Ribosomally synthesized peptides with antimicrobial properties

Research review paperRibosomally synthesized peptides with antimicrobialproperties:biosynthesis,structure,function,and applicationsMaria Papagianni *Department of Hygiene and Technology of Food of Animal Origin,School of Veterinary Medicine,Aristotle University of Thessaloniki,Thessaloniki 54006,GreeceReceived 3June 2003;accepted 7June 2003AbstractRibosomally synthesized peptides with antimicrobial properties (antimicrobial peptides—AMPs)are produced by eukaryotes and prokaryotes and represent crucial components of their defense systems against microorganisms.Although they differ in structure,they are nearly all cationic and very often amphiphilic,which reflects the fact that many of them attack their target cells by permeabilizing the cell membrane.They can be roughly categorized into those that have a high content of a certain amino acid,most often proline,those that contain intramolecular disulfide bridges,and those with an amphiphilic region in their molecule if they assume an a -helical structure.Most of the known ribosomally synthesized peptides with antimicrobial functions have been identified and studied during the last 20years.As a result of these studies,new knowledge has been acquired into biology and biochemistry.It has become evident that these peptides may be developed into useful antimicrobial additives and drugs.The use of two-peptide antimicrobial peptides as replacement for clinical antibiotics is promising,though their applications in preservation of foods (safe and effective for use in meat,vegetables,and dairy products),in veterinary medicine,and in dentistry are more immediate.This review focuses on the current status of some of the main types of ribosomally synthesized AMPs produced by eucaryotes and procaryotes and discusses the novel antimicrobial functions,new developments,e.g.heterologous production of bacteriocins by lactic acid bacteria,or construction of multibacteriocinogenic strains,novel applications related to these peptides,and future research paradigms.D 2003Elsevier Inc.All rights reserved.Keywords:Antimicrobial peptides;Bacteriocins;Defensins;Peptide antibiotics0734-9750/03/$-see front matter D 2003Elsevier Inc.All rights reserved.doi:10.1016/S0734-9750(03)00077-6*Fax:+30-2310-999829.E-mail address:mp2000@vet.auth.gr (M.Papagianni)./locate/biotechadvBiotechnology Advances 21(2003)465–4991.IntroductionGene-encoded,ribosomally synthesized antimicrobial peptides (AMPs)are widely distributed in nature,being produced by mammals,birds,amphibians,insects,plants,and microorganisms (Cammue et al.,1994;Sahl,1994;Boman,1995).Although they form a diverse group of peptides as judged by their primary structures,they are often cationic and amphiphilic,and most of them kill bacteria by permeabilizing their cell membranes.Their positive charge presumably facilitates interactions with the negatively charged bacterial phospholipid-containing membranes and or acidic bacterial cell walls,whereas their amphiphilic character enables membrane permeabilization.They can be roughly categorized into those that have a high content of a certain amino acid,most often proline,those that contain intramolecular disulfide bridges,and those with an amphiphilic region in their molecule if they assume an a -helical structure (Table 1).In mammals,AMPs are expressed in phagocytes and mucosal epithelial cells (Lehler et al.,1993;Boman,1995;Martin et al.,1995)and represent crucial components of the innate immune system.Their broad-spectrum antibacterial activity,allied to their strategic location in phagocytes or epithelial surfaces,makes them central effector molecules of innate immunity (Zasloff,2002).In insects,bacterial infection induces the release of antibacterial peptides into the haemolymph (Boman,1995).Amphibian skin is an extraordinary rich source of AMPs.Amphibian AMPs display a remarkable range of microbicidal activity versus both Gram-positive and Gram-negative bacteria,yeast and fungal strains,protozoa and viruses (Zasloff,1987;Kreil,1994;Barra and Simmaco,1995;Rinaldi,2002).Thus,antimicrobial peptides in animals are thought to be key effector molecules in the innate immunity that are particularly important in early defense against invading microorganisms.As pointed out by Boman (1995,1996),antimicrobial peptides are an ideal first line of defense since peptides are made much more rapidly than proteins:antibacterial peptides may be made more than 100times faster than IgM,assuming a constant rate of peptide bond formation.Moreover,small peptides diffuse more rapidly than large protein molecules and immune cells.The production of ribosomally synthesized antimicrobial peptides by bacteria as a defense mechanism Table 1Examples of AMPs grouped according to structural characteristicsHigh content of a certain amino acidIntramolecular disulfide bridges Amphiphilic a -helical structure PeptideAmino acid Source Peptide Source Peptide Source Abaecin Pro Insects a -Defensins Humans,rabbits,miceMagainins Frogs Apidaecins Pro,Arg Insects h -defensins CattleDermaseptins Frogs Bac-5Pro,Arg Sheep,goat Insect defensins InsectsBombinin Frogs Bac-7Pro,Arg Cattle,sheep Plant defensins PlantsCecropin Insects Drosocin Pro Insects Protegrins PigsEsculentins-1Frogs Prophenin Pro,Phe Pigs Drosomycin Insects Esculentins-2FrogsM.Papagianni /Biotechnology Advances 21(2003)465–499466M.Papagianni/Biotechnology Advances21(2003)465–499467 against other organisms is well documented and represents an intensive area of research (Garneau et al.,2002;Riley and Wertz,2002).Most ribosomally synthesized AMPs presently known have been identified during the past20years,the insect cecropins and the defensins isolated in the early1980s being the first animal AMPs thouroughly characterized(Steiner et al.,1981;Selsted et al.,1983). Since then,there has been an increasing interest in ribosomally synthesized AMPs and several novel molecules,often with unprecedented structural features,have been discov-ered.A ribosomally synthesized AMP,the lantibiotic bacteriocin nisin produced by some strains of lactococci,is being presently widely used as a food preservative(Delves-Broughton et al.,1996;Riley and Wertz,2002;Ross Paul et al.,2002).Several novel peptides are identified each year from frog species that exhibit antiparasitic and anticancer properties(Rinaldi,2002).Peptide-based antibiotics are widely considered a potential answer to the growing problem of resistance to conventional antibiotics.The interest in AMPs is also due to their general biological and biochemical importance.By studying these peptides,insight has been gained into host defense systems,membrane-protein interactions,and protein modification and secretion.Some AMPs contain D-amino acids (Mignogna et al.,1993;Skaugen et al.,1994;Nissen-Meyer and Nes,1997),and they consequently represent an excellent model system for studying how D-amino acids may be formed in ribosomally synthesized polypeptides.This paper presents an overview of some of the main types of ribosomally synthesized AMPs produced by eucaryotes and procaryotes and discusses the novel antimicrobial functions,new developments,e.g.heterologous production of bacteriocins by lactic acid bacteria,construction of multibacteriocinogenic strains,and novel applications related to these peptides.2.AMPs in mammals2.1.DefensinsThe defensins belong to the largest group of AMPs,the cationic peptides,which are widely distributed in animals and plants.They are characterized by complex structural motifs and form a huge group of4-kDa open-ended cysteine-and arginine-rich,amphiphilic peptides.Defensins are arranged in families,based on their structural differences.Invertebrate(Bulet et al.,1999;Andreu and Rivas,1998;Dimarcq et al., 1998)and plant(Garcia-Olmedo et al.,1998)defensins are characterized by three and four disulfide bridges,respectively,and show a common structure comprising an a-helix linked to a h-sheet by two disulfide bridges(CS a h-motif).Their predominant h-sheet structure stabilized by the disulfide bonds is a unifying feature that distinguishes them from other antimicrobial peptides,many of which form amphiphilic a-helixes. NMR and crystallographic studies revealed that defensins may exist as noncovalently linked dimers shaped baskets that have a hydrophobic bottom and a polar top(White et al.,1995).Mammalian defensins are quite different from the arthropod and plant defensins in their sequence and structure and they represent themselves an important group of ribosomallysynthesized AMPs.They have been divided into a -defensins (or classical defensins)and the h -defensins (White et al.,1995),which are characterized by an antiparallel h -sheet structure,stabilized by three disulfide bridges (Zasloff,2002).They differ in number of residues,location and connectivities of their cysteine residues,as well as in their unique consensus sequences.The a -defensins are 29–35residues long,while the h -defensins 38–42residues long.Upon optimal alignment,the a -defensins and h -defensins have identical residues at eight positions,including the six cysteines.The defensins are synthesized as preproteins containing approximately 95residues including a typical 19-residue signal sequense at the N-terminus followed by a 40–45-residue anionic prosequense (Lehler et al.,1993;Ganz,1994;Martin et al.,1995;Selsted and Quellette,1995).The signal sequense functions to target the prepro-protein to the endoplasmic reticulum,whereas the biological role of the prosequense is not known.It may function to mask the membrane-permeabilizing activity during intracellular process-ing and transport,assist in proper folding,and/or help target the proprotein to the appropriate organelle (Nissen-Meyer and Nes,1997).The a -defensins have been found in azurophil granules of neutrophils and of some macrophages in humans,monkeys,and several rodent species and may account for more than 5%of the total cellular protein (Selsted and Quellette,1995).These defensins presumably kill phagocytized microorganisms when the defensin containing granules fuse with phagocytic vacuoles containing the ingested microorganisms.In humans and mice,a -defensins are also expressed and secreted into the intestine by Paneth cells,secretory epithelial cells of the small intestinal crypts with a presumed host defense function (Selsted and Quellette,1995;Quellette and Selsted,1996).Every mammalian species explored so far has h -defensins.They have been found in neutrophiles and the respiratory track and tongue of cattle (Bevins,1994;Schonwetter et al.,1995;Diamond et al.,1996),and in the leucocytes of chickens (Harwig et al.,1994).However,in other species,h -defensins are more often produced by epithelial cells lining various organs (e.g.the epidermis,bronchial tree and genitourinary tract)and they play an important role in host defense of the respiratory track.The genes encoding the neutrophil defensins from human,guinea pigs,and rabbits all span about 3kb and contain three exons.The last exon encodes the mature defensin peptide,and the second-to-last encodes the prepro-domain (Ganz,1994;Martin et al.,1995;Selsted and Quellette,1995).The human and mouse enteric defensin genes contain only two exons that correspond to the last two exons of the neutrophil defensin genes (Ganz,1994).Defensins are produced constitutively and/or in response to microbial products,e.g.bacterial lipolysaccharides (LPS)or proinflammatory cytokines (Ganz and Lehler,1998;Lehler and Ganz,1999;Schro ¨der,1999).The defensins in phagocytic cells and mucosal tissues are effector molecules in innate immunity.At micromolar concentrations,the defensins show antimicrobial activity against a wide range of Gram-positive and Gram-negative bacteria,fungi,and some enveloped viruses (Lehler et al.,1993;Martin et al.,1995;Selsted and Quellette,1995;Nissen-Meyer and Nes,1997).Defensins exhibit remarkable antiviral activity against recombinant adeno-associated virus and Herpes simplex virus.Direct binding of defensins appears to prevent envelope virus infectivity (Ganz,1999;Virella-Lowell et al.,2000).The initial step in the permeabilization of theM.Papagianni /Biotechnology Advances 21(2003)465–499468M.Papagianni/Biotechnology Advances21(2003)465–499469 target-cell membrane involves electrostatic interactions between the positively charged arginine residues and the negatively charged moieties on the target-cell surface.The hydrophobic part of the peptide is then probably pulled into the membrane by the trans-membrane potential,after which several dimers or monomers form a membrane-spanning pore estimated to have a diameter of25A˚(Selsted and Quellette,1995;White et al., 1995).Human natural killer(NK),B and g y T cells have recently been shown to produce neutrophil-derived a-defensins(HNP1-3,human neutrophil-derived a-defensins),partic-ularly when stimulated with cytokines(Agerberth et al.,2000),raising the possibility that defensins,in addition to functioning as effectors of innate immunity,might also be a component of the effector arsenal of cell-mediated adaptive immunity.The earliest clues that defensins might play a role in adaptive immunity were the observations that HNP1 and HNP2are chemotactic for human monocytes(Territo et al.,1989)and T cells(Chertov et al.,1996).Subsequent studies have revealed that human a-defensins are selectively chemotactic for resting CD4/CD45RA+and CD8+T cells(Yang et al.,2000),whereas hBD2is chemotactic for resting CD45R0+memory T cells(Yang et al.,1999).h-defensin gene expression in vertebrate epithelia,including that of humans,and their up-regulation in the presence of bacterial LPS and TNF-a(tumor necrosis factor)emphasize that these molecules might function to protect the host against microbial pathogenesis at the critical confrontation sites.These studies provide convincing evidence that defensins play a key role in directing and augmenting an adaptive immune response.The role of defensins in innate and adaptive immunity is discussed in detail in the recent reviews by Yang et al. (2002)and Raj and Dentino(2002).Additional important biological functions establish that defensins are multifunctional cell-effector molecules in addition to their role in integrating the innate and adaptive immune responses.Human a-defensins inhibit fibrinolysis by modulating tissue-type plasminogen activator and plasminogen binding to fibrin and endothelial cells,which could have significant implications in thrombotic pathology(Risso,2000).In vitro studies have shown that neutrophil defensins interact with