006). It is worth mentioning that a few AMPs play a role as anti-endotoxins (Scott et al., 2002; Bowdish et al., 2005) and chemokines in vertebrates (Durr and Peschel, 2002; 132 http://aps.unmc.edu/AP/main.php mailto:Klara.Stensvag@uit.no Davidson et al., 2004) and might also induce production of cytokines and chemokines (Bals and Wilson, 2003). These immunomodulatory functions do not directly kill microbes, but recruitment and activation of immune cells and signalling molecules improves the host defence system. In this review we will present an overview of AMPs identified and characterized from echinoderms and present their characteristics. In particularly, we will describe their antimicrobial activities, their primary structure, and posttranslational modifications of these molecules. Finally, we will address some important questions regarding future AMP research on echinoderms. Antimicrobial activities in echinoderms The phylum echinodermata is a large phylum of deuterostomes containing approximately 7,000 living species divided into the groups Crinoidea (sea lilies), Ophiuroidea (brittle stars), Asteroidea (starfishes), Echinoidea (sea urchins) and Holothuroidea (sea cucumbers). The adult echinoderms have a few major organs such as mouth parts, intestine, nerve ring, and gonad. These organs are located in the cavity, named celomic cavity. The remaining space of the celomic cavity is filled with celomic fluid which contains various “blood cells”, the celomocytes. These cells are though to mediate the main immune functions in echinoderms, including antimicrobial activities. Many studies have been conducted to examine the activity of celomocytes lysates and celomic fluid against bacteria, fungi and even tumour cells. Celomic fluid from Echinus esculentus possess bactericidal activity against marine Pseudomonas sp (Wardlaw and Unkles, 1978). Gerardi et al. (1990) found that the highest bacterial growth inhibition against several Vibrio sp. is shown by phagocytes (called amoebocytes) and red spherule cells of Paracentrotus lividus.. Stabili et al. (1996) also discovered antibacterial activity against V. alginolyticus in celomocyte lysates and celomic fluid of P. lividus. Recently, it was reported that celomocytes of P. lividus show a cytotoxic activity against rabbit erythrocytes and the K562 tumour cell line (Arizza et al., 2007). Antibacterial activity was detected in extracts of several tissues from the green sea urchin S. droebachiensis, the common sea star Asterias rubens, and the sea cucumber Cucumaria frondosa (Haug et al., 2002). Some of the activities detected (in extracts of celomocytes and body walls) were caused by compounds of protein nature. Several drug discovery projects have screened echinoderms for antibiotic activities. An early study by Rinehart et al (1981) showed that antimicrobial activity was present in 43 % of 83 unidentified species of echinoderms (collected from the west coast of Baja California and the Gulf of California) and 58 % of 36 unidentified Caribbean species displayed antimicrobial activities. In the northern Gulf of Mexico, 80 % of 22 echinoderm species showed antimicrobial activity (Bryan et al., 1994). Body wall extracts of echinoderms displayed activity against marine bacteria, but did also inhibit settlement of marine larvae (Bryan et al., 1996). Several antimicrobial molecules (other than AMPs) have been isolated from echinoderms, including echinochrome A (Kuwahara et al., 2009; Service and Wardlaw, 1984), steroidal glycosides (Andersson et al., 1989; Chludil et al., 2002; Levina et al., 2009), and polyhydroxylated sterols (Iorizzi et al., 1995). Although these results indicate that echinoderms present remarkable activity against microbes, only a few AMPs have been reported. Antimicrobial peptides in echinoderms Lysozymes are widely distributed throughout the animal kingdom and catalyze the hydrolysis of peptidoglycans of bacterial cell walls of especially Gram-positive bacteria, and acts as a nonspecific innate immunity molecules against the invasion of bacterial pathogens (Jollès and Jollès, 1984). They are characterized as basic proteins of low molecular weight (15-25 kDa) with an isoelectric point of 10.5– 11.0, stable at acidic pH and even at high temperatures. There are several lysozymes or lysozyme-like proteins identified from celomic fluid, celomocytes and other tissues of echinoderms (Jollès and Jollès, 1975; Canicatti and Roch, 1989; Canicatti, 1990; Canicatti and D´Ancona, 1990; Gerardi et al., 1990; Stabili et al., 1994; Stabili and Canicatti, 1994; Stabili and Pagliara, 1994, 2009; Shimizu et al., 1999; Bachali et al., 2004; Cong et al., 2009) (Table 1). A study by Beauregard and co-workers indicated the presence of AMPs in the celomic fluid of the orange-footed sea cucumber, C. frondosa (Beauregard et al., 2001). The celomic fluid extract was purified by molecular sieve chromatography and antibacterial activity was detected in separate fractions. A peptide of approximately 6 kDa was identified, but no sequence was reported (Table 1). In a celomocyte extract from the starfish A. rubens showing antimicrobial activity, several protein/peptides with molecular mass around 2 kDa were isolated (Maltseva et al., 2004; Maltseva et al., 2007) (Table 1). Two peptides were identified as part of the histone molecule, H2A. Two other peptides were identified as fragments of actin, while one peptide was a fragment of filamin A. In addition, four other AMPs were detected, but not characterized. It had been known for long time that histones have antibacterial properties (Hirsch 1958). Histone-derived fragments (H1, from H2A and H2B) have shown to possess antimicrobial activity (From et al., 1996; Robinette et al., 1998; Patrzykat et al., 2001; Richards et al., 2001; Fernandes et al., 2002). Although Maltseva et al. (2004, 2007) also identified fragments of actin and filamin A in the active extract, the antimicrobial activity of these fragments need to be further investigated. Recently, Schillaci et al. (2009) reported that a 5 kDa peptide fraction from the celomocytes of P. lividus presents activity against both Gram-positive and Gram-negative bacteria and fungi (Table 1). The peptide fraction also showed the ability to inhibit the formation of Staphylococcus biofilms. The authors suggested that the antimicrobial and antistaphylococcal biofilm activity of this peptide 133 Table 1 Antimicrobial peptides and proteins characterized in echinoderms Class and genus Origin (tissue/cell type) Compound Mw (kDa) Reference Asteroidea Asterias forbesi Cell-free celomic fluid Complement-like Leonard et al., 1990 A. rubens Body wall Peptides/proteins <20 Haug et al., 2002 Celomocytes Fragments of actin, histone H2A, and filamin A 1.8- 2.4 Maltseva et al., 2007; Maltseva et al., 2004 Celomocytes Peptides 2.0-4.7 Maltseva et al., 2007; Maltseva et al., 2004 Lysozyme 15.5 Jollès and Jollès 1975; Bachali et al., 2004 Marthasterias glacialis Eggs Lysozyme-like Stabili and Pagliara, 1994, 2009 Body wall mucus Lysozyme-like Canicatti and D´Ancona, 1990 Echinoidea Holothuria scabra Celomic fluid Lectin >10 Gowda et al., 2008 H. polii Celomocytes Lysozyme-like Canicatti and Roch, 1989 Various tissues Lysozyme-like Canicatti, 1990 Paracentrotus lividus Celomocytes Lysozyme-like Gerardi et al., 1990 Celomocytes Protein ~ 60 Stabili et al., 1996 Celomocytes Peptide ~ 5 Schillaci et al., 2009 Larvae Lysozyme-like Stabili et al., 1994 Seminal plasma Lysozyme-like Stabili and Canicatti, 1994 Various tissues Lysozyme-like Canicatti, 1990 Strongylocentrotus droebachiensis Celomocytes Stongylocins 5.6- 5.8 Li et al., 2008 Celomocytes Centrocins 4.4-4.5 Li et al., 2010b S. intermedius Celomic fluid Lysozyme 14 Shimizu et al., 1999 S. purpuratus Celomocyte cDNA SpStrongylocinsa 5.6-6.1 Li et al., 2010a Holothuroidea Cucumaria echinata Whole body Fragments of lectin CEL-III 2.0- 4.2 Hatakeyama et al., 2004; Hisamatsu et al., 2008 C. frondosa Celomic fluid Peptides ~ 6 Beauregard et al., 2001 Various tissues Peptides/proteins <20 Haug et al., 2002 Parastichopus califormicus Celomic cavity LPS-binding compound Dybas and Fankboner, 1986 Stichopus japonicus Intestine cDNA Lysozymea 14 Cong et al., 2009 a Recombinantly produced and tested for activity. fraction is associated with beta-thymosin like fragments. Beta thymosins have been reported to induce the production of metalloproteinases, display chemotactic and anti-inflammatory activities (Huff et al., 2001) and even function as AMPs released by human platelets (Tang et al., 2002). Several AMPs have been isolated and characterized from the green sea urchin, S. droebachiensis. One group is the cysteine rich peptides named strongylocins (Li et al., 2008). Homologous genes, named SpStrongylocins, were also identified in the sister species S. purpuratus (Li et al., 2010a). All these native peptides or recombinant equivalents show antibacterial activity against both Gram positive and Gram negative bacteria (Table 2). In addition, there are several heterodimer structured peptides, named centrocins, identified from S. droebachiensis (Li et al, 2010b). They show strong potent activity, not only against Gram-positive and Gram-negative bacteria, but the longest peptide chain of the dimers also show strong activity against fungi and yeasts. In silico analysis of strongylocins and comparison of the native peptide sequences and the ones deduced from cDNA sequences, show that these peptides contain three regions: a signal peptide, a prosequence and a native sequence (Fig. 1). Analysis , using both the neutral network model and the hidden Markov model (Bendtsen et al., 2004; http://www.cbs.dtu.dk/services/SignalP/), indicates that a signal peptide cleavage site is located between the amino acid number 22 and 23 for all these peptides. 134 http://www.cbs.dtu.dk/services/SignalP/ Table 2 Susceptibility of bacterial strains to the antibacterial peptides, strongylocins and centrocins, isolated from S. droebachiensis celomocytes, and recombinant SpStrongylocins (Li et al., 2008; Li et al. 2010a, b) Minimal inhibitory concentration (μM) Peptide Listonella anguillarum Escherichia coli Corynebacterium glutamicum Staphylococcus aureus Strongylocin 1 a 2.5 5.0 2.5 2.5 Strongylocin 2 a 1.3 5.0 2.5 2.5 Recombinant SpStrongylocin 1 b 15.0 7.5 7.5 15.0 Recombinant SpStrongylocin 2 b 15.0 7.5 3.8 15.0 Centrocin 1 a 2.5 1.3 1.3 2.5 Centrocin 2 a 2.5 2.5 1.3 5.0 a Minimal inhibitory concentration (MIC) was determined as the lowest concentration of peptide causing an optical density less than 50 % of the growth control without any peptide present. b Minimal inhibitory concentration (MIC) was determined as the lowest concentration of peptide causing 100 % of the growth inhibition of the test organism compared to the growth control. It is not surprising that strongylocin 1 and SpStrongylocin 1 contain an identical signal peptide sequence since they share high amino acid sequence similarity. However, SpStrongylocin 2 does not share an identical signal peptide with strongylocin 2, but with strongylocin 1. Although no experimental data show which mechanism assists the migration of these precursors of strongylocins and SpStrongylocins within the cells, the diversity of the signal peptides suggests that strongylocin 2 likely employs a divergent intracellular trafficking pathway than the other sea urchin AMPs. Analysis of strongylocins and SpStrongylocins show that their prosequences are located at the N- terminal region, following the signal peptide. It is common that prosequences are located at the N- terminal or the C-terminal region, or even in between parts of the mature sequences. The prosequence is considered to act as an intracellular steric chaperone during the folding process (Inouye, 1991), and it has been shown that prosequence- supported protein folding is crucial for processing of the proper protein having antifungal activity (Reichhart and Achstetter, 1990). In addition, it has been shown that the prosequences in some proteolytic enzyme precursors inhibit the activity of the mature proteins (Neurath, 1989). The prosequences of strongylocins and SpStrongylocins are negatively charged sequences which may neutralize the positive charges of the mature sequences (Li et al., 2008; Li et al., 2010a, b). Therefore, the precursor molecules are likely less toxic to the host cells than the mature peptides. The peptides will obtain their activity during their maturation once the prosequences are removed. Strongylocins contain six cysteine residues, which form three disulfide bridges (Li et al., 2008; Li et al., 2010a). Cysteine-rich AMPs are found in various phyla, such as the defensin families in both vertebrates and invertebrates, tachystatins in horse shoe crabs and circulin A in plants (Daly et al., 1999; Wang et al., 2009). Table 3 shows that strongylocins and SpStrongylocins display a novel cysteine location pattern compared to other known cysteine-rich peptides containing 6 cysteines. This indicates that these AMPs may not form the same disulfide bridge linkages as the other peptides containing six cysteines, since proteins that have the same cysteine residue pattern tend to have identical disulfide connections (Bania et al., 1999; Bontems et al., 1991; Pallaghy et al., 1994). Disulfide bridges are crucial for the antimicrobial activity of most cysteine-containing AMPs (Daher et al., 1986; Mandal and Nagaraj, 2002), and they may also stabilize the conformation of the molecules (Selsted and Ouellette, 2005). Therefore, the disulfide linkages may aid strongylocins and SpStrongylocins to resist proteolysis within the celomocytes, and/or after their release into protease-containing environments. Strongylocin 2 contains a tryptophan in the mature sequence which is likely brominated (Li et al., 2008). Several AMPs with bromotryptophan have also been isolated from other marine organisms, for example styelin D from the tunicate, Styela clavata (Taylor et al., 2000), cathelicidin from the hagfish, Myxine glutinosa (Shinnar et al., 2003), and hedistin from the annelid, Nereis diversicolor (Tasiemski et 135 Fig. 1 Alignment of strongylocins from S. droebachiensis and SpStrongylocins from S. purpuratus. The predicted cleavage site between the signal peptide and the proregion is shown (▼). The first amino acid in active strongylocin 2 and SpStrongylocin 2 are likely a modified tryptophan (♦). Identical amino acids are shown in boxes, similar amino acids are shaded in grey, and cysteines are highlighted in black. al., 2007). Brominated amino acids may protect the peptides from proteolysis, and/or increase the biological activity of peptides (Bittner et al., 2007). Although SpStrongylocin 2 also contains a tryptophan residue in the same position as strongylocin 2, the recombinantly produced SpStrongylocins, having a non-brominated Trp residue, are still antimicrobial active (Li et al., 2010a). Therefore, we speculate that the brominated tryptophan affects the peptides by enhancing their stability, but does not affect their antimicrobial activity (Li et al., 2010a). This suggestion is supported by results showing no difference in the antimicrobial activity between the heavy chain of the dimeric peptide centrocin 1, with or without the brominated modification (Stensvåg, unpublished). Future studies on AMPs in echinoderms AMPs are important immune defence molecules for echinoderms that lack a vertebrate- type adaptive immune system (reviewed by Smith et al., 2010). It is therefore interesting to know whether these AMPs are secreted into the celomic fluid or stored in the granula of certain cell types. It has been documented that neutrophilic granules packed with human cathelicidin and defensins are fused with phagocytic vacuoles, where they contain certain concentrations of AMPs to eliminate phagocytosed pathogens (reviewed by Ganz, 2003; Lehrer, 2004; Brogden, 2005). For penaeidins in shrimp, it seems that haemocytes migrate towards the infection sites and then release the AMPs presumably by lysis of the cells (Munoz et al., 2002). Echinoderms have a large celomic cavity filled with circulating fluid which could dilute AMPs if they were secreted or released by the cells. Therefore, further investigation on the distribution of AMPs in echinoderms might uncover whether the peptides are involved in 1) intracellular elimination of pathogens; 2) local release of AMPs at the infection sites; or 3) massive release of AMPs to regulate other immune activities. Although several studies have documented mammalian AMPs as immune regulatory molecules (Reviewed by Hancock et al., 2006 and Diamond et al., 2009), unfortunately, there are not many investigations on invertebrate AMPs. It has been reported that Tachypleus tridentatus haemocyte granules can release tachyplesin after 136 Table 3 Comparison of cysteine location patterns in AMPs containing six cysteines Peptide family Cysteine location patterns 1 Animals Strongylocins C – C – C – CC – C S. droebachiensis SpStrongylocins C – C – C – CC – C S. purpuratus Βeta-defensins C – C – C – C – CC Bos taurus Alpha-defensins C – C – C – C – CC Homo sapiens Tachystatins C – C – CC – C – C T. tridentatus Knottin-type AMPs C – C – CC – C – C Phytolacca americana Thionins type III and IV AMPs CC – C – C – C – C Sorghum bicolor Insect defensins C – C – C – C – C – C Rhodnius prolixus 1 Adjacent