Eur J Biochem 270, 2068–2081 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03584.x Antimicrobial peptides from hylid and ranin frogs originated from a 150-million-year-old ancestral precursor with a conserved signal peptide but a hypermutable antimicrobial domain Damien Vanhoye, Francine Bruston, Pierre Nicolas and Mohamed Amiche Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, Paris, France The dermal glands of frogs produce antimicrobial peptides that protect the skin against noxious microorganisms and assist in wound repair The sequences of these peptides are very dissimilar, both within and between species, so that the 5000 living anuran frogs may produce  100 000 different antimicrobial peptides The antimicrobial peptides of South American hylid frogs are derived from precursors, the preprodermaseptins, whose signal peptides and intervening sequences are remarkably conserved, but their C-terminal domains are markedly diverse, resulting in mature peptides with different lengths, sequences and antimicrobial spectra We have used the extreme conservation in the preproregion of preprodermaseptin transcripts to identify new members of this family in Australian and South American hylids All these peptides are cationic, amphipathic and a-helical They killed a broad spectrum of microorganisms and acted in synergy 42 preprodermaseptin gene sequences from 10 species of hylid and ranin frogs were analyzed in the context of their phylogeny and biogeography and of geophysical models for the fragmentation of Gondwana to examine the strategy that these frogs have evolved to generate an enormous array of peptide antibiotics The hyperdivergence of modern antimicrobial peptides and the number of peptides per species result from repeated duplications of a  150million-year-old ancestral gene and accelerated mutations of the mature peptide domain, probably involving a mutagenic, error-prone, DNA polymerase similar to Escherichia coli Pol V The presence of antimicrobial peptides with such different structures and spectra of action represents the successful evolution of multidrug defense by providing frogs with maximum protection against infectious microbes and minimizing the chance of microorganisms developing resistance to individual peptides The hypermutation of the antimicrobial domain by a targeted mutagenic polymerase that can generate many sequence changes in a few steps may have a selective survival value when frogs colonizing a new ecological niche encounter different microbial predators Frogs and toads have developed a successful strategy for surviving microbe-laden hostile environments The skin secretions of these animals not only produce huge amounts of biologically active peptides that are very similar to mammalian neuropeptides and hormones [1–7], they also contain a rich arsenal of broad-spectrum, cytolytic antimicrobial peptides These defend the naked skin against noxious microorganisms and assist in wound repair [8–11] The peptides are small, 10–50 amino acid residues long, cationic, and act in a variety of ways, although disrupting and permeabilizing the target cell membrane is the most frequent [12–15] This prevents a target organism from developing resistance to the peptide Hence, these peptides have been recognized as potential therapeutic agents [16,17] The sequences of these antimicrobial peptides differ considerably from one amphibian to another [9,10] The skin of a frog may have 10–20 antimicrobial peptides of differing sizes, sequences, charges, hydrophobicity, tridimensional structures and spectrum of action, and this armament differs between frogs belonging to different families, genera, species or even subspecies, so that no two species have yet been found that have the same panoply of peptide antibiotics [18] This impressive divergence between and within species means that there may be as many as 100 000 different peptides produced by the dermatous glands of the  5000 anuran amphibians [19] Advanced frogs (suborder: Neobatrachia) are by far the most important source of antimicrobial peptides and tens of peptide antibiotics have been found in only a few different frog species [18] These include peptides from European, Asian and North American frogs of the genus Rana (family: Ranidae; subfamily: Raninae) that are 10–47 residues long and have a 6- to 9-membered disulfide-bridged, cyclic region at their C-terminal end (Table 1) A total of 13 distinct peptide families have been identified based on sequence similarities [20,21] These are the brevinin-1 and brevinin-2 families [22], the esculentins-1 and -2 [23,24], ranatuerins-1 and -2 [25], ranalexins [26,27], palustrins-1, -2 and -3 [2], tigerinins [28] and the japonicins-1 and -2 [21] The peptides Correspondence to M Amiche, Laboratoire de Bioactivation des Peptides, Institut Jacques Monod, Place Jussieu, 75251 Paris Cedex 05, France Fax: +33 44 27 59 94, Tel.: +33 44 27 69 52, E-mail: amiche@ijm.jussieu.fr Abbreviations: DRP, dermaseptin-related peptide; MIC, minimal inhibitory concentration; Ma, million years ago (Received 12 February 2003, revised 11 March 2003, accepted 19 March 2003) Keywords: antimicrobial peptides; frog skin; dermaseptin; hypermutation; gene family Ó FEBS 2003 Evolution of amphibian antimicrobial peptides (Eur J Biochem 270) 2069 Table Origins and amino acid sequence of antimicrobial peptides for all frog species in this study Cysteine residues in bold letters form a disulfide bridge; a, amide Peptide Amphibian Family Sub-family Genus Species Name Sequence Hylidae Phyllomedusinae Phyllomedusa bicolor Dermatoxin Phylloxin DRS B1 DRS B2 DRS B3 DRS B4 DRS B6 PBN2 PBN1 DRP-AC1 DRP-AC2 DRP-AC3 DRP-AA11 DRP-AA2-5 DRP-AA3-1 DRP-AA3-3 DRP-AA3-4 DRP-AA3-6 DRP-PD1-5 DRP-PD2-2 DRP-PD3-3 DRP-PD3-6 DRP-PD3-7 Caerin 1.1 Caerin 1.11 Caerin 1.12 Caerin 1.13 Caerin 1.14 Caerin 1.15 Ranalexin Brevinin-1 E Brevinin-2 Ef Esculentin 1B SLGSFLKGVGTTLASVGKVVSDQFGKLLQAGQa GWMSKIASGIGTFLSGMQQa Agalychnis callydryas Agalychnis annae Pachymedusa Pelodryadinae Ranidae Raninae Litoria Rana dacnicolor caerulea catesbeiana esculenta rugosa temporaria pipiens Gaegurin-4 Gaegurin-5 Temporin B Temporin H Temporin G Brevinins ) 2Ta Brevinins ) 2Tb Ranatuerin-2P Ranatuerin-2 Pa in another family, the temporins, are short linear sequences of 10–13 residues [29] The South American hylid frogs of the Phyllomedusinae subfamily (family: Hylidae) also produce a rich array of linear a-helical antimicrobial peptides that are 19–34 residues long (Table 1) They include the dermaseptins B and dermaseptins S [30–33], phylloxin [34] and dermatoxin [35] from frogs of the Phyllomedusa genus and 24–33 residues peptides called dermaseptin-related peptides DRP-AA and DRP-PD from Agalychnis annae and Pachymedusa dacnicolor, respectively [36] Analysis of cDNA clones of antimicrobial peptides from South American hylids [32,34–37] and Asian, European AMWKDVLKKIGTVALHAGKAALGAVADTISQa GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAVa ALWKNMLKGIGKLAGQAALