antimicrobial peptides from marine invertebrates: challenges and perspectives in marine...

Biotechnology Advances 29 (2011) 519–530

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Research review paper

Antimicrobial peptides from marine invertebrates: Challenges and perspectives inmarine antimicrobial peptide discovery

Sigmund V. Sperstad, Tor Haug, Hans-Matti Blencke, Olaf B. Styrvold, Chun Li, Klara Stensvåg ⁎Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Tromsø, N-9037 Tromsø, Norway

⁎ Corresponding author. Tel.: +47 77 64 45 12; fax:E-mail address: [email protected] (K. Stensv

0734-9750/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.biotechadv.2011.05.021

a b s t r a c t
a r t i c l e i n f o
Available online 13 June 2011

Keywords:BiotechnologyBioprospectingSalt tolerantPurificationCrustaceaTunicataMollusca

The emergence of pathogenic bacteria resistance to conventional antibiotics calls for an increased focus on thepurification and characterization of antimicrobials with new mechanisms of actions. Antimicrobial peptidesare promising candidates, because their initial interaction with microbes is through binding to lipids. Theinterference with such a fundamental cell structure is assumed to hamper resistance development. In thepresent review we discuss antimicrobial peptides isolated from marine invertebrates, emphasizing theisolation and activity of these natural antibiotics. The marine environment is relatively poorly explored interms of potential pharmaceuticals, and it contains a tremendous species diversity which evolved in closeproximity to microorganisms. As invertebrates rely purely on innate immunity, including antimicrobialpeptides, to combat infectious agents, it is believed that immune effectors from these animals are efficient andrapid inhibitors of microbial growth.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5192. Diversity and general characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5203. Discovery of antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522

3.1. Considerations regarding sampling and sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5223.2. Extraction and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5223.3. Primary structure elucidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5243.4. Genetic and in silico approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524

4. Activity of AMPs from marine invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5254.1. Screening and antimicrobial potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5254.2. Antimicrobial activity in regimes specific to the marine environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526

4.2.1. The effect of the temperature on AMP activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5264.2.2. The effect of high hydrostatic pressure on AMP activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5274.2.3. AMP activity in high saline environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527

5. Miscellaneous peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5276. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528

1. Introduction

Antimicrobial peptides (AMPs) are a group of molecules exhibitingantimicrobial activity in vitro. In nature, they constitute an important

part of the innate immune system in animals, where they participatein the neutralization and elimination of intruding microorganisms(Zasloff, 2002). Since the purification of the first AMP, isolated fromHyalophora cecropia and named cecropin, (Hultmark et al., 1980;Steiner et al., 1981), more than 1.500 sequences which encode forAMPs or putative AMPs are today published in databases (http://aps.unmc.edu/AP/main.php). Due to their ubiquitous presence in nature,within both unicellular and multicellular organisms, they are

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520 S.V. Sperstad et al. / Biotechnology Advances 29 (2011) 519–530

considered crucial and effective immune effectors that probably haveevolved through positive selection (Fernandes et al., 2010; Tennessen,2005; Viljakainen and Pamilo, 2008). During the last decades, thesenatural antibiotics have attracted an increasing amount of attentiondue to their promising role as therapeutics or drug leads (Giuliani etal., 2007; Hadley and Hancock, 2010; Hughes and Fenical, 2010). Theemergence and increase of bacterial strains resistant to conventionalantibiotics have called for supplementary antimicrobials. This concernhas been addressed in several papers (Dantas et al., 2008; Fischbachand Walsh, 2009; Grundmann et al., 2006), and it is evident that thisissue has to be taken seriously to maintain an efficient responseagainst life-threatening microorganisms. From the pharmaceuticalpoint of view, the development of new antibiotics during the lastdecades has been focused on modifications of already existingpharmaceuticals (e.g., β-lactams, macrolides, quinolones) (Newmanand Cragg, 2007). This strategy may improve the efficiency andpotency, but it also exposes the bacteria to a limited arsenal ofantibacterial “strategies”. The development of antibiotic-resistanceamong pathogenic bacteria, as seen in methicillin-resistantStaphylococcus aureus and vancomycin-resistant Enterococcus, maybe counteracted with molecules exhibiting novel mechanism ofactions. In this regard, AMPs provide alternative ways of eliminatinginvading pathogens. The majority of AMPs share two features thatenable them to interact with microbes: i) they have a net positivecharge, which enables them to interact through electrostatic forceswith bacterial membranes which in turn are predominately anionic,and ii) they can form amphiphatic structures in hydrophobicenvironments and thus penetrate into the bacterial phospholipidbilayer (Brogden, 2005). These characteristics are assumed to bedirectly linked to their antibacterial potential. Their mechanism ofaction can grossly be divided into two main categories: i) peptidesthat bind to the bacterial membrane and create irreversible pores ordestabilize the membrane, causing an efflux of cytoplasma. Thismembrane-disruptive strategy kills bacteria within minutes. ii)Peptides that cross the bacterial membrane, and subsequently inhibitbacterial growth by binding intracellular components. This mem-brane-non-disruptive antibacterial action has been reported forseveral proline–arginine rich peptides, including pyrrhocoricin,bactenecin-7, apidaecin, and drosocin (Nicolas, 2009). This strategyis less prone to cause release of endotoxins, and it is considered moreselective towards microorganisms. Regardless of the mechanism ofaction, the first step is interaction with bacterial membranes ormembrane components. There is mounting evidence that bacteria areless likely to develop resistance against compounds that target suchfundamental structures (Hancock and Patrzykat, 2002) than conven-tional antibiotics which target specific motifs in molecules. Purifica-tion of novel AMPs, and in-depth characterizations of theirantimicrobial mechanism of action, may be a sound way to gainmore knowledge about alternative modes of antimicrobial action.

The marine environment contains tremendous species richness. Itis estimated to comprise approximately half of the total globalbiodiversity, while estimates of the marine macro fauna alone rangebetween 0.5 and 10×106 different species (Bouchet, 2006; de Vriesand Hall, 1994). The marine environment differs substantially fromterrestrial and sweet water habitats. This difference is reflected by thefact that insects as the most diverse group of terrestrial animals arevirtually absent from marine habitats, while potential niches seem tobe occupied by crustaceans (Ruxton and Humphries, 2008). Sinceboth groups rely on their innate immunity as the only means ofimmunological defense against microorganisms, habitat specificadaptations of AMPs are likely to exist. Interestingly, the expressionof AMPs in insects and crustaceans differ markedly, as insects activatethe production of AMPs after bacterial exposure (Lemaitre et al.,1997), while crustaceans seem to express their natural antibioticsconstitutively (Muñoz et al., 2002; Smith et al., 2008; Sperstad et al.,2010). Compared to the number of AMPs isolated from terrestrial

invertebrates, however, the number of registered AMPs from marineinvertebrates is low. Several arguments call for an increasedawareness of the potential found in the ocean. First of all, the marineenvironment is relatively unexplored. The ocean contains uniquehabitats which harbor animals that through evolution have developedmolecules for defense against pathogens and predators, and forparalyzing/killing of prey (Krug, 2006). Furthermore, marine animalsare in close proximity with microbes. The estimated density ofbacteria in sea water and sediment is in the order 105–107/ml and108–1010/g, respectively (Austin, 1988). This constant pressure frompotentially harmful microbes, combined with the evolutionarysuccess among marine invertebrates, suggests that the immuneeffectors found in these animals are highly effective in bacterialkilling/inhibition.

Additionally, one major difference between the marine environ-ment and terrestrial environments is the relative stability of thephysical factors especially in the deep pelagic waters. Life there isusually confronted with low temperature, elevated pressure, absolutedarkness and high salinity. In return it can depend on the relativestability of these environmental parameters (Lauro and Bartlett,2008). These factors drive the evolution of all marine organisms,including marine prokaryotic pathogens. As a result microorganismsfrom the deep sea differ in membrane and protein composition(Simonato et al., 2006). Enzymes of many marine organisms showspecific adaptations. Bacterial and archeal proteins adapted to lowtemperature (Feller and Gerday, 2003), high hydrostatic pressure(Simonato et al., 2006) and high salinity (Mevarech et al., 2000) havebeen characterized. However, the aspect of AMP co-evolution to theseselective pressures is poorly understood and has not beeninvestigated.

This review aims to discuss AMPs from marine invertebrates,mainly emphasizing on the challenges and perspectives of purifyingsuch peptides. In addition, different aspects concerning theirantimicrobial potential are discussed. For a more general descriptionof these molecules, recent review articles are available (Otero-González et al., 2010; Smith et al., 2010). Antimicrobial peptides arein this paper considered as peptides/proteins between 1 and 12 kDa,exhibiting a profound activity against microorganisms in vitro, andwhich are coded by single genes and ribosomally synthesized. Manylow molecular weight, extensively modified, and cyclic peptides havebeen characterized from sponges and tunicates. These peptides arepresumably produced non-ribosomally or by associated microorgan-isms and will therefore not be implemented in this review. Peptideswith antimicrobial activity, but obtained from larger proteins orclassified with other functions, are also omitted. However, thedistinction between AMPs and some other peptides showingantimicrobial activity is rather unclear. In the end we will brieflymention some of these peptides which truly are antimicrobial, but notare considered in this review.

2. Diversity and general characteristics

To date, around 40 different AMPs or AMP-families have beencharacterized from marine invertebrates (Table 1). Of these, only oneAMP belongs to an AMP family already characterized in terrestrialspecies, namely defensin (Charlet et al., 1996). The vast majority hasnovel primary structures and is either species-specific or confined tocertain taxa. Antimicrobial peptides have been characterized fromnumerous marine invertebrates from several taxa, including Cnidaria(Ovchinnikova et al., 2006), Annelida (Ovchinnikova et al., 2004),Chelicerata (Iwanaga, 2002), Tunicata (Lee et al., 1997b), Mollusca(Mitta et al., 2000), Crustacea (Smith et al., 2008), and Echinodermata(Li et al., 2008). The sequence diversity is considerable, and singlespecies may express several different types of peptides, in addition toisoforms. Considering the different types of habitat that these animalsare living in and the high density of bacteria in their environment

Table 1Overview of gene-encoded AMPs from marine invertebrates, including some key features.

Phyla AMPs/AMP-families Size Distribution # Cys Activity spectruma Origin Key reference

Arthropoda Crustins 56–201 aa Decapoda (order) 12 G+ (G−, F) Hemocytes Smith et al., 2008Penaeidins 47–67 aa Penaeidae (family) 6 G+, F, (G−) Hemocytes Tassanakajon et al., 2010Bactenecin-like 6.5 kDa Carcinus maenas N.D. G+, G−b Hemocytes Schnapp et al., 1996Homarin N.D. Homarus americanus N.D. G−b Hemocytes Battison et al., 2008Callinectin 32 aa C. sapidus 4 G−b Hemocytes Noga et al., 2011Ls-Stylicin 1 82 aa L. stylirostris 13 F (G−) Hemocytes Rolland et al., 2010Hyastatin 114 aa Hyas araneus 6 G+, G−, F Hemocytes Sperstad et al., 2009bArasin 1 37 aa H. araneus 4 G+, G−, F Hemocytes Stensvåg et al., 2008Tachyplesins 17 aa Limulidae (family) 4 G+, G−, F Hemocytes Nakamura et al., 1988Tachycitin 73 aa Tachypleus tridentatus 10 G+, G−, F Hemocytes Kawabata et al., 1996Tachystatins 41–44 aa T. tridentatus 6 G+, G−, F Hemocytes Osaki et al., 1999Big defensin 79 aa T. tridentatus 6 G+, G−, F Hemocytes Saito et al., 1995Polyphemusins 18 aa Limulus polyhemus 4 G+, G−, F Hemocytes Miyata et al., 1989Scygonadin 102 aa Scylla serrata 2 G+ (G−) Seminal plasma Huang et al., 2006SSAP 102 aa S. serrata 2 G+, G−b Hemocytes Yedery and Reddy, 2009Arasin-likeSp 41 aa Scylla paramamosain 4 G+, G−b Hemocytes Imjongjirak et al., 2011GRPSp 29 aa S. paramamosain 2 G+b Hemocytes Imjongjirak et al., 2011

Tunicata Clavanins 23 aa Styela clava 0 G+, G−, F Hemocytes Lee et al., 1997cClavaspirin 23 aa S. clava 0 G+, G−, F Pharyngeal Lee et al., 2001bStyelins 31–32 aa S. clava 0 G+, G−b Hemocytes/pharyngeal Lee et al., 1997bDicynthaurinc 30/30 aa Halocynthia aurantium 2 G+, G− Hemocytes Lee et al., 2001aHalocidinc 18/15 aa H. aurantium 2 G+, G−, Fd Hemocytes Jang et al., 2002Halocyntin 26 aa Halocynthia papillosa 0 G+, G−b Hemocytes Galinier et al., 2009Papillosin 34 aa H. papillosa 0 G+, G−b Hemocytes Galinier et al., 2009Ci-MAM-A N.D. Ciona intestinalis 0 G+, G−, F Hemocytes Fedders et al., 2008Ci-PAP-A N.D. C. intestinalis 0 G+, G−, F Hemocytes Fedders and Leippe, 2008

Mollusca Defensins 39–43 aa Pteriomorpha (subclass) 6–8 G+, G− Hemocytes/gills/mantle Charlet et al., 1996Big defensins 84–94 aa Bivalvia (class) 6 G+, G−, Fe Hemocytes Zhao et al., 2007Mytilins 32–34 aa Bivalvia (class) 8 G+, G−, F Hemocytes Mitta et al., 2000Myticins 40 aa Bivalvia (class) 8 G+ (G−, F) Hemocytes Mitta et al., 1999Mytimycin 54 aa Mytilus (genus) 12 F Hemocytes Charlet et al., 1996Dolabellanin-B2 33 aa Dolabella auricularia 4 G+, G−, F Body wall Iijima et al., 2003

Annelida Hedistin 22 aa Nereis diversicolor 0 G+, G−b Coelomocytes Tasiemski et al., 2007Arenicins 21 aa Arenicola marina 2 G+, G−, F Coelomocytes Ovchinnikova et al., 2004Perinerin 51 aa Perinereis aibuhitensis Grube 4 G+, G−, F Homogenate Pan et al., 2004

Echinodermata Strongylocins 48–52 aa Strongylocentrotus droebachiensis 6 G+, G−, F Coelomocytes Li et al., 2008Centrocinsc 30/12 aa S. droebachiensis G+, G−, F Coelomocytes Li et al., 2010b

Cnidaria Aurelin 40 aa Aurelia aurita 6 G+, G−b Ectoplasma Ovchinnikova et al., 2006

N.D.: not determined.a G+: gram positive bacteria, G−: gram negative bacteria, F: fungi. Brackets indicate microorganisms with considerably lower susceptibility towards the AMP.b The AMP has not been screened against all groups of microorganisms included in this table.c Dimeric AMPs.d A synthetic analoge of halocidin shows antifungal activity (Jang et al., 2006).e Not screened against fungi, but showed fungicidal activity against the expression host, Pichia pastoris (Zhao et al., 2007).

521S.V. Sperstad et al. / Biotechnology Advances 29 (2011) 519–530

(Austin, 1988), it makes sense that the peptides are diversified duringthe evolution. Also the immune effectors found in these animals arerelatively poorly explored, particularly animals with no directeconomical importance. When comparing AMPs from the differenttaxa, however, some characteristics can be ascertained. Except forAMPs from Tunicata, Cys-containing peptides seem to dominate inmarine invertebrates (Table 1). The peptide precursors from all phyla,except those from crustaceans, usually have an anionic propiece and/or a C-terminal extension which is cleaved off to release the maturepeptide. The function of the anionic propeptides is still unclear, buttheymay act by neutralizing the positive charge of themature AMP, orthey may display some immunological/physiological function whencleaved off. Post-translational modifications are common, usuallywith cleavage of an amidation signal and a subsequent amidation ofthe C-terminus, but also more complex and extensive modificationsare apparent among some AMPs (Lee et al., 1997b; Li et al., 2010b;Noga et al., 2011; Taylor et al., 2000). And lastly, with very fewexceptions, most marine invertebrates appear to express their AMPswithout experimental challenge, in sharp contrast to insects and forinstance the fresh water Metazoan Hydra (Bosch et al., 2009; Lemaitreand Hoffmann, 2007).

Many of the AMPs from marine invertebrates are produced asisoforms. Both penaeidins and crustins, the two major AMP familieswithin Crustacea, are expressed as isoforms in several species

(Bartlett et al., 2002; Cuthbertson et al., 2002; Sperstad et al.,2009a; Tassanakajon et al., 2010). Interestingly, penaeidins andcrustins have only been purified once and twice (Destoumieux etal., 1997; Relf et al., 1999; Sperstad et al., 2009a), respectively, and onthe basis of these amino acid sequences, numerous isoforms oftranscripts and putative peptides are now identified (Smith et al.,2008; Tassanakajon et al., 2010). Isoforms are also evident in thestyelin- and clavanin-families, AMPs from the tunicate Styela clava(Lehrer et al., 2003), in tachystatins from the horseshoe crabTachypleus tridentatus (Osaki et al., 1999), and in mytilins from themolluskMytilus galloprovincialis (Mitta et al., 2000), to mention a few.

In addition to isoforms, one single species may produce an arrayof different AMPs, similar to insects which induce the expression ofseveral different AMPs when challenged with bacteria (Lemaitreet al., 1997). The spider crab Hyas araneus, the horseshoe crab T.tridentatus, the tunicate S. clava, and the two mollusks Mytilusedulis and M. galloprovincialis, are all representatives of speciesexpressing several AMPs. The simultaneous presence of diverseAMPs, probably acting in synergy or complementary to each other,may provide the organisms with an extended defense against awider range of pathogenic microorganisms. Such interactions havebeen reported, at least in vitro, between different AMPs isolatedfrom the horseshoe crab (Iwanaga et al., 1998) and the oyster,Crassostrea gigas (Gueguen et al., 2009).


Considering the high species diversity in the ocean, relatively fewspecies have been investigated for the expression of AMPs. At thephylum level, AMPs have been isolated from only 6 invertebrate phyla(Tunicata included) of the N30 animal phyla containing marinespecies (Bouchet, 2006). Also within the different phyla, there is a lackof comparative data between taxa. For example, among marinecrustaceans all AMPs have been characterized from decapods (Rosaand Barracco, 2010). In Tunicata, a subphylum containing anestimated of 4900 marine species (Bouchet, 2006), AMPs have onlybeen detected in 5 species, all belonging to the class Ascidiacea. Asmost species investigated so far seem to contain one or more novelAMP structures, this may either be due to the lack of data from awidertaxonomic range, or that species have evolved unique moleculesadapted to their specific habitat. Mytilin and mollusk defensin, asexamples, were first discovered in themolluskM. edulis (Charlet et al.,1996), and are now characterized in several other mollusk species(Gestal et al., 2007; Mitta et al., 2000; Seo et al., 2005). A similarpicture is shown for crustin (Smith et al., 2008), penaeidin(Tassanakajon et al., 2010), and big defensin (Saito et al., 1995;Zhao et al., 2007), all of which expand over several taxa. In contrast,the tunicate genus Halocynthia may contain species-specific AMP.Antimicrobial peptides have been isolated from two species, H.papillosa and H. aurantium, and while H. papillosa contain two α-helical AMPs named halocyntin and papillosin (Galinier et al., 2009),H. aurantium express two dimeric AMPs, dicynthaurin and halocidin(Jang et al., 2002; Lee et al., 2001a). This indicates that two closelyrelated species can synthesize a different array of antimicrobialcompounds. However, as long as extensive sequence information stillis not available from these organisms, no conclusions can be madewhether these AMPs are species-specific or not. For instance, theantifungal peptidemytimycinwas isolated and partially characterizedfrom the mollusk Mytilus edulis in the mid 90's (Charlet et al., 1996),but was not detected in the closely related speciesM. galloprovincialis(Mitta et al., 2000). Recently, a normalized EST library from M.galloprovincialis was constructed, and mytimycin transcripts wereidentified and successfully amplified (Sonthi et al., in press). Thus, toelucidate the true reservoir of AMPs in any species, it is necessary toconduct comprehensive studies, both by biochemical and geneticmeans. Today, several different approaches are used to isolatebioactive peptides. Below we will discuss some aspects of thedifferent methods used to isolate AMPs from marine invertebrates,the challenges encountered in extraction and purification, and howgenetic tools may be used to discover AMPs not detected by bioassay-guided purification.

3. Discovery of antimicrobial peptides

3.1. Considerations regarding sampling and sample preparation

One major problem for discovery and analysis of naturalcompounds from the marine environment is supply of bioactivematerial (Cragg et al., 1997). The concentrations of active peptides inmarine invertebrates are often less than 10−6% of the wet weight, andtheir yield from traditional extraction procedures is even lower. As anexample, Pettit et al.(1987) managed to isolate only 28.7 mg of theanticancer peptide Dolastatin 10 from around 1000 kg of the sea hareDolabella auricularia. The procurement of enough material forstructure elucidation, wide-spectrum bioassays and clinical studiesis therefore an obstacle for comprehensive pharmacological evalua-tion of the active compounds. Furthermore, many of the marinepeptides have complex structures (cysteine-rich, post-ribosomallymodified), making chemical synthesis of these compounds difficultand expensive.

Normally, limited numbers of organisms can be collected forextraction and isolation of the active peptides. A restricted naturalabundance of the source organisms will not support isolation with

such low yield based on wild harvest (Pomponi, 1999). Most marineAMPs have therefore been isolated from common, abundant in-vertebrates or aquaculture species that would be easy to recollect ifmore material was needed (Table 1). However, multiple factors mayaffect the outcome of AMP discovery. Examples are: seasonal andgeographical variations (sampling site), different life stages (pelagic,benthic), age, sex, and physiological status (disease, breeding,moulting). In addition, taxonomic identification of many marineinvertebrates is challenging (Suzuki et al., 2005), and incorrecttaxonomic or incomplete assignments can lead to difficulties duringpeptide isolation from recollected samples. The anionic AMP,scygonadin, is only found in males of the crab Scylla serrata (Huanget al., 2006). In our laboratory, two novel families of AMPs wereisolated and characterized from the coelomocytes of 66 individuals ofthe green sea urchin, Strongylocentrotus droebachiensis (Li et al., 2008,2010b). Recollection and attempts to purify higher amounts of thepeptides from ca. 500 individuals (using identical purificationprocedures) was unsuccessful. None of the peptides were evendetected (unpublished results).

Due to difficulties in obtaining sufficient quantities of bioactivepeptides, which may occur in organisms as parts per million amongthousands of other substances, it may be wise to dissect the largestinvertebrates into different tissues/organs and fluids before extraction(Haug et al., 2002a, 2002b, 2004). If the peptide of interest isproduced/stored in a specific tissue or organ, it would be unwise toextract the whole organism and thereby add thousands of inactive,interfering compounds to the extract. Dividing the organisms intodifferent parts or selecting the appropriate tissue/sample could alsoprovide an indication of whether the animal produces the activepeptide itself, or if it is derived from the diet or from associatedparasites or microorganisms. For instance, cecropin P1 was in 1989isolated and characterized from the small intestine of a pig, and it wasassumed that the peptide was synthesized by bovine cells (Lee et al.,1989). Fourteen years later, the true producer of the peptide wasdiscovered, namely a parasitic nematode (Andersson et al., 2003). It isreasonable to believe that an antibacterial peptide detected in a bloodcell extract is due to factors of the animal's own immune system,whereas antibacterial compounds detected in tissues exposed to theoutside environment (e.g. skin, gills, etc.) or in gastrointestinal tissueshas a higher probability to be diet-derived or produced by symbiontsor parasites.

Certainly, if the organisms are too small to dissect into smaller parts,whole body extraction is a necessity. However, only two marine AMPs,dolabellanin-B2 from the sea hare D. auricularia (Iijima et al., 2003) andperinerin from the clamworm Perinereis aibuhitensis (Pan et al., 2004),have been discovered by this procedure. Most marine AMPs have in factbeen isolated from the blood compartment, either from the hemolymph/coelomic fluid (whole blood) or hemocytes/coelomocytes (blood cells).A few peptides have been discovered and isolated from other tissues.However, in marine invertebrates the tissues are surrounded byhemocytes/coelomocytes, and these cells infiltrate the organs. Therefore,AMPs isolated from other tissues than the blood cells, may very welloriginate from hemocytes/coelomocytes.

3.2. Extraction and purification

Purification of AMPs is usually a necessity for further character-ization. Biochemical structure elucidation requires pure, homogenouspeptides in adequate amounts. Based on the heterogeneous nature ofthe marine AMPs, a standardized purification protocol is notestablished and various methods have been used. The purificationstrategy will also be determined by the amount of material availableand the concentration of active peptide(s). Appropriate preparativesample handling plays a crucial role in the outcome of the isolationprocess, especially for minute samples. Care should be taken to avoiddegranulation of blood cells, which may cause the release of peptide-


degrading cytosolic proteases and/or coagulation factors. Numerouspre-analytical variables have a potential influence on the quantitativeand qualitative outcome, stability of the sample, and reproducibility ofthe procedure. Among these variables are the sample preservationand storage conditions (elapsed time and temperature beforeextraction/separation, freezing, and freeze drying), type of bloodcollection device and tube used (glass or plastic), separation of cellsfrom blood (centrifugation speed, duration, and temperature), use ofbuffer (type, pH, ionic strength, and temperature), and use and type ofanticoagulants and protease inhibitory cocktails. Note that someanticoagulants and protease inhibitors display antimicrobial activitythemselves (causing false positives) and may intervene duringpurification of natural peptides. Most purification schemes for marineAMPs are based on multistep strategies (Table 2) with methodologybased on the features of most AMPs; relatively small size, cationic

Table 2Overview of purification strategies used in the isolation of marine AMPs. The starting materipreparation methods, see references.

AMP type AMP source Purification strate

Mytilus defensins, mytilins,mytimycin

Mussel hemocytes 1) Extraction: ace2) SPE (C18)3) RP-HPLC4) SEC5) 2×RP-HPLC

Clavanins, styelins, halocidin,dicynthaurin, aurelin

Tunicates hemocytes orpharyngeal tissue, jellyfishmesoglea

1) Extraction: 5%2a) Ultrafiltration2b) SEC and/or2c) AU-PAGE3) RP-HPLC

Tachyplesins, tachystatins, tachycitin,polyphemusins, big defensin

Horseshoe crab hemocytes 1) Extraction: 302) SEC3a) IEC (cation) a3b) RP-HPLC or3c) SEC

Dolabellanin B2, arasin, hyastatin,crustins , centrocins, strongylocins

Sea hare body wall, spider crabhemocytes, sea urchincoelomocytes

1) Extraction: 602a) Ultrafiltration2b) SPE (C18)3) 1–2×RP-HPLC

Penaeidins, myticin, mytilins, Ap-S,hedistin, halocyntin, papillosin

Shrimp, mollusc, polychaete ortunicate hemocytes

1) Extraction: 5%2a) SPE (C18) an2b) SEC3) 1–4×RP-HPLC

Crustin (carcinin) Shore crab hemocytes 1) Extraction: 202) Dialysis3) IEC (cation)4) RP-HPLC5) SEC

Perinerin Clamworm whole body 1) Extraction: 1 M2) SPE (C18)3) Affinity chrom4) RP-HPLC

Callinectin Blue crab hemocytes 1) Extraction: 0.02) IEC (cation)3) RP-HPLC

SSAP Mud crab hemocytes 1) Extraction: 102) Dialysis3) IEC (cation)4) Ultrafiltration5) 2×RP-HPLC

Arenicins Lugworm coelomocytes 1) Extraction: 102) Ultrafiltration3) AU-PAGE4) RP-HPLC

Scygonadin Mud crab seminal plasma 1) Extraction: 152) IEC (cation)3) RP-HPLC

Bactenecin-like (6.5 kDa) Shore crab hemocytes 1) SPE (C18)2) SEC3) 2×RP-HPLC

Homarin Lobster hemocytes 1) Extraction: 102) 2×SPE (HLB)3) IEC (cation)4) SDS-PAGE/AU-

charge and amphipathic nature. In general, most purification schemescontain; i) an extraction/precipitation step combined with centrifu-gation and/or ultrafiltration, removing particulate matter and largerproteins; ii) a preparative purification step, using methods like sizeexclusion chromatography (SEC, also known as gel permeationchromatography or gel filtration), ion exchange chromatography(IEC) and solid phase extraction (SPE), removing inorganic (salts),anionic proteins, fatty material and other bioassay-interferingcompounds, and finally; iii) one or several analytical separationsteps using reversed-phase high performance liquid chromatography(RP-HPLC), separating the bioactive peptides from inactive ones basedon differences in hydrophobicity. The presence of bioactive peptidesin the fractions obtained is usually tested after each step in theseprotocols, a procedure which has led to the term “bioassay-guidedpurification”.

al prior purification is usually a cell/organ lysate of the sample. Regarding prior sample

gy References

tic acid Charlet et al., 1996

acetic acidand/or

Jang et al., 2002; Lee et al., 2001a; Lehrer et al.,2001; Ovchinnikova et al., 2006

% acetic acid or 20 mM HCl

nd/or

Kawabata et al., 1996; Miyata et al., 1989;Nakamura et al., 1988; Osaki et al., 1999; Saito etal., 1995

% acetonitrile+0.1% TFAand/or

Iijima et al., 2003; Li et al., 2008, 2010b; Sperstad etal., 2009a,b; Stensvåg et al., 2008

or 2 M acetic acidd/or

Arenas et al., 2009; Destoumieux et al., 1997;Galinier et al., 2009; Mitta et al., 1999, 2000;Tasiemski et al., 2007

% acetic acid Relf et al., 1999

HCl+5% formic acid+1% TFA

atography (heparin)

Pan et al., 2004

5 M Tricine–Tris buffer Khoo et al., 1999

% acetic acid Yedery and Reddy, 2009

% acetic acid Ovchinnikova et al., 2004

% acetic acid+0.1% TFA Huang et al., 2006

Schnapp et al., 1996

% acetic acid

PAGE

Battison et al., 2008


As an example of the first purification step, an efficient extractionmedium has been 60% (v/w) acetonitrile (ACN), containing 0.1%trifluoroacetic acid (TFA). This mixture has proved to be effective inextracting antibacterial compounds from diverse marine organisms(Haug et al., 2002a, 2002b, 2004; Tadesse et al., 2008). In addition, thisextraction medium has previously been used to isolate both peptides(Clark et al., 1994) and compounds of non-protein nature (Moore etal., 1993). In our laboratory, a modified procedure has beendeveloped. Incubation of the extracts at −20 °C for 1–2 h producestwo liquid phases, an ACN-rich phase and an aqueous phase. Thistemperature-dependent separation is caused by the high salt contentin the invertebrate tissues, which is immiscible with ACN (Gu andShih, 2004). Most marine invertebrates have salt concentrations intheir body fluids equal or close to that of the surrounding seawater(Schmidt-Nielsen, 1990). In many cases, the ACN-rich phase containlittle, but colorful material (pigments, lipids and other lipophiliccompounds) with low water solubility and high UV absorbance,characteristics that cause problems in many bioassays. The AMPs areusually detected in the aqueous phase whereas proteins larger than15 kDa are precipitated due to a combination of solvent, low pH andsalts.

The high salt content in aqueous extracts from marine organismsmay lead to false positives during bioactivity screening and difficultiesduring HPLC and mass spectrometry (MS) analysis. Solid phaseextraction, using cartridges with reversed-phase packing materials, isa widely used and effective desalting technique (see Table 2). MostAMPs contain 30–50%, with an average of 42.6% hydrophobic aminoacids, according to the APD2 database (http://aps.unmc.edu/AP/main.php) (Wang andWang, 2004;Wang et al., 2009). This characteristic isexploited during purification: on reversed-phase C18 SPE cartridges(and HPLC columns), AMPs usually elute with 20–50% acetonitrile.Stepwise elution from SPE with increasing concentration of acetoni-trile has therefore successfully been used in separating and concen-trating marine AMPs (Defer et al., 2009; Haug et al., 2002a).Ultrafiltration through membranes with low molecular weight(MW) cut-off usually gives imperfect separation of salts (Shimizu,1985).

The technical improvements within liquid chromatography (LC)and especially HPLC methodology during the last decades haveresulted in highly efficient purification procedures for naturalproducts, including peptides. The methods used for purifying marineAMPs are largely based on general methodology developed forpeptide purification, and detailed protocols have been published(Bulet, 2008; Conlon, 2007; Hetru and Bulet, 1997; Schröder, 2010;Selsted, 1997). Regarding the final purification steps, RP-HPLC usingselected elution conditions (soft slope solvent gradients, based on thehydrophobic nature of the peptides) is the method of choice in moststudies. The high resolving power of reversed-phase columnscontaining octadecyl (C18) or octyl (C8) packing materials haveproven superior in obtaining pure peptide fractions for furtherpeptide characterization.

3.3. Primary structure elucidation

Primary structure elucidation of isolated AMPs is generallyperformed using traditional techniques, including reduction andalkylation, amino acid analysis, proteolytic treatment followed byRP-HPLC purification of peptide fragments, Edman degradation, and/ormass spectrometry of the obtained fragments. Marine peptidesmaybe challenging for all these methods. Dimeric peptides, like thecentrocins isolated from the green sea urchin S. droebachiensis(Li et al., 2010b), and dicynthaurin and halocidin, isolated from thetunicate H. aurantium (Jang et al., 2002; Lee et al., 2001a), will displayduplicate signals of similar intensity during Edman degradation. Suchresults may be misjudged to be due to impure peptides, but can easilybe resolved by cleavage of the two peptide chains followed by

separate purification and sequencing. Peptides containing blocked N-termini or otherwise modified residues may block or interrupt thesequencing processes during Edman degradation, and may not bedetected at all during amino acid analysis, unless specified gradientsand unusual amino acid standards are used. Post-translationalmodifications are found in several AMPs isolated from marineinvertebrates, and labor-intensive and time-consuming MS-analysesare necessary to obtain complete structure elucidation. Styelin D, anAMP isolated from the hemocytes of the solitary ascidian, S. clavawasshown to have remarkably extensive post-translational modifications,containing unusual amino acids like dihydroxyarginine, dihydroxyly-sine, 6-bromotryptophan and 3,4-dihydroxyphenylalanine. In addi-tion, the peptide exhibited microheterogeneity because of differentialmono- and dihydroxylation of several lysine residues and a C-terminal amidation (Taylor et al., 2000). The elucidation of theprimary structure of callinectin, a 3.7 kDa AMP originally isolatedfrom the hemocytes of the blue crab, Callinectes sapidus, proved to bedifficult during sequencing (Khoo et al., 1999). It was later shown tocontain an oxidized tryptophan N-terminally, either hydroxy-N formylkynurenine, N-formylkynurenine, or hydroxyl-tryptophan(Noga et al., 2011). A number of marine peptides contain brominatedtryptophans. Examples are the above mentioned styelin D (Tayloret al., 2000), hedistin (Tasiemski et al., 2007), centrocins (Li et al.,2010b), and strongylocin 2 (Li et al., 2008). Although the biologicalrole of post-translational modifications in marine AMPs is generallyunknown, the processes would probably make the peptides intopoorer substrates for endogenous proteolytic enzymes, and therebyincrease their lifetime in vivo (Shinnar et al., 2003), a feature that is anadvantage for peptide drug candidates.

3.4. Genetic and in silico approach

Isolation of cDNA transcripts or EST-clones showing homology totranscripts of already characterized AMP sequences is an alternativestrategy for AMP discovery (Fig. 1). This is by far the most frequentlyused method today, and it has expanded several AMP-families basedon sequences from purified peptides (e.g., penaeidin, crustin,defensin, cathelicidin). The advantage of this strategy is the relativeease of performance, and the high rate of success for finding newAMPs (Patrzykat and Douglas, 2003). Using this strategy, the diversityof AMP sequences can be explored, and conserved motifs can berevealed. In addition, it has proven useful for studies of AMPs that actas immune effectors in economically important species, such asdecapods and molluscs (Li et al., 2009a; Rosa and Barracco, 2010).However, the isolated transcript will probably not code for any novelAMPmotif (see Fig. 1), and it canbe assumed that itwill exhibit a similarantimicrobial profile andmechanism of action as the originally purifiedpeptide. To be certain that the sequence codes for an AMP, the putativepeptide must be synthesized and tested for antimicrobial activity.

Another approach using sequence information was employed toidentify the novel AMP Ls-Stylicin 1 from the shrimp speciesLitopenaeus stylirostris (Rolland et al., 2010). Here, shrimps able tosurvive an infection with the pathogen Vibrio penaeicidae wereinvestigated in terms of their transcripts involved in immunity (deLorgeril et al., 2005). Of these, an abundantly expressed transcriptcoded for a putative peptide containing 13 cysteine residues and ashort region enriched in proline residues. When this peptide wasrecombinantly expressed in Escherichia coli cells and tested forbioactivities, it showed antifungal activity against Fusariumoxysporum and antibacterial activity against Vibrio sp. The exis-tence of 13 cysteine residues indicates that the peptide exists as adimer in vivo, and the recombinant expression produced both amonomeric and a dimeric form, both showing similar antimicrobialactivity (Rolland et al., 2010).

In silico discovery of AMPs has also been employed in the tunicateCiona intestinalis. Due to the difficulties of purifying and characterizing



Species 1

Species 2

Peptide extraction

Bioassay-guided purification

cDNA library

cDNAsequences

Bioinformatics

Genome

Gene sequences

In silicoapproach

Cloning

EST/cDNA sequencesGenetic approach

Partial peptide sequences

Bioinformatics

Bioassay-guided purification• Unknown peptides can be identified• Based on activity• High probability for novel AMP motifs• Fewer AMPs • Concentration dependent• Labour intensive • Time-consuming

Genetic approach• Low probability for identification of unknown AMPs

• Based on motif from known peptide• Many homologues• Easy and rapidly done• Probes and primers can be designed immediately

• Unknown activity, peptide must be recombinantly produced/ synthesized

Outcome

In silico approach• Unknown peptides can be identified• Based on known structural features of AMPs like charge and hydrophobicity

• High probability of novel AMP motifs• Unknown activity, peptide must be recombinantly produced/ synthesized

AMP

Synthesis/ Recombinantproduction


Biological testing

Isolation

Species 1

Species 2

Peptide extraction

Bioassay-guided purification

cDNA library

cDNAsequences

Bioinformatics

Genome

Gene sequences

In silicoapproach

Cloning

EST/cDNA sequencesGenetic approach

Partial peptide sequences

Bioinformatics

Bioassay-guided purification• Unknown peptides can be identified• Based on activity• High probability for novel AMP motifs• Fewer AMPs • Concentration dependent• Labour intensive • Time-consuming

Genetic approach• Low probability for identification

of unknown AMPs• Based on motif from known peptide• Many homologues• Easy and rapidly done• Probes and primers can be designed

immediately• Unknown activity, peptide must be recombinantly produced/ synthesized

In silico approach• Unknown peptides can be identified• Based on known

structural features of AMPs like charge and hydrophobicity

• High probability of novel AMP motifs• Unknown activity, peptide must be

recombinantly produced/ synthesized

AMP



Biological testing

Isolation

A

B

CA

B

C

Fig. 1. Overview of differences and challenges of isolating marine antimicrobial peptides with the traditional bioassay-guided purification approach (A), the genetic approach(B), and the in silico approach (C).The listed outcome is modified from Patrzykat and Douglas (2003).


AMPs from hemocyte extracts by bioassay-guided purification,Fedders and Leippe, (2008) searched the EST- and genome-databasesderived from C. intestinalis hemocytes for the presence of antimicro-bials. First of all, no sequences in the databases had similarities withknown AMPs from other tunicates, such as styelins or clavanins. Toidentify potential candidates, the following criteria were set for thesearch: i) the EST was abundant in the database, ii) the EST containeda short open reading frame, iii) the putative peptide possessed atentative signal sequence and a cationic region, and lastly iv) theputative peptide showed no similarity with already known peptides/proteins (Fedders and Leippe, 2008). This strategy for AMP discoveryrevealed about 20 peptide sequences which were defined aspotentially antimicrobial. Synthesis of the cationic, amphiphaticregion of two of these, Ci-PAP-A and Ci-MAM-A, showed bothfragments to exhibit broad spectrum antimicrobial activity (Feddersand Leippe, 2008; Fedders et al., 2008, 2010). This purely in silicoapproach shows how existing information about AMP characteristicscan be used to discover novel antimicrobials from sequence data. Byconsidering size, net charge, and the arrangement of the peptideprecursor, it is possible to discover potential AMPs from complexdatabases.

The development of third generation sequencing techniques willin the near future provide a tremendous amount of sequenceinformation. Emerging fields, such as system biology, transcriptomics,and peptidomics, will in addition yield comprehensive data from awider taxonomic range of animals. In silico isolation of AMPs andother antimicrobials will thus probably increase highly in the nextyears. It is important, however, to be aware of the challenges relatedto such an approach. First of all, it is not possible to predict if thetranslated peptide in vivo contains post-ribosomal modifications,

or in the case of cysteine residues, to assign the correct disulfideconnections. Candidates must be synthesized or expressed recombi-nantly, and subsequently screened against selected microorganisms,to truly assign them as antimicrobials. Furthermore, this strategyrelies on already known characteristics of AMPs (e.g. size, cationicity,amphipathicity), which methodically will exclude peptides that haveother features, such as a negative net charge (Harris et al., 2009).Therefore, bioassay-guided purification will continue to be ofimportance in the discovery of novel marine AMPs.

4. Activity of AMPs from marine invertebrates

4.1. Screening and antimicrobial potential

The antimicrobial potential of any compound is reflected by thetest strains selected for activity measurements. When choosing thestrategy of bioassay-guided purification, the amount of purifiedmaterial usually does not permit a broad primary screening, andthus the test strains have to be selected carefully. Of these, E. coli andPseudomonas aeruginosa are common representatives of Gramnegative strains, while Micrococcus luteus, Bacillus megaterium and S.aureus often are selected as Gram positive strains. Clearly, the selectedstrains for the primary screening determine the outcome. There is noguarantee that fractions not showing bacterial inhibition are devoid ofantimicrobial substances, as it all relies on the susceptibility of a veryfew, user-selected microorganisms. Ls-Stycilin 1, for example, wasscreened against numerous microorganisms, but showed a clearbiased preference for the fungi F. oxysporum (Rolland et al., 2010).Considering the lack of activity against strains usually used in primaryscreening, this peptide would not have been detected by a bioassay-


guided purification strategy. This example shows the importance ofselecting the appropriate microorganisms for screening. In terms ofspecies related to aquaculture, such as decapods, the main emphasizeis on microbial strains directly linked to diseases affecting the animalsin aquaculture (de Lorgeril et al., 2005; Hauton et al., 2006). Themotivation for searching for antimicrobial compounds in theseanimals is mainly immunological, and thus AMPs have been studiedextensively in their role as immune effectors (Bachère et al., 2004; Liet al., 2009a). Species belonging to Annelida or Tunicata, on the otherside, have AMPs that are mainly studied for their mechanism of actionand antimicrobial activity against human pathogens (Andrä et al.,2008; Fedders et al., 2010; Lee et al., 1997b; Park and Lee, 2009; vanKan et al., 2003).

In general, the antimicrobial potential of AMPs from marineinvertebrates does not differ from AMPs isolated from terrestrialsources. Examples show that some marine AMPs may be specific tocertain marine bacteria (Relf et al., 1999), or simply requiring highersalt concentration to exhibit activity, but in terms of potency,antimicrobial peptides from marine environments have the samepotential as terrestrial AMPs. The general features of an AMP, itscationicity and amphipathicity, suggest a broad-spectrum activityrange, where electrostatic forces ensure interaction between peptideand lipid, and the amphipathicity enables the peptide to penetrate thecytoplasmic membrane. However, several peptides show a clearpreference to certain types of microorganisms. Both crustins andpenaeidins act more efficiently against Gram positive bacteria(Destoumieux et al., 1999; Relf et al., 1999), and the mentioned Ls-Stylicin 1 is highly active against the shrimp pathogen F. oxysporum,has low activity against Vibrio sp., but shows no activity against otherfungal or bacteria species (Rolland et al., 2010). In the mollusk M.galloprovincialis, the different AMPs act complementarily, withmytimycin being a purely anti-fungal agent, defensins and myticinsare essentially active against Gram positive bacteria, and the mytilinsdisplay a broader activity spectrum depending on the isoform (Mittaet al., 2000). This differentiated antimicrobial spectrum may beexplained by differential lipid composition in the membrane of themicroorganisms, or an inability to penetrate the outer membrane ofcertain Gram negative microorganisms. Nevertheless, a narrowerantimicrobial spectrum of AMPs shows that cationicity or amphi-pathicity alone are not sufficient for microbial killing/inhibition. Thisis further supported by the relatively few, but still highly active,anionic AMPs which have been characterized in later years (Harriset al., 2009).

In contrast to AMPs from amphibians, which are in the size rangeof 1.3–5 kDa (Pukala et al., 2006), some AMPs from marineinvertebrates are relatively long and might be considered poly-peptides. By studying the effect of deletion analogs, it is apparent thatmany of these have their antimicrobial potential confined to smallerregions of the native peptide. Penaeidin consists of two distinctlydifferent domains, a Pro–Arg rich N-terminal region and a Cys-containing C-terminal region, and it has been shown that the Pro–Argrich region alone not is sufficient to maintain full activity(Cuthbertson et al., 2004). Hyastatin, which has sequence similaritieswith penaeidin with the addition of a Gly-rich N-terminal region,showed an equal dependence on the Cys-containing region to exhibitantimicrobial activity (Sperstad et al., 2009b). In contrast, arasin 1which also contains a Pro–Arg rich N-terminal region and a Cys-richC-terminal region, has the antimicrobial potential located in the N-terminal region (unpublished results). The antimicrobial activity ofcrustins is believed to be related to the whey-acidic-protein (WAP)domain, as this alone has shown to inhibit bacterial growth (Jia et al.,2008). A crustin-like AMP from Fenneropenaeus chinensis lacking theWAP domain had no effect on selected test bacteria, while the WAP-containing CruFc showed activity against Gram-positive bacteria(Zhang et al., 2007). The centrocins, purified from the sea urchinS. droebachiensis, are hetero-dimeric AMPs consisting of a heavy chain

and a light chain connected with one disulphide bridge (Li et al.,2010b). Here, the heavy chain alone exhibits the same antimicrobialactivity as the native molecule. Furthermore, big defensin isolatedfrom the horseshoe crab, consists of an N-terminal hydrophobicregion and a C-terminal region resembling defensin. The formerregion shows higher inhibitory activity against Gram positive bacteriathan Gram negative bacteria, and the latter shows higher activityagainst Gram negative bacteria than Gram positive bacteria (Saitoet al., 1995). These examples clearly show that although some marineinvertebrates express longer peptides, the pharmacophores arelocated in defined regions of the native molecule. While amphibianAMPs mostly are located in the skin and seem to act purely as naturalantibiotics, most AMPs discovered from marine invertebrates areproduced in circulating blood cells and may well have other roles inthe animal than as antimicrobials. These longer AMPs can thus containregions responsible for other activities (Destoumieux et al., 2000;Iwanaga, 2002; Sperstad et al., 2009b), giving the molecule amultifunctional character.

4.2. Antimicrobial activity in regimes specific to the marine environment

4.2.1. The effect of the temperature on AMP activityThere is very little known about the influence of temperature on

the expression and activity of antimicrobial peptides in general andeven less about peptides isolated frommarine organisms in particular.In some cases temperature dependent expression patterns of AMPhave been proposed. Shore crabs, which tend to be subjected toshifting temperatures more than organisms living in pelagic waters,seem to upregulate the expression of carcinin at both 5 and 20 °Cwhich represent their lower and upper temperature limit, respec-tively (Brockton and Smith, 2008). Expression of an AMP in thefreeze-tolerant frog Rana sylvatica, not a marine species, seems to beinduced only at high temperatures, though the expression in responseto temperatures below 4 °C has not been tested (Matutte et al., 2000).Although a seasonal variation of AMP expression in the mussel M.galloprovincialis was observed, no clear correlation to environmentalparameters including temperature could be assigned (Li et al., 2009b).These findings suggest that changes in temperature and otherphysical parameters in the growth environment might affectexpression of AMPs and be partly responsible for failed re-isolationof previously identified compounds as well as obscure the existence offurther AMPs in organisms already subjected to activity basedantimicrobial screening (Li et al., 2010a). Yet there is no example ofmarine peptides described in the literature where an adaption to lowtemperature environments is obvious. However, it seems that alsopeptides like lactoferricin, originating frommammals, are more activeat lower temperatures than at 37 °C (Vorland et al., 1999). In contrastis the peptide arenicin, originating from the lugworm Arenicolamarinaequally active at 4 °C and at 37 °C (Andrä et al., 2008).

Microorganisms, on the other hand, can sense and extensivelyadapt to low environmental temperatures. They can change mem-brane composition as well as protein expression in response totemperature shifts (Shivaji and Prakash, 2010) and use differentcompatible solutes to inhibit freezing of the cytoplasm. Metagenomicstudies confirmed that an altered use of amino acids in enzymes ofcold adapted microorganisms is a common feature (Casanueva et al.,2010). Since antimicrobial peptides always interact with membranesin order to either destroy membrane integrity or transfer to thecytoplasm (Brogden, 2005), changes in membrane compositionmightaffect AMP sensitivity of microorganisms. It is assumed that AMPshave not lost their activity throughout evolution, because bothbacteria and AMPs are co-evolving and thereby in a balance (Pescheland Sahl, 2006). This gives rise to the hypothesis that organisms livingat low temperatures adapted their peptide based defense tomatch themembrane composition and protein repertoire of the pathogens oftheir respective environment. However, in spite of the discovery of a


fair amount of AMPs from marine invertebrates, there is noexperimental proof for cold adapted AMPs. This does by no meansexclude their existence, but merely reflect that often screening isbased on mesophilic and psychrotolerant bacterial strains at optimalor nearly optimal growth temperature, and that activity is not testedat different temperatures or the prevalent temperature of the hostorganism (see Table 3). Though AMPs with low temperature optimamight not be in the focus of medical interest, there are reports thatshow that addition of the bacterial peptide bactenecin can improvekidney survival under cold storage e.g. during transportation inadvance of transplantation, probably by controlled permeabilizationof cell membranes which protects against ischemic injury (McAnultyet al., 2004). Peptides might also find application in food processingand storage at low temperatures as described for bacteriocins whichwere shown to decontaminate vegetables before storage to decreasethe number of potentially harmful bacilli and to inhibit regrowth ofbacilli during storage at low temperatures (CoboMolinos et al., 2008).AMPs evolved in a cold marine environment might show improvedcapabilities for these applications. However, in order to uncovercold adapted AMPs it is likely that screening conditions have to beadjusted.

4.2.2. The effect of high hydrostatic pressure on AMP activityWe found no mentioning of marine AMP activity affected by high

hydrostatic pressure. High hydrostatic pressure affects membranesand protein activity (Simonato et al., 2006) and thereby modifies thevery targets of antimicrobial peptide activity. Though pressure haslittle relevance for pharmaceuticals and medicine so far, it is used infood preservation and has became relevant in other biotechnologicalapproaches (Demazeau and Rivalain, 2010). Experiments conductedon the bacteriocin nisin and the AMP lactoferricin suggest that highpressure increases their antimicrobial activity and spectrum againstGram-negative bacteria at 155–600 MPa (Masschalck et al., 2001).Though no data has been published onmarine AMPs and their activityin response to pressure, peptides from organisms adapted to highpressure environments (b110 MPa) might be interesting for foodpreservation purposes in combination with high pressure treatment.

Table 3Overview of salt tolerant AMPs with respect to activity range and assay temperature.

AMP Range of activitya

(mM NaCl)Temperature(°C)

Reference

Arenicinb [0–500] 4, 37 Andrä et al., 2008Carcinin ]514] 20 (35c) Relf et al., 1999Cg-Defensin [85–1000] 30 Gueguen et al., 2006Ci-MAM-A24 [0–450] 28, 37 Fedders et al., 2008Ci-PAP-A22 [0–150] 37 Fedders and Leippe, 2008Clavanin [0–145[ 25 Løfgren et al., 2009Clavanin A [100–200[ 37 Lee et al., 1997aClavanin AK [100–300] 37 Lee et al., 1997aHalocidin [0–300] 37 Jang et al., 2003Homarin [514] n.m. Battison et al., 2008Mytilin [0–450] 25 Løfgren et al., 2009Mytilin A [86, 450] 37 Charlet et al., 1996Penaeidin [0–145[ 25 Løfgren et al., 2009Perinerin [86] 37 Pan et al., 2004Styelin A, B [0–400] n.m. Lee et al., 1997bStyelin D [100–200] n.m. Taylor et al., 2000Tachyplesin-1 [0–450] 25 Løfgren et al., 2009

n.m.: not mentioned.Brackets indicate the activity intervall. Closed brackets indicate activity at all testedconcentrations whereas an open bracket indicates inhibition below/above the indicatedconcentration.

a If differences between test strains, the salt concentration for the least affectedstrains are shown.

b Reduced activity in presence of salt but still comparably high overall activity.c No activity for non-marine bacteria which were only grown at 35 °C.

4.2.3. AMP activity in high saline environmentsMarine organisms seem to be the ideal candidates for the

discovery of AMPs active at elevated salt concentrations, becausemarine organisms evolved in an environment with an average salinityof 600 mM. However, studies have shown that marine organisms,much like terrestrial organisms, produce AMPs which are not equallyactive at elevated salt concentrations, e.g. dicynthaurin isolated fromthe tunicate H. aurantium hemocytes, is not active at 100 mM NaCland above (Lee et al., 2001a). However, this is not astonishing, sinceAMPs are most often isolated from hemocytes (Table 1) and might bepresent in phagolysosomes or vacuoles with very low NaCl content tokill phagocytosed pathogens. On the other hand, carcinin, an AMPisolated from the shore crab C. maenas, loses its activity at low saltconcentrations, while it is active at 500 mM NaCl (Relf et al., 1999).The salt-dependence of carcinin illustrates that conventional screen-ing methods generally based on low salt media like Muller Hintonbroth might inhibit the activity of peptides which are selectivelyactive at the high salt concentrations of the marine environment theyhave evolved in.

A number of AMPs isolated from different marine invertebrates isactive around physiological NaCl concentration and above. A list ofpeptides exhibiting antimicrobial activity in the presence of NaCl isshown in Table 3. Though NaCl obviously influences the activity ofsome AMPs, there are certainly other factors involved in AMP activitymodulation. It has been shown that e.g. the buffer compositionstrongly influences the effect of NaCl in in vitro assays and that acarbonate buffer system can neutralize salt inhibition (Dorschneret al., 2006). In contrast, did the use of peptonated sea water asgrowthmedium for V. anguillarum inhibit AMP activity at similar NaClconcentrations as in the assay system, where the peptides were active(Løfgren et al., 2008). It has been shown that salt tolerance alsodepends on the conformational stability of peptides. Linear de-rivatives of arenicin lost its pore forming activity in the presence ofsalt, indicating that the disulfide bridge facilitates salt tolerance(Andrä et al., 2009).

Generally one can expect a higher probability of isolating salttolerant or dependent compounds from marine invertebrates thanfrom terrestric species. Choosing salt water exposed tissue andadjustment of screening conditions to high salt media will mostprobably further increase the probability to isolate interestingcompounds.

Medical application of AMPs highly depends on their activity atphysiological salt concentration. Activity at even higher salt concen-trations might be required, e.g. for topical application where high saltconcentrations due to perspiration and evaporation are present on thesurface of human skin. Salt tolerance is not a general feature of AMPs.However, human dermcidins, which are constitutively secreted byhuman sweat glands, are salt tolerant and present a natural protectionagainst skin infections (Schittek et al., 2001). Another application forsalt tolerant AMPsmight be their use in marine aquaculture instead ofconventional antibiotics (Løfgren et al., 2009).