double cysteine residues are highlighted in yellow. Information regarding cysteine arrangements in the different peptides was obtained from the Antimicrobial Peptide Database (Wang et al., 2009). encountering microbial endotoxins and form a complex (Iwanaga et al., 1998; Hirakura et al., 2002). This complex will then block the activation of factor C, which is crucial for haemolymph coagulation (Nakamura et al., 1988). Pancer et al. (1999) identified an NF-κB homologue, transcription factor in S. purpuratus celomocytes According to analysis of the immune related gene repertoire of S. purpuratus (Hibino et al., 2006; Rast et al., 2006), Toll-like receptor (TLR) genes, interleukin (IL)-17 genes, IL receptors, and tumor necrosis factor (TNF) family members are present in the sea urchin genome. It is possible that AMPs are involved in the endotoxin-induced signalling pathway from the TLR to NF-κB, and/or suppress inflammation. Therefore, it is important to keep in mind that AMPs may play multiple immune roles in the echinoderm´s immunity as in other species (Hancock and Diamond, 2000; Lehrer, 2004; Durr et al., 2006; Hancock et al., 2006; Mookherjee and Hancock, 2007; Cuthbertson et al., 2008; Smith et al., 2008). The discovery of AMPs in echinoderms has attracted attention from both pharmaceutical and academic sectors. However, there should be more focus on searching for new AMPs in Crinoidea and Ophiuroidea by newly developed high-throughput screening systems or other proteomic techniques since there is limited information about antimicrobial compounds from these echinoderm classes. In summary, AMPs are important host defence molecules in invertebrates. Investigations of antimicrobial peptides/proteins in echinoderms have revealed two novel families of AMPs in Strongylocentrotus, lysozymes in various species and fragments of larger proteins having antibacterial activity. These AMPs do not only attract interest as potent drugs or drug leads, but may also become useful in studying the echinoderm immune system. Acknowledgements Writing this review was supported by grants from the University of Tromsø, The Marine Biotechnology in Tromsø (MABIT) research program (No. BS0034) and the Norwegian Research Council (Nos. 178214/S40 and 184688/S40). Oleg Sokolov is appreciated for translating the Russian papers. References Andersson L, Bohlin L, Iorizzi M, Riccio R, Minale L, Moreno-López W. Biological activity of some saponins and saponin-like compounds from starfish and brittle-stars. Toxicon 27: 179-188, 1989. Arizza V, Giaramita FT, Parrinello D, Carnmarata M, Parrinello N. Cell cooperation in coelomocyte cytotoxic activity of Paracentrotus lividus, coelomocytes. Comp. Biochem. Physiol. 147A: 389-394, 2007. Bachali S, Bailly X, Jolles J, Jolles P, Deutsch JS. The lysozyme of the starfish Asterias rubens. Eur. J. Biochem. 271: 237-242, 2004. Bals R, Wilson JM, Cathelicidins--a family of multifunctional antimicrobial peptides. Cell. Mol. Life Sci. 60: 711-720, 2003. Bania J, Stachowiak D, Polanowski A. Primary structure and properties of the cathepsin G/chymotrypsin inhibitor from the larval hemolymph of Apis mellifera. Eur. J. Biochem. 262: 680-687, 1999. Beauregard KA, Truong NT, Zhang H, Lin W, Beck G. The detection and isolation of a novel antimicrobial peptide from the echinoderm, Cucumaria frondosa. Adv. Exp. Med. Biol. 484: 55-62, 2001. Bendtsen JD, Nielsen H, von Heijne G, Brunak S. Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340: 783-795, 2004. Bittner S, Scherzer R, Harlev E. The five bromotryptophans. Amino Acids 33: 19-42, 2007. Bontems F, Roumestand C, Gilquin B, Menez A, Toma F. Refined structure of charybdotoxin: common motifs in scorpion toxins and insect defensins. Science 254: 1521-523, 1991. Bowdish DM, Davidson DJ, Lau YE, Lee K, Scott MG, Hancock RE. Impact of LL-37 on anti- infective immunity. J. Leukoc. Biol. 77: 451-459, 2005. Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3: 238-250, 2005. 137 Bryan PJ, McClintock JB, Watts SA, Marion KR, Hopkins TS. Antimicrobial activity of ethanolic extracts of echinoderms from the northern Gulf of Mexico. In: B David, A Guille, J-P Feral, M Roux (eds), Echinoderms Through Time, Balkema, Rotterdam pp 17-23, 1994. Bryan PJ, Rittschof D, McClintock JB. Bioactivity of echinoderm ethanolic body-wall extracts: an assessment of marine bacterial attachment and macroinvertebrate larval settlement. J. Exp. Mar. Biol. Ecol. 196: 79-96, 1996. Canicatti C, Distribution d´une activite lysozymiale dans un echinoderme holothuroide, Holothuria polii, et dans les oeufs et les larves d´un echinoderme echinoide, Paracentrotus lividus. Eur. Arch. Biol. (Bruxelles) 101: 309-318, 1990. Canicatti C, D´Ancona G. Biological protective substances in Marthasterias glacialis (Asteroidea) epidermal secretion. J. Zool. (Lond.) 222: 445-454, 1990. Canicatti C, Roch P, Studies on Holothuria polii (Echinodermata) antibacterial proteins. I. Evidence for and activity of a coelomocyte lysozyme. Experientia 45: 756-759, 1989. Chludil HD, Seldes AM, Maier MS. Antifungal steroidal glycosides from the Patagonian starfish Anasterias minuta: Structure-activity correlations. J. Nat. Prod. 65: 153-157, 2002. Cong L, Yang X, Wang X, Tada M, Lu M, Liu H, et al. Characterization of an i-type lysozyme gene from the sea cucumber Stichopus japonicus, and enzymatic and nonenzymatic antimicrobial activities of its recombinant protein. J. Biosci. Bioeng. 107: 583-588, 2009. Cuthbertson BJ, Deterding LJ, Williams JG, Tomer KB, Etienne K, Blackshear PJ, et al, Diversity in penaeidin antimicrobial peptide form and function. Dev. Comp. Immunol. 32:167-181, 2008. Daher KA, Selsted ME, Lehrer RI. Direct inactivation of viruses by human granulocyte defensins. J. Virol. 60: 1068-1074, 1986. Daly N L, Koltay A, Gustafson KR, Boyd MR, Casas-Finet JR, Craik DJ. Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV activity. J. Mol. Biol. 285: 333-345, 1999. Davidson DJ, Currie AJ, Reid GS, Bowdish DM, MacDonald KL, Ma RC, et al. The cationic antimicrobial peptide LL-37 modulates dendritic cell differentiation and dendritic cell-induced T cell polarization. J. Immunol. 172: 1146-1156, 2004. Diamond G, Beckloff N, Weinberg A, Kisich KO. The roles of antimicrobial peptides in innate host defense. Curr. Pharm. Des. 15: 2377-2392, 2009. Durr M, Peschel A. Chemokines meet defensins: the merging concepts of chemoattractants and antimicrobial peptides in host defense. Infect. Immun. 70: 6515-6517, 2002. Durr UH, Sudheendra US, Ramamoorthy A. LL-37, the only human member of the cathelicidin family of antimicrobial peptides. Biochim. Biophys. Acta 1758: 1408-1425, 2006. Dybas L, Fankboner PV. Holothurian survival strategies: Mechanisms for the maintenance of a bacteriostatic environment in the coelomic cavity of the sea cucumber. Dev. Comp. Immunol. 10: 311-330, 1986. Fernandes JMO, Kemp GD, Molle G, Smith JV. Novel antimicrobial properties of histone H2A from skin secretions of rainbow trout, Oncorhynchus mykiss. Biochem. J. 368: 611- 620, 2002. From M, Gunne H, Bergman AC, Agerberth B, Bergman T, Boman A, et al. Biochemical and antibacterial analysis of human wound and blister fluid. Eur. J. Biochem. 237: 86-92, 1996. Ganz T. Defensins: Antimicrobial peptides of innate immunity. Nat. Rev. Immunol. 3: 710-720, 2003. Gerardi P, Lassegues M, Canicatti C. Cellular distribution of sea urchin antibacterial activity. Biol. Cell 70: 153-157, 1990. Gowda NM, Goswami U, Khan MI. T-antigen binding lectin with antibacterial activity from marine invertebrate, sea cucumber (Holothuria scabra): Possible involvement in differential recognition of bacteria. J. Invert. Pathol. 99: 141-145, 2008. Hancock RE, Brown KL, Mookherjee N. Host defence peptides from invertebrates--emerging antimicrobial strategies. Immunobiology 211: 315-322, 2006. Hancock R E, Diamond G, The role of cationic antimicrobial peptides in innate host defences. Trends Microbiol. 8: 402-410, 2000. Hatakeyama T, Suenaga T, Eto S, Niidome T, Aoyagi H. Antibacterial activity of peptides derived from the C-terminal region of a hemolytic lectin, CEL-III, from the marine invertebrate Cucumaria echinata. J. Biochem. 135: 65-70, 2004. Haug T, Kjuul AK, Styrvold OB, Sandsdalen E, Olsen MO, Stensvåg K. Antibacterial activity in Strongylocentrotus droebachiensis (Echinoidea), Cucumaria frondosa (Holothuroidea), and Asterias rubens (Asteroidea). J. Invert. Pathol. 81: 94-102, 2002. Hibino T, Loza-Coll M, Messier C, Majeske AJ, Cohen AH, Terwilliger DP, et al, The immune gene repertoire encoded in the purple sea urchin genome. Dev. Biol. 300: 349-365, 2006. Hirakura Y, Kobayashi S, Matsuzaki M Specific interactions of the antimicrobial peptide cyclic B-sheet tachyplesin I with lipopolysaccharides. Biochim. Biophys. Acta 1562: 32-36, 2002. Hirsch JG. Bactericidal action of histone, J. Exp. Med. 108: 925-944, 1958. Hisamatsu K, Tsuda N, Goda S, Hatakeyama T. Characterization of the alpha-helix region in domain 3 of the haemolytic lectin CEL-III: Implications for self-oligomerization and haemolytic processes. J. Biochem. 143: 79-86, 2008. Huff T, Muller CS, Otto AM, Netzker R, Hannappel E. beta-Thymosins, small acidic peptides with multiple functions. Int. J. Biochem. Cell Biol. 33: 205-220, 2001. Inouye M. Intramolecular chaperone: the role of the pro-peptide in protein folding. Enzyme 45: 314- 321, 1991. Iorizzi M, Bryan P, McClintock J, Minale L, Palagiano E, Maurelli S, et al. Chemical and 138 biological investigation of the polar constituents of the starfish Luidia clathrata, collected in the Gulf of Mexico. J. Nat. Prod. 58: 653-671, 1995. Iwanaga S, Kawabata S. Evolution and phylogeny of defense molecules associated with innate immunity in horseshoe crab. Front. Biosci. 3, D973-984, 1998. Jollès J, Jollès P. The lysozyme from Asterias rubens. Eur. J. Biochem. 54: 19-23, 1975. Jollès P, Jollès J. What's new in lysozyme research? Always a model system, today as yesterday, Mol. Cell. Biochem. 63: 165-189, 1984. Kuwahara R, Hatate H, Yuki T, Murata H, Tanaka R, Hama Y. Antioxidant property of polyhydroxylated naphthoquinone pigments from shells of purple sea urchin Anthocidaris crassispina. Lwt-Food Sci. Technol. 42: 1296- 1300, 2009. Lehrer RI. Primate defensins. Nat. Rev. Microbiol. 2: 727-38, 2004. Leonard LA, Strandberg JD, Winkelstein JA. Complement-like activity in the sea star, Asterias forbesi. Dev. Comp. Immunol. 14: 19- 30, 1990. Levina EV, Kalinovsky AI, Dmitrenok PV. Steroid compounds from two pacific starfish of the genus Evasterias. Russian J. Bioorg. Chem. 35: 123-130, 2009. Li C, Haug T, Styrvold OB, Jørgensen TØ, Stensvåg K. Strongylocins, novel antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis. Dev. Comp. Immunol. 32:1430- 1440, 2008. Li C, Blencke HM, Smith LC, Karp MT, Stensvåg K. Two recombinant peptides, SpStrongylocins 1 and 2, from Strongylocentrotus purpuratus, show antimicrobial activity against Gram- positive and Gram-negative bacteria. Dev. Comp. Immunol. 34: 286-292, 2010a. Li C, Haug T, Moe KM, Styrvold OB, Stensvåg K. Centrocins: isolation and characterization of novel dimeric antimicrobial peptides from the green sea urchin, Strongylocentrotus droebachiensis. Dev. Comp. Immunol. DOI: 10.1016/j.dci.2010.04.004, 2010b [in press]. Maltseva AL, Aleshina GM, Kokryakov VN, Krasnodembsky EG. Diversity of antimicrobial peptides in acidic extracts from coelomocytes of starfish Asterias rubens L. Vestnik Sankt- Peterburgskogo Universiteta, Seriya 3, Biologiya, 1: 85-94, 2007 (in Russian). Maltseva AL, Aleshina GM, Kokryakov VN, Krasnodembsky EG, Ovchinnikova TV. New antimicrobial peptides from coelomocytes of sea star Asterias rubens L. Vestnik Sankt- Peterburgskogo Universiteta, Seriya 3, Biologiya 4: 101-108, 2004 (in Russian). Mandal M, Nagaraj R. Antibacterial activities and conformations of synthetic alpha-defensin HNP- 1 and analogs with one, two and three disulfide bridges. J. Pept. Res. 59: 95-104, 2002. Mookherjee N, Hancock RE. Cationic host defence peptides: innate immune regulatory peptides as a novel approach for treating infections. Cell. Mol. Life Sci. 64: 922-933, 2007. Munoz M, Vandenbulcke F, Saulnier D, Bachere E. Expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp. Eur. J. Biochem. 269: 2678-2689, 2002. Nakamura T, Furunaka H, Miyata T, Tokunaga F, Muta T, Iwanaga S. Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus). J. Biol. Chem. 263: 16709-16713, 1988. Neurath H, Proteolytic processing and physiological regulation. Trends Biochem. Sci. 14: 268-271, 1989. Pallaghy PK, Nielsen KJ, Craik DJ, Norton RS. A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides. Protein Sci. 3: 1833-839, 1994. Pancer Z, Rast JP, Davidson EH. Origins of immunity: transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes. Immunogenetics 49: 773-786, 1999. Patrzykat A, Zhang L, Mendoza V, Iwama G, Hancock R. Synergy of histone-derived peptides of coho salmon with lysozyme and flounder pleurocidin. Antimicrob. Agents Chemother. 45: 1337-1342, 2001. Rast JP, Smith LC, Loza-Coll M, Hibino T, Litman GW. Genomic insights into the immune system of the sea urchin. Science 314: 952-956, 2006. Reichhart JM, Achstetter T. Expression and secretion of insect immune peptides in yeast. Res. Immunol. 141: 943-946, 1990. Richards RC, O'Neil DB, Thibault P, Ewart KV. Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar). Biochem. Biophys. Res. Commun. 284: 549-555, 2001. Riley MAR, Chavan MA. Bacteriocins: ecology and evolution. Chapter 5, Peptide and protein antibiotics from the domain Archaea: Halocins and sulfolobicins, pp 93-109, 2007. Rinehart KL, Shaw PD, Shield LS, Gloer JB, Harbour GC, Koker MES, et al. Marine natural- products as sources of anti-viral, anti-microbial, and anti-neoplastic agents. Pure Appl. Chem. 53: 795-817, 1981. Robinette D, Wada S, Arroll T, Levy MG, Miller WL, Noga EJ. Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins. Cell. Mol. Life Sci. 54: 467-475, 1998. Schillaci D, Arizza V, Parrinello N, Di Stefano V, Fanara S, Muccilli V, et al. Antimicrobial and antistaphylococcal biofilm activity from the sea urchin Paracentrotus lividus. J. Appl. Microbiol. 108: 17-24, 2009. Scott M G, Davidson DJ, Gold MR, Bowdish D, Hancock RE. The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses. J. Immunol. 169: 3883- 3891, 2002. Selsted ME, Ouellette AJ. Mammalian defensins in the antimicrobial immune response. Nat. Immunol. 6: 551-557, 2005. Service M, Wardlaw AC. Echinochrome A as a bactericidal substance in the coelomic fluid of 139 Echinus esculentus (L.). Comp. Biochem. Physiol. 79B: 161-165, 1984. Shimizu M, Kohno S, Kagawa H, Ichise N. Lytic activity and biochemical properties of lysozyme in the coelomic fluid of the sea urchin Strongylocentrotus intermedius. J. Invert. Pathol. 73: 214-222,1999. Shinnar AE, Butler KL, Park HJ. Cathelicidin family of antimicrobial peptides: proteolytic processing and protease resistance. Bioorg. Chem. 31: 425-436, 2003. Smith LC, Ghosh J, Buckley MK, Clow AL, Dheilly MN, Haug T, et al. Echinoderm Immunity. In: Soderhall K (ed), Invertebrate Immunology, Landes Bioscience, Inc. 2010 [in press]. Smith VJ, Fernandes JM, Kemp GD, Hauton C. Crustins: enigmatic WAP domain-containing antibacterial proteins from crustaceans. Dev. Comp. Immunol. 32: 758-772, 2008. Stabili L, Canicatti C. Antibacterial activity of the seminal plasma of Paracentrotus lividus. Can. J. Zool. 72: 1211-1216, 1994. Stabili L, Licciano M, Pagliara P. Evidence of antibacterial and lysozyme-like activity in different planktonic larval stages of Paracentrotus lividus. Mar. Biol. 119: 501-505, 1994. Stabili L, Pagliara P. Antibacterial protection in Marthasterias glacialis eggs - characterization of lysozyme-like activity. Comp. Biochem. Physiol. 109B: 709-713, 1994. Stabili L, Pagliara P. Effect of zinc on lysozyme-like activity of the seastar Marthasterias glacialis (Echinodermata, Asteroidea) mucus. J. Invert. Pathol. 100: 189-192, 2009. Stabili L, Pagliara P, Roch P, Antibacterial activity in the coelomocytes of the sea urchin Paracentrotus lividus. Comp. Biochem. Physiol. 113B: 639-644, 1996. Tang YQ, Yeaman MR, Selsted ME. Antimicrobial peptides from human platelets. Infect. Immun. 70: 6524-6533, 2002. Tasiemski A, Schikorski D, Le Marrec-Croq F, Pontoire-Van Camp C, Boidin-Wichlacz C, Sautiere PE. Hedistin: A novel antimicrobial peptide containing bromotryptophan constitutively expressed in the NK cells-like of the marine annelid, Nereis diversicolor. Dev. Comp. Immunol. 31: 749-762, 2007. Taylor SW, Craig AG, Fischer WH, Park M, Lehrer RI, Styelin D. An extensively modified antimicrobial peptide from ascidian hemocytes. J. Biol. Chem. 275: 38417-38426, 2000. Villasin J, Pomory CM. Antibacterial activity of extracts from the body wall of Parastichopus parvimensis (Echinodermata: Holothuroidea). Fish Shellfish Immunol. 10: 465-467, 2000. Wang G, Li X, Wang Z. APD2: the updated antimicrobial peptide database and its application in peptide design. Nucleic Acids Res. 37: D933-937, 2009. Wardlaw AC, Unkles SE. Bactericidal activity of celomic fluid from sea-urchin Echinus esculentus. J. Invert. Pathol. 32: 25-34, 1978. Yang L, Harroun TA, Weiss TM, Ding L, Huang HW. Barrel-stave model or toroidal model? A case study on melittin pores. Biophys. J. 81: 1475- 1485, 2001. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 415: 389-395, 2002. 140