ACTH receptors,inhibiting ACTH-induced steroidogenesis.They also induce histamine secretion from mast cells mediated by G-protein-dependent response,which is disctinctly different from antigen-IgE-mediated activation(Risso,2000).a-Defensins have been found to induce proteoglycan-dependent catabolism of low-density lipoprotein(LDL)by vascular cells, leading to a new class of inflammatory apolipoprotein,suggesting that they could possibly contribute to atherogenesis(Raj and Dentino,2002).They also play a role in the dynamic regulation of the antiprotease secretion in the lung at the site of inflammation by regulating the secretory leukocyte protease inhibitor and elafin release from bronchial epithelial cells(Risso,2000).Defensins have been found to be involved in wound healing by increasing proliferation of epithelial cells(Van Wetering et al., 1999).2.2.Cathelin-associated AMPsThe cathelin-associated peptides share a common N-terminal prepro-sequense (Zanetti et al.,1995),but otherwise constitute a very diverse group of AMPs differinggreatly in sequense,structure,and number of residues.The conserved proregion consists of about 100amino acid residues,and it is homologous to the cysteine protease inhibitor,cathelin.Proteins with this proregion and a highly variable C-terminal cationic antimicrobial domain are consequently termed cathelicidins (Zanetti et al.,1995).The C-terminal antimicrobial domain is cleaved off upon formation of the mature antimicrobial peptide,which is referred to as a cathelin-associated antimicrobial peptide.The cathelin-associated antimicrobial peptides that will be discussed below may be categorized into three groups:(1)those that have a high content of one or two amino acids,often proline;(2)those that contain intramolecular disulfide bonds;and (3)those with amphiphilic regions if they assume an a -helical structure.The group of cathelin-associated peptides that are rich in one or two amino acids includes Bac-5(bactenecin,MW 5kDa,42residues),Bac-7(bactenecin,MW 7kDa,59residues),PR-39(Pro-Arg-rich peptide,39residues),and prophenin (Pro-Phe-rich peptide,79residues),which all have a high content of proline (>45%)(Frank et al.,1990;Agerberth et al.,1991;Harwig et al.,1995;Nissen-Meyer and Nes,1997).Moreover,Bac-5,Bac-7,and PR-39are also rich in arginine,whereas prophenin is rich in phenylalanine.It is suggested that the amphiphilic structure of these peptides is responsible for their interaction with membranes,which is consistent with the observa-tion that the peptides permealize the Escherichia coli membrane (Nissen-Meyer and Nes,1997).A polyproline helical type-II structure that may interact with cell membranes has been suggested for PR-39(Cabiaux et al.,1994).Indolicidin,a cationic peptide with a high content of tryptophan,also forms a polyproline type-II helix that disrupts the cytoplasmic membrane by channel formation (Falla et al.,1996).PR-39and prophenin are found in the intestine and/or leucocytes of pigs,whereas Bac-5and Bac-7are found in the large cytoplasmic granules present in neutrophils of cattle,goat,and sheep,but not in neutrophils of most other animals (Agerberth et al.,1991;Harwig et al.,1995).In contrast to defensins,Bac-5and Bac-7are stored in the granules as inactive precusors with MWs of 20and 16kDa,respectively,and they become activated by proteases in the azurophil granules when these granules fuse with the probactenecin-containing granules.The group of cathelin-associated antimicrobial peptides that contain two or more cysteines joined by intramolecular disulfide bonds includes cyclic dodecapeptide (found in bovine neutrophil granules)and the protegrins (found in porcine leuco-cytes).Cyclic dodecapeptide contains only 12residues,four of which are arginines,linked with two cysteines by an intramolecular disulfide bond,while the remaining residues are the hydrophobic amino acids Val,Ile and Leu (Romeo et al.,1988).Its mode of action is not known.The protegrins contain 16–18amino acid residues,four of which are cysteines joined by two intramolecular disulfide bonds (Kokryakov et al.,1993).The protegrins have hydrophobic and hydrophilic clusters that are of importance in their interaction with target-cell membranes (Nissen-Meyer and Nes,1997).The third group of cathelin-associated AMPs mentioned above are those that will become amphiphilic if they assume an a -helical structure.This group of peptidesM.Papagianni /Biotechnology Advances 21(2003)465–499470M.Papagianni/Biotechnology Advances21(2003)465–499471 includes:C18,a21-residue sequense derived from the C-terminal region of CAP18,a cathelicidin expressed in rabbit neutrophils(Tossi et al.,1994);PMAP-36and PMAP-37 (porcine myeloid antibacterial peptide with36and37residues,respectively)expressed in pig myeloid cells(Mahoney et al.,1995);and LL-37(N-terminal residues are L-L, contains37residues),present in human granulocytes(Gudmundsson et al.,1996). Although these peptides differ in primary structures,they all have a high content of the basic amino acids arginine and lysine(25–35%)and apparently kill cells by permea-bilizing cell membranes.The genes encoding prophenin,PR-39,the protegrins,and LL-37are organized in a similar way and this may possibly be the case for all genes coding for cathelicidins (Nissen-Meyer and Nes,1997).They all contain four exons,the first three of which code for the conserved signal peptide and the cathelin proregion,whereas the fourth exon codes for processing sites and the variable C-terminal antimicrobial domain that is cleaved off upon formation of the mature antimicrobial peptide(Zhao et al.,1995a,b; Gudmudsson et al.,1995,1996).The promoter regions of these genes contain recognition sites that are also found in the promoter regions of genes that participate in immune responses,and this suggests that the expression of cathelicidin-encoding genes responds to cytokines,such as IL-6,that are produced early during infection(Zhao et al.,1995a,b;Gudmudsson et al.,1995,1996).Cathelin-associated AMPs are bactericidal at micromolar concentrations and are,with few exceptions,active against both Gram-positive and Gram-negative bacteria.3.AMPs in amphibiansAmphibian AMPs are synthesized in the skin of a single species as structurally related members of a family,usually named after the source frog and toad species.This probably provides the producing organism with maximum coverage against a wider range of invading microbes at a minimum metabolic cost.The AMPs from amphibian skin isolated so far(about500)share some main characteristics,as that of bearing a net cationic charge at physiological pH,due to the presence of Lys and/or Arg residues.On the other hand,there are large differences in features such as the size and sequence. Structurally,amphibian AMPs belong to two distinct classes:linear a-helical peptides without cysteines;and peptides with one disulfide bridge forming a C-terminal loop. In any case,peptides can adopt an amphiphilic a-helical structure in hydrophobic environments.The skin of Anurans(frogs and toads)amphibians is an extraordinarily rich source of AMPs,to the extent that a large part of all reported molecules has this origin. The first AMP was found in the skin of the European frog Bombina variegata some 30years ago.The subsequent discovery of the potent magainins(from the Hebrew ‘‘magain’’,shield)in skin secretions of the African clawd frog Xenopus laevis was a new,decisive spur to further research.Acting as wide-spectrum microbicides against a variety of bacteria,protozoa and fungi,amphibian peptides have stimulated increasing interest because of their unique characteristics and potential therapeutic usefulness.3.1.MagaininsThe discovery of magainins was triggered by the observation that frogs with wounds thrive in waters dense with bacteria (Zasloff,1987).Initially the magainins were isolated from the frog skin,but have since also been found in the stomach (Moore et al.,1991).They contain 21–27residues,none of which are cysteines,and are synthesized as polyprotein precursors consisting of several magainin segments that are cleaved off to yield the mature peptides (Zasloff,1987).Upon binding to membranes,the magainins form an amphiphilic a -helix of which one side going down the helix is hydrophobic and the other side is hydrophilic (Zasloff,1987).In this respect the magainins are similar to some of the cathelin-associated AMPs and to other AMPs found in the skin secretions of frogs,the cecropins produced by insects,and a number of bacterial AMPs.Although the detailed mechanism by which AMPs with a -helical structures permealize cell membranes is not completely elucidated,there is a general agreement that upon interacting with membranes,amphiphilic a -helical peptides may initially lie parallel to the plane of the membrane with the hydrophobic side of the helix facing towards and shallowly penetrating the membrane.Biophysical studies conducted by Matsuzaki et al.(1995)on the interaction of magainins with phosphlipid bilayers suggest that membrane pores may transiently form through a ‘‘barrel-stave’’mechanism in which several magainin peptides form a bundle of membrane-spanning helices.The a -helical peptides are oriented perpendicularly to the plane of the membrane,with the hydrophilic side of the helices facing into the transmembrane channel and the hydrophobic side facing the hydrophobic lipids surrounding the channel.Consistent with the view that membrane lipids are the main target is the observation that chemically synthesized all-D magainin 2has the same antimicrobial activity as natural all-L magainin 2(Wade et al.,1990).Thus,the magainins do not need to interact with chiral centers such as enzymes,classical receptors,or other membrane proteins in order to exert their antimicrobial activity.However,evidence exists that selected amphibian AMPs might work by hitting multiple targets in bacteria, e.g.cytoplasmic membrane and intracellular locations.Experimental evidence is indeed accumulating that peptides from various sources might actually act by binding to intracellular targets,such as the DNA and RNA,presumably interfering with their metabolic functions.As for amphibian AMPs,the intracellular localization of immu-nolabelled magainin 2in E.coli was recently demonstrated by transmission electron microscopy (Haukland et al.,2001).3.2.Other amphibian AMPsDermaceptins (28–34residues),a large family of AMPs expressed in the skin of Phillomedusa tree frogs (Mor and Nicolas,1994),are widely used in structural require-ments studies.Bombinins (25–27residues)from Bombina species (Gibson et al.,1991;Simmaco et al.,1991;Rinaldi,2002),form another group that have sequence similarities neither with dermaceptins nor with the magainins.However,like the magainins,they are linear cationic peptides that can assume an amphiphilic a -helical conformation uponM.Papagianni /Biotechnology Advances 21(2003)465–499472M.Papagianni/Biotechnology Advances21(2003)465–499473 interaction with cell membranes.A detailed investigation of the structure,membrane-binding properties and cytotoxic activity of seven analogues of the haemolytic and antimicrobial dermaseptin S4,revealed that active derivatives had a common amphiphilic structure with a clear separation of two lobes of positive and negative electrostatic potential surfaces(Kustanovich et al.,2002).Brevinins-2,esculentins-1,and esculentins-2(up to46residues),all isolated from Rana species,also have a sequence in their N-terminal region that can form an amphiphilic a-helix presumably involved in membrane permeabilization(Kreil,1994).Temporin-1Od and temporin-1Ja,isolated from Rana ornativentris and R.japonica,respectively,are structurally atypical in the sense that they lack basic residue in the sequence,and therefore bear only the positive charge associated with the a-amino group(Isaacson et al.,2002; Kim et al.,2001).Brevinins-1(24residues)and ranalexin(20residues),both isolated from Rana species(Clark et al.,1994;Barra and Simmaco,1995;Rinaldi,2002),seem to permealize cell membranes by a mechanism different from that used by the amphiphilic helical peptides.Although their C-terminal region contains two cysteine residues that are separated by five residues and joined by a disulfide bridge,forming a motif(the‘‘Rana box’’)common to many other AMPs isolated from Rana species,their N-terminal region is predominantly hydrophobic and cannot form an amphiphilic a-helix.This must also be the case for the predominantly hydrophobic bombinins H isolated from Bombina species (Nissen-Meyer and Nes,1997).As is the case for protegrins,tachyplesins,and indolicidin, the C-terminal residues of bombinins H,bombinins and dermaceptins are amidated(Kreil, 1994;Shalev et al.,2002),thereby increasing their possitive charge.In the work of Shalev et al.(2002),carboxyamidation of dermaseptin S4was found to have significant structural consequences on the solution NMR structure of the peptide,causing the amidated peptide to be more rigid and more extended than its non-amidated analogue.Because amidating the C-terminal carboxyl is one of the means of enchancing antimicrobial activity of a given peptide,and this is usually attributed to the increased overall positive charge,this study revealed that the effects of amidation may be more diversified and less predictable that previously thought.More recently described amphibian AMPs include japonicin-1and japonicin-2 isolated from R.japonica(Isaacson et al.,2002);nigrocin1and2,from R. nigromaculata(Park et al.,2001);brevinin-20a,from R.ornaventris(Kim et al., 2001);tigerinin1,from R.tigerina(Sai et al.,2001);pseudin-2,from Pseudis paradoxa (Olson et al.,2001);maximim1,from Bombina maxima(Lai et al.,2002);XT-1,from Xenopus tropicalis(Ali et al.,2002),and distinctin,isolated from Phylomedusa distincta (Batista et al.,2001).Regulation of the synthesis of AMPs in frog skin has not been studied extensively. However,recent research demonstrates the induction of the synthesis of AMPs in ranid frogs in responce to a range of stimuli.The level of an AMP in skin extracts of Rana sylvatica increases when the animal is in an environment that promotes the growth of microorganisms(Mattute et al.,2002).This work,the first in vivo demonstration of the induction of defence peptides in a vertebrate,also indicated that frogs regulate their peptide antibiotic synthesis by an NF-n B/I n B a-like mechanism.The regulation of the expression of a gene coding a bombinin-like peptide in Bombina orientalis was studied by assesing the functionality of its promoter in the tumourous blood cell line mbn-2from。