5. Miscellaneous peptides

Antimicrobial peptides are, as mentioned earlier, defined as suchdue to their ability to inhibit microbial growth in vitro. In order to limitthe extent of this review, several peptides which may be defined asAMPs have not been considered. These include plicatamide (Tincuet al., 2000) and the halocyamines (Azumi et al., 1990), having 8 and 4aa residues, respectively. These AMPs from tunicates show antimi-crobial activity, but it is still uncertain whether these very shortpeptides are encoded by a single gene, originated by proteolyticcleavage, produced non-ribosomally, or produced by associatedmicroorganisms. Additionally, anti-lipopolysaccharide factors (ALFs)that not always are considered as AMPs, are also exhibitingantimicrobial activity. When Tanaka et al.(1982) isolated a compound


with a molecular weight less than 10 kDa from the horseshoe crabsLimulus polyphemus and T. tridentatus, it was shown to bindlipopolysaccharide and thus inhibit the LPS-mediated activation ofthe coagulation system. Today, ALFs have been characterized fromseveral marine arthropods (Tassanakajon et al., 2010). Later in-vestigations have revealed that ALFs can inhibit the growth of Grampositive bacteria and fungi (Somboonwiwat et al., 2005), microor-ganisms that lack LPS. Thus, from being considered as a LPS-bindingmolecule, ALFs now show many of the features assigned toantimicrobial peptides, i.e. the appropriate size, the presence of ashort signal sequence, and a net positive charge (Tassanakajon et al.,2010).

These examples clearly show that a designation given to a certainmolecule may not reveal the whole functional spectrum of thatcompound. This is equally true for peptides that today are defined asantimicrobial peptides, in which penaeidins have been suggested toact as opsonins (Muñoz et al., 2002), crustins as protease inhibitors(Amparyup et al., 2008), and most AMPs exhibiting activity towardsGram negative bacteria probably have the ability to bind LPS. It is clearthat in the near future the classification of peptides interacting withbiological membranes should be comprehensively revised to givesound and clear classes for membrane-acting peptides.

6. Concluding remarks

Marine invertebrates, representing an enormous genetic andbiological diversity, have proven to be a rich source for discoveringpotent AMPs with novel and unique structural motifs. However, manymarine invertebrates generate small samples for peptide isolation andthe most potent/promising peptides are usually present in minuteconcentrations. These facts make them difficult to detect and identify.The development and use of sensitive and selective bioassays andtarget-based assays, and high-resolution purification/characterizationmethodology will enable the discovery of many more marine AMPs inthe future. Additionally, the development of third-generation se-quencing will in combination with bioinformatics allow extensive insilico mining of AMPs. This strategy will particularly be important formarine invertebrates which are not easily collected in sufficientamounts for extraction of compounds.

Screening procedures in the bioassay-guided purification ap-proaches today tend to be biased, favoring conditions with low saltcontent and inhibition of mesophilic microorganisms. Though theseconditions clearly have resulted in the discovery of numerousinteresting AMPs, the full potential of marine peptide diversity isnot exploited because of this biased focus of screening conditions.Therefore, future screening activities should also target antimicrobialpeptides which are specifically adapted to the marine environment.This change of focus may result in the purification of novel AMPs withproperties relevant to biotechnological applications.

Acknowledgements

This work was supported by grants from the University of Tromsøand grants from the Norwegian Research Council (no. 184688/S40).

References

Amparyup P, Donpudsa S, Tassanakajon A. Shrimp single WAP domain (SWD)-containing protein exhibits proteinase inhibitory and antimicrobial activities. DevComp Immunol 2008;32:1497–509.

AnderssonM, Boman A, Boman HG. Ascaris nematodes from pig and humanmake threeantibacterial peptides: isolation of cecropin P1 and two ASABF peptides. Cell MolLife Sci 2003;60:599–606.

Andrä J, Jakovkin I, Grötzinger J, Hecht O, Krasnosdembskaya AD, Goldmann T, et al.Structure and mode of action of the antimicrobial peptide arenicin. Biochem J2008;410:113–22.

Andrä J, Hammer MU, Grötzinger J, Jakovkin I, Lindner B, Vollmer E, et al. Significance ofthe cyclic structure and of arginine residues for the antibacterial activity of

arenicin-1 and its interaction with phospholipid and lipopolysaccharide modelmembranes. Biol Chem 2009;390:337–49.

Arenas G, Guzman F, Cardenas C, Mercado L, Marshall SH. A novel antifungal peptidedesigned from the primary structure of a natural antimicrobial peptide purifiedfrom Argopecten purpuratus hemocytes. Peptides 2009;30:1405–11.

Austin B. Marine microbiology. Cambridge: Cambridge University Press; 1988.Azumi K, Yokosawa H, Ishii S. Halocyamines — novel antimicrobial tetrapeptide-like

substances isolated from the hemocytes of the solitary ascidianHalocynthia-Roretzi.Biochemistry 1990;29:159–65.

Bachère E, Gueguen Y, Gonzalez M, de Lorgeril J, Garnier J, Romestand B. Insights intothe anti-microbial defense of marine invertebrates: the penaeid shrimps and theoyster Crassostrea gigas. Immunol Rev 2004;198:149–68.

Bartlett TC, Cuthbertson BJ, Shepard EF, Chapman RW, Gross PS, Warr GW. Crustins,homologues of an 11.5-kDa antibacterial peptide, from two species of penaeidshrimp, Litopenaeus vannamei and Litopenaeus setiferus. Mar Biotechnol 2002;4:278–93.

Battison AL, Summerfield R, Patrzykat A. Isolation and characterisation of twoantimicrobial peptides from haemocytes of the American lobster Homarusamericanus. Fish Shellfish Immunol 2008;25:181–7.

Bosch TCG, Augustin R, Anton-Erxleben F, Fraune S, Hemmrich G, Zill H, et al.Uncovering the evolutionary history of innate immunity: the simple metazoanHydra uses epithelial cells for host defence. Dev Comp Immunol 2009;33:559–69.

Bouchet P. Themagnitude of marine biodiversity. In: Duarte CM, editor. The explorationof marine biodiversity: scientific and technological challenges. Bilbao, Spain:Fundación BBVA; 2006. p. 31–62.

Brockton V, Smith VJ. Crustin expression following bacterial injection and temperaturechange in the shore crab, Carcinus maenas. Dev Comp Immunol 2008;32:1027–33.

Brogden KA. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?Nat Rev Microbiol 2005;3:238–50.

Bulet P. Strategies for the discovery, isolation, and characterization of natural bioactivepeptides from the immune system of invertebrates. Methods Mol Biol 2008;494:9–29.

Casanueva A, Tuffin M, Cary C, Cowan DA. Molecular adaptations to psychrophily: theimpact of ‘omic’ technologies. Trends Microbiol 2010;18:374–81.

Charlet M, Chernysh S, Philippe H, Hetru C, Hoffmann JA, Bulet P. Innate immunity.Isolation of several cystein-rich antimicrobial peptides from the blood of a mollusc,Mytilus edulis. J Biol Chem 1996;271:21808–13.

Clark DP, Durell S, Maloy WL, Zasloff M. Ranalexin. A novel antimicrobial peptide frombullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic,polymyxin. J Biol Chem 1994;269:10849–55.

Cobo Molinos A, Abriouel H, Lucas Lopez R, Ben Omar N, Valdivia E, Galvez A. Inhibitionof Bacillus cereus and Bacillus weihenstephanensis in raw vegetables by applicationof washing solutions containing enterocin AS-48 alone and in combination withother antimicrobials. Food Microbiol 2008;25:762–70.

Conlon JM. Purification of naturally occurring peptides by reversed-phase HPLC. NatProtoc 2007;2:191–7.

Cragg GM, Newman DJ, Snader KM. Natural products in drug discovery anddevelopment. J Nat Prod 1997;60:52–60.

Cuthbertson BJ, Shepard EF, Chapman RW, Gross PS. Diversity of the penaeidinantimicrobial peptides in two shrimp species. Immunogenetics 2002;54:442–5.

Cuthbertson BJ, Bullesbach EE, Fievet J, Bachère E, Gross PS. A new class (penaeidin class4) of antimicrobial peptides from the Atlantic white shrimp (Litopenaeus setiferus)exhibits target specificity and an independent proline-rich-domain function.Biochem J 2004;381:79–86.

Dantas G, Sommer MOA, Oluwasegun RD, Church GM. Bacteria subsisting onantibiotics. Science 2008;320:100–3.

de Lorgeril J, Saulnier D, Janech MG, Gueguen Y, Bachère E. Identification of genes thatare differentially expressed in hemocytes of the Pacific blue shrimp (Litopenaeusstylirostris) surviving an infection with Vibrio penaeicida. Physiol Genomics2005;21:174–83.

de Vries DJ, Hall MR. Marine biodiversity as a source of chemical diversity. Drug Dev Res1994;33:161–73.

Defer D, Bourgougnon N, Fleury Y. Detection and partial characterisation of anantimicrobial peptide (littorein) from the marine gastropod Littorina littorea. Int JAntimicrob Agents 2009;34:188–90.

Demazeau G, Rivalain N. High hydrostatic pressure and biology: a brief history. ApplMicrobiol Biotechnol 2010;89:1305–14.

Destoumieux D, Bulet P, Loew D, van Dorsselaer A, Rodriguez J, Bachère E. Penaeidins, anew family of antimicrobial peptides isolated from the shrimp Penaeus vannamei(Decapoda). J Biol Chem 1997;272:28398–406.

Destoumieux D, Bulet P, Strub JM, van Dorsselaer A, Bachère E. Recombinant expressionand range of activity of penaeidins, antimicrobial peptides from penaeid shrimp.Eur J Biochem 1999;266:335–46.

Destoumieux D, Muñoz M, Cosseau C, Rodriguez J, Bulet P, Comps M, et al. Penaeidins,antimicrobial peptides with chitin-binding activity, are produced and stored inshrimp granulocytes and released after microbial challenge. J Cell Sci 2000;113:461–9.

Dorschner RA, Lopez-Garcia B, Peschel A, Kraus D, Morikawa K, Nizet V, et al. Themammalian ionic environment dictates microbial susceptibility to antimicrobialdefense peptides. FASEB J 2006;20:35–42.

Fedders H, Leippe M. A reverse search for antimicrobial peptides in Ciona intestinalis:identification of a gene family expressed in hemocytes and evaluation of activity.Dev Comp Immunol 2008;32:286–98.

Fedders H, Michalek M, Grötzinger J, Leippe M. An exceptional salt-tolerantantimicrobial peptide derived from a novel gene family of haemocytes of themarine invertebrate Ciona intestinalis. Biochem J 2008;416:65–75.


Fedders H, Podschun R, Leippe M. The antimicrobial peptide Ci-MAM-A24 is highlyactive against multidrug-resistant and anaerobic bacteria pathogenic for humans.Int J Antimicrob Agents 2010;36:264–6.

Feller G, Gerday C. Psychrophilic enzymes: hot topics in cold adaptation. Nat RevMicrobiol 2003;1:200–8.

Fernandes JMO, Ruangsri J, Kiron V. Atlantic cod piscidin and its diversification throughpositive selection. PLoS One 2010;5:e9501.

Fischbach MA, Walsh CT. Antibiotics for emerging pathogens. Science 2009;325:1089–93.

Galinier R, Roger E, Sautiere P-E, Aumelas A, Banaigs B, Mitta G. Halocyntin andpapillosin, two new antimicrobial peptides isolated from hemocytes of the solitarytunicate, Halocynthia papillosa. J Pep Sci 2009;15:48–55.

Gestal C, Costa MM, Figueras A, Novoa B. Analysis of differentially expressed genesin response to bacterial stimulation in hemocytes of the carpet-shell clamRuditapes decussatus: identification of new antimicrobial peptides. Gene2007;406:134–43.

Giuliani A, Pirri G, Nicoletto S. Antimicrobial peptides: an overview of a promising classof therapeutics. Cent Eur J Biol 2007;2:1–33.

Grundmann H, Aires-De-Sousa M, Boyce J, Tiemersma E. Emergence and resurgence ofmeticillin-resistant Staphylococcus aureus as a public-health threat. Lancet2006;368:874–85.

Gu YS, Shih PH. Salt-induced phase separation can effectively remove the acetonitrilefrom the protein sample after the preparative RP-HPLC. Enzyme Microb Technol2004;35:592–7.

Gueguen Y, Herpin A, Aumelas A, Garnier J, Fievet J, Escoubas JM, et al. Characterizationof a defensin from the oyster Crassostrea gigas — recombinant production, folding,solution structure, antimicrobial activities, and gene expression. J Biol Chem2006;281:313–23.

Gueguen Y, Romestand B, Fievet J, Schmitt P, Destoumieux-Garzon D, Vandenbulcke F,et al. Oyster hemocytes express a proline-rich peptide displaying synergisticantimicrobial activity with a defensin. Mol Immunol 2009;46:516–22.

Hadley EB, Hancock RE. Strategies for the discovery and advancement of novel cationicantimicrobial peptides. Curr Top Med Chem 2010:1872–81.

Hancock RE, Patrzykat A. Clinical development of cationic antimicrobial peptides: fromnatural to novel antibiotics. Curr Drug Targets Infect Disord 2002;2:79–83.

Harris F, Dennison SR, Phoenix DA. Anionic antimicrobial peptides from eukaryoticorganisms. Curr Protein Pept Sci 2009;10:585–606.

Haug T, Kjuul AK, Stensvåg K, Sandsdalen E, Styrvold OB. Antibacterial activity in fourmarine crustacean decapods. Fish Shellfish Immunol 2002a;12:371–85.

Haug T, Kjuul AK, Styrvold OB, Sandsdalen E, Olsen OM, Stensvåg K. Antibacterialactivity in Strongylocentrotus droebachiensis (Echinoidea), Cucumaria frondosa(Holothuroidea), and Asterias rubens (Asteroidea). J Invertebr Pathol 2002b;81:94–102.

Haug T, Stensvåg K, Olsen OM, Sandsdalen E, Styrvold OB. Antibacterial activities invarious tissues of the horse mussel, Modiolus modiolus. J Invertebr Pathol 2004;85:112–9.

Hauton C, Brockton V, Smith VJ. Cloning of a crustin-like, single whey-acidic-domain,antibacterial peptide from the haemocytes of the European lobster, Homarusgammarus, and its response to infection with bacteria. Mol Immunol 2006;43:1490–6.

Hetru C, Bulet P. Strategies for the isolation and characterization of antimicrobialpeptides of invertebrates. Methods Mol Biol 1997;78:35–49.

Huang WS, Wang KJ, Yang M, Cai JJ, Li SJ, Wang GZ. Purification and partcharacterization of a novel antibacterial protein Scygonadin, isolated from theseminal plasma of mud crab, Scylla serrata (Forskål, 1775). J Exp Mar Biol Ecol2006;339:37–42.

Hughes CC, Fenical W. Antibacterials from the sea. Chem-Eur J 2010;16:12512–25.Hultmark D, Steiner H, Rasmuson T, Boman HG. Insect immunity. Purification and

properties of three inducible bactericidal proteins from hemolymph of immunizedpupae of Hyalophora cecropia. Eur J Biochem 1980;106:7–16.

Iijima R, Kisugi J, Yamazaki M. A novel antimicrobial peptide from the sea hare Dolabellaauricularia. Dev Comp Immunol 2003;27:305–11.

Imjongjirak C, Amparyup P, Tassanakajon A. Two novel antimicrobial peptides, arasin-likeSp and GRPSp, from the mud crab Scylla paramamosain, exhibit the activityagainst some crustacean pathogenic bacteria. Fish Shellfish Immunol 2011;30:706–12.

Iwanaga S. The molecular basis of innate immunity in the horseshoe crab. Curr OpinImmunol 2002;14:87–95.

Iwanaga S, Kawabata S, Muta T. New types of clotting factors and defense moleculesfound in horseshoe crab hemolymph: their structures and functions. J Biochem1998;123:1–15.

JangWS, Kim KN, Lee YS, NamMH, Lee IH. Halocidin: a new antimicrobial peptide fromhemocytes of the solitary tunicate, Halocynthia aurantium. FEBS Lett 2002;521:81–6.

JangWS, Kim CH, Kim KN, Park SY, Lee JH, Son SM, et al. Biological activities of syntheticanalogs of Halocidin, an antimicrobial peptide from the tunicate Halocynthiaaurantium. Antimicrob Agents Chemother 2003;47:2481–6.

Jang WS, Kim HK, Lee KY, Kim SA, Han YS, Lee IH. Antifungal activity of syntheticpeptide derived from halocidin, antimicrobial peptide from the tunicate,Halocynthia aurantium. FEBS Lett 2006;580:1490–6.

Jia Y-P, Sun Y-D, Wang Z-H, Wang Q, Wang X-W, Zhao X-F, et al. A single whey acidicprotein domain (SWD)-containing peptide from fleshy prawn with antimicrobialand proteinase inhibitory activities. Aquaculture 2008;284:246–59.

Kawabata S, Nagayama R, Hirata M, Shigenaga T, Agarwala KL, Saito T, et al. Tachycitin, asmall granular component in horseshoe crab hemocytes, is an antimicrobial proteinwith chitin-binding activity. J Biochem 1996;120:1253–60.

Khoo L, Robinette DW, Noga EJ. Callinectin, an antibacterial peptide from blue crab,Callinectes sapidus, hemocytes. Mar Biotechnol 1999;1:44–51.

Krug PJ. Defense of benthic invertebrates against surface colonization by larvae: achemical arms race. Prog Mol Subcell Biol 2006;42:1–53.

Lauro FM, Bartlett DH. Prokaryotic lifestyles in deep sea habitats. Extremophiles2008;12:15–25.

Lee J-Y, Boman A, Chuanxin S, Andersson M, Jörnvall H, Mutt V, et al. Antibacterialpeptides from pig intestines: isolation of a mammalian cecropin. Proc Natl Acad SciU S A 1989;86:9159–62.

Lee IH, Cho Y, Lehrer RI. Effects of pH and salinity on the antimicrobial properties ofclavanins. Infect Immun 1997a;65:2898–903.

Lee IH, Cho Y, Lehrer RI. Styelins, broad-spectrum antimicrobial peptides from solitarytunicate, Stylea clava. Comp Biochem Physiol 1997b;118B:515–21.

Lee IH, Zhao CQ, Cho Y, Harwig SSL, Cooper EL, Lehrer RI. Clavanins, α-helicalantimicrobial peptides from tunicate hemocytes. FEBS Lett 1997c;400:158–62.

Lee IH, Lee YS, Kim CH, Kim CR, Hong T, Menzel L, et al. Dicynthaurin: an antimicrobialpeptide from hemocytes of the solitary tunicate, Halocynthia aurantium. BiochemBiophys Acta 2001a;1527:141–8.

Lee IH, Zhao C, Nguyen T, Menzel L, Waring AJ, Sherman MA, et al. Clavaspirin, anantibacterial and haemolytic peptide from Styela clava. J Pept Res 2001b;58:445–56.

Lehrer RI, Lee IH, Menzel L, Waring A, Zhao CQ. Clavanins and styelins, alpha-helicalantimicrobial peptides from the hemocytes of Styela clava. Adv Exp Med Biol2001;484:71–6.

Lehrer RI, Tincu JA, Taylor SW, Menzel LP, Waring AJ. Natural peptide antibiotics fromtunicates: structures, functions and potential uses. Integr Comp Biol 2003;43:313–22.

Lemaitre B, Hoffmann J. The host defense of Drosophila melanogaster. Annu RevImmunol 2007;25:697–743.

Lemaitre B, Reichhart JM, Hoffmann JA. Drosophila host defense: differential inductionof antimicrobial peptide genes after infection by various classes of microorganisms.Proc Natl Acad Sci U S A 1997;94:14614–9.

Li C, Haug T, Styrvold OB, Jørgensen TO, Stensvåg K. Strongylocins, novel antimicrobialpeptides from the green sea urchin, Stronglyocentrotus droebachiensis. Dev CompImmunol 2008;32:1430–40.

Li CH, Zhao JM, Song LS. A review of advances in research on marine molluscanantimicrobial peptides and their potential application in aquaculture. MolluscanRes 2009a;29:17–26.

Li H, Toubiana M, Monfort P, Roch P. Influence of temperature, salinity and E. coli tissuecontent on immune gene expression in mussel: results from a 2005–2008 survey.Dev Comp Immunol 2009b;33:974–9.

Li C, Blencke H-M, Paulsen V, Haug T, Stensvåg K. Powerful workhorses forantimicrobial peptide expression and characterization. Landes Bioscience2010a;1:217–20.

Li C, Haug T, Moe MK, Styrvold OB, Stensvåg K. Centrocins: isolation andcharacterization of novel dimeric antimicrobial peptides from the green sea urchin,Strongylocentrotus droebachiensis. Dev Comp Immunol 2010b;34:959–68.

Løfgren SE, Miletti LC, Steindel M, Bachère E, Barracco MA. Trypanocidal andleishmanicidal activities of different antimicrobial peptides (AMPS) isolated fromaquatic animals. Exp Parasitol 2008;118:197–202.

Løfgren SE, Smania A, Smania EDA, Bachère E, Barracco MA. Comparative activity andstability under salinity conditions of different antimicrobial peptides isolated fromaquatic animals. Aquac Res 2009;40:1805–12.

Masschalck B, Van Houdt R, Michiels CW. High pressure increases bactericidal activityand spectrum of lactoferrin, lactoferricin and nisin. Int J Food Microbiol 2001;64:325–32.

Matutte B, Storey KB, Knoop FC, Conlon JM. Induction of synthesis of an antimicrobialpeptide in the skin of the freeze-tolerant frog, Rana sylvatica, in response toenvironmental stimuli. FEBS Lett 2000;483:135–8.

McAnulty JF, Foley JD, Reid TW, Heath TD, Waller KR, Murphy CJ. Suppression of coldischemic injury in stored kidneys by the antimicrobial peptide bactenecin.Cryobiology 2004;49:230–40.

Mevarech M, Frolow F, Gloss LM. Halophilic enzymes: proteins with a grain of salt.Biophys Chem 2000;86:155–64.

Mitta G, Hubert F, Noël T, Roch P. Myticin, a novel cysteine-rich antimicrobial peptideisolated from haemocytes and plasma of the mussel Mytilus galloprovincialis. Eur JBiochem 1999;265:71–8.

Mitta G, Hubert F, Dyrynda EA, Boudry P, Roch P. Mytilin B and MGD2, twoantimicrobial peptides of marine mussels: gene structure and expression analysis.Dev Comp Immunol 2000;24:381–93.

Miyata T, Tokunaga F, Yoneya T, Yoshikawa K, Iwanaga S, Niwa M, et al. Antimicrobialpeptides, isolated from horseshoe crab hemocytes, tachyplesin II, and polyphe-musins I and II: chemical structures and biological activities. J Biochem 1989;106:663–8.

Moore KS, Wehrli S, Roder H, Rogers M, Forrest Jr JN, McCrimmon D, et al. Squalamine:an aminosterol antibiotic from the shark. Proc Natl Acad Sci U S A 1993;90:1354–8.

Muñoz M, Vandenbulcke F, Saulnier D, Bachère E. Expression and distribution ofpenaeidin antimicrobial peptides are regulated by haemocyte reactions inmicrobial challenged shrimp. Eur J Biochem 2002;269:2678–89.

Nakamura T, Furunaka H, Miyata T, Tokunaga F, Muta T, Niwa M, et al. Tachyplesin, aclass of antimicrobial peptide from the hemocytes of the horseshoe crab(Tachypleus tridentatus). J Biol Chem 1988;263:16709–13.

Newman DJ, Cragg GM. Natural products as sources of new drugs over the last 25 years.J Nat Prod 2007;70:461–77.

Nicolas P. Multifunctional host defense peptides: intracellular-targeting antimicrobialpeptides. FEBS J 2009;276:6483–96.


Noga EJ, Stone KL, Wood A, Gordon WL, Robinette D. Primary structure and cellularlocalization of callinectin, an antimicrobial peptide from the blue crab. Dev CompImmunol 2011;35:409–15.

Osaki T, Omotezako M, Nagayama R, Hirata M, Iwanaga S, Kasahara J, et al. Horseshoecrab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequencesimilarity to spider neurotoxins. J Biol Chem 1999;274:26172–8.

Otero-González AJ, Magalhães BS, Garcia-Villarino M, López-Abarrategui C, Sousa DA,Dias SC, et al. Antimicrobial peptides from marine invertebrates as a new frontierfor microbial infection control. FASEB J 2010;24:1320–34.

Ovchinnikova TV, Aleshina GM, Balandin SV, Krasnosdembskaya AD, Markelov ML,Frolova EI, et al. Purification and primary structure of two isoforms of arenicin, anovel antimicrobial peptide from marine polychaeta Arenicola marina. FEBS Lett2004;577:209–14.

Ovchinnikova TV, Balandin SV, Aleshina GM, Tagaev AA, Leonova YF, KrasnodembskyED, et al. Aurelin, a novel antimicrobial peptide from jellyfish Aurelia aurita withstructural features of defensins and channel-blocking toxins. Biochem Biophys ResCommun 2006;348:514–23.

Pan W, Liu X, Ge F, Han J, Zheng T. Perinerin, a novel antimicrobial peptide purifiedfrom the clamworm Perinereis aibuhitensis grube and its partial characterization.J Biochem (Tokyo) 2004;135:297–304.

Park C, Lee DG. Fungicidal effect of antimicrobial peptide arenicin-1. Biochim BiophysActa-Biomembranes 2009;1788:1790–6.

Patrzykat A, Douglas SE. Gone gene fishing: how to catch novel marine antimicrobials.Trends Biotechnol 2003;21:362–9.

Peschel A, Sahl HG. The co-evolution of host cationic antimicrobial peptides andmicrobial resistance. Nat Rev Microbiol 2006;4:529–36.

Pettit GR, Kamano Y, Herald CL, Tuinman AA, Boettner FE, Kizu H, et al. The isolation andstructure of a remarkable marine animal antineoplastic constituent: Dolastatin 10.J Am Chem Soc 1987;109:6883–5.

Pomponi SA. The bioprocess-technological potential of the sea. J Biotechnol 1999;70:5–13.

Pukala TL, Bowie JH, Maselli VM, Musgrave IF, Tyler MJ. Host-defence peptides from theglandular secretions of amphibians: structure and activity. Nat Prod Rep 2006;23:368–93.

Relf JM, Chisholm JRS, Kemp GD, Smith VJ. Purification and characterization of acysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes of theshore crab, Carcinus maenas. Eur J Biochem 1999;264:350–7.

Rolland JL, AbdelouahabM, Dupont J, Lefevre F, Bachère E, Romestand B. Stylicins, a newfamily of antimicrobial peptides from the Pacific blue shrimp Litopenaeusstylirostris. Mol Immunol 2010;47:1269–77.

Rosa R, BarraccoM. Antimicrobial peptides in crustaceans. Invertebr Surv 2010;7:262–84.Ruxton GD, Humphries S. Can ecological and evolutionary arguments solve the riddle of

the missing marine insects? Mar Ecol 2008;29:72–5.Saito T, Kawabata S, Shigenaga T, Takayenoki Y, Cho J, Nakajima H, et al. A novel big

defensin identified in horseshoe crab hemocytes: isolation, amino acid sequence,and antimicrobial activity. J Biochem 1995;117:1131–7.

Schittek B, Hipfel R, Sauer B, Bauer J, Kalbacher H, Stevanovic S, et al. Dermcidin: a novelhuman antibiotic peptide secreted by sweat glands. Nat Immunol 2001;2:1133–7.

Schmidt-Nielsen K. Animal physiology: adaption and enviroment. Fourth edition. NewYork: Cambridge University Press; 1990.

Schnapp D, Kemp GD, Smith VJ. Purification and characterization of a proline-richantibacterial peptide, with sequence similarity to bactenecin-7, from thehaemocytes of the shore crab, Carcinus maenas. Eur J Biochem 1996;240:532–9.

Schröder J-M. Purification of antimicrobial peptides from human skin. Methods MolBiol 2010;618:15–30.

Selsted ME. HPLC methods for purification of antimicrobial peptides. Methods Mol Biol1997;78:17–33.

Seo JK, Crawford JM, Stone KL, Noga EJ. Purification of a novel arthropod defensin fromthe American oyster, Crassostrea virginica. Biochem Biophys Res Commun2005;338:1998–2004.

Shimizu Y. Bioactive marine natural products, with emphasis on handling of water-soluble compounds. J Nat Prod 1985;48:223–35.

Shinnar AE, Butler KL, Park HJ. Cathelicidin family of antimicrobial peptides: proteolyticprocessing and protease resistance. Bioorg Chem 2003;31:425–36.

Shivaji S, Prakash JSS. How do bacteria sense and respond to low temperature? ArchMicrobiol 2010;192:85–95.

Simonato F, Campanaro S, Lauro FM, Vezzi A, D'Angelo M, Vitulo N, et al. Piezophilicadaptation: a genomic point of view. J Biotechnol 2006;126:11–25.

Smith VJ, Fernandes JMO, Kemp GD, Hauton C. Crustins: enigmatic WAP domain-containing antibacterial proteins from crustaceans. Dev Comp Immunol 2008;32:758–72.

Smith VJ, Desbois AP, Dyrynda EA. Conventional and unconventional antimicrobialsfrom fish, marine invertebrates and micro-algae. Mar Drugs 2010;8:1213–62.

Somboonwiwat K, Marcos M, Tassanakajon A, Klinbunga S, Aumelas A, Romestand B,et al. Recombinant expression and anti-microbial activity of anti-lipopolysaccha-ride factor (ALF) from the black tiger shrimp Penaeus monodon. Dev Comp Immunol2005;29:841–51.

Sonthi M, Toubiana M, Pallavicini A, Venier P, Roch P. Diversity of coding sequences andgene structures of the antifungal peptide mytimycin (MytM) from theMediterranean mussel, Mytilus galloprovincialis. Mar Biotechnol. in press. doi:10.1007/s10126-010-9345-4.

Sperstad SV, Haug T, Paulsen V, Rode TM, Strandskog G, Solem ST, et al. Characterizationof crustins from the hemocytes of the spider crab, Hyas araneus, and the red kingcrab, Paralithodes camtschaticus. Dev Comp Immunol 2009a;33:583–91.

Sperstad SV, Haug T, Vasskog T, Stensvåg K. Hyastatin, a glycine-rich multi-domainantimicrobial peptide isolated from the spider crab (Hyas araneus) hemocytes. MolImmunol 2009b;46:2604–12.

Sperstad SV, Smith VJ, Stensvåg K. Expression of antimicrobial peptides from Hyasaraneus haemocytes following bacterial challenge in vitro. Dev Comp Immunol2010;34:618–24.

Steiner H, Hultmark D, Engström Å, Bennich H, Boman HG. Sequence and specificity oftwo antibacterial proteins involved in insect immunity. Nature 1981;292:246–8.

Stensvåg K, Haug T, Sperstad SV, Rekdal Ø, Indrevoll B, Styrvold OB. Arasin 1, a proline-arginine-rich antimicrobial peptide isolated from the spider crab,Hyas araneus. DevComp Immunol 2008;32:275–85.

Suzuki MM, Nishikawa T, Bird A. Genomic approaches reveal unexpected geneticdivergence within Ciona intestinalis. J Mol Evol 2005;61:627–35.

Tadesse M, Gulliksen B, Strom MB, Styrvold OB, Haug T. Screening for antibacterialand antifungal activities in marine benthic invertebrates from northern Norway.J Invertebr Pathol 2008;99:286–93.

Tanaka S, Nakamura T, Morita T, Iwanaga S. Limulus anti-LPS factor — an anticoagulantwhich inhibits the endotoxin-mediated activation of Limulus coagulation system.Biochem Biophys Res Commun 1982;105:717–23.

Tasiemski A, Schikorski D, Le Marrec-Croq F, Pontoire-Van Camp C, Boidin-Wichlacz C,Sautière P-E. Hedistin: a novel antimicrobial peptide containing bromotryptophanconstitutively expressed in the NK cells-like of the marine annelid, Nereisdiversicolor. Dev Comp Immunol 2007;31:749–62.

Tassanakajon A, Amparyup P, Somboonwiwat K, Supungul P. Cationic antimicrobialpeptides in penaeid shrimp. New York: Mar Biotechnol: Springer; 2010.p. 487–505.

Taylor SW, Craig AG, Fischer WH, Park M, Lehrer RI. Styelin D, an extensively modifiedantimicrobial peptide from ascidian hemocytes. J Biol Chem 2000;275:38417–26.

Tennessen JA. Molecular evolution of animal antimicrobial peptides: widespreadmoderate positive selection. J Evol Biol 2005;18:1387–94.

Tincu JA, Craig AG, Taylor SW. Plicatamide: a lead to the biosynthetic origins of thetunichromes? Biochem Biophys Res Commun 2000;270:421–4.

van Kan EJM, Ganchev DN, Snel MME, Chupin V, van der Bent A, de Kruijff B. The peptideantibiotic clavanin A interacts strongly and specifically with lipid bilayers.Biochemistry 2003;42:11366–72.

Viljakainen L, Pamilo P. Selection on an antimicrobial peptide defensin in ants. J MolEvol 2008;67:643–52.

Vorland LH, Ulvatne H, Andersen J, Haukland HH, Rekdal Ø, Svendsen JS, et al.Antibacterial effects of lactoferricin B. Scand J Infect Dis 1999;31:179–84.

Wang Z,Wang GS. APD: the Antimicrobial Peptide Database. Nucleic Acids Res 2004;32:D590–2.

Wang GS, Li X, Wang Z. APD2: the updated antimicrobial peptide database and itsapplication in peptide design. Nucleic Acids Res 2009;37:D933–7.

Yedery RD, Reddy KVR. Purification and characterization of antibacterial proteins fromgranular hemocytes of Indian mud crab, Scylla serrata. Acta Biochim Pol 2009;56:71–82.

Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389–95.Zhang J, Li F,Wang Z, Xiang J. Cloning and recombinant expression of a crustin-like gene

from Chinese shrimp, Fenneropenaeus chinensis. J Biotechnol 2007;127:605–14.Zhao JM, Song LS, Li CH, Ni DJ, Wu LT, Zhu L, et al. Molecular cloning, expression of a big

defensin gene from bay scallop Argopecten irradians and the antimicrobial activityof its recombinant protein. Mol Immunol 2007;44:360–8.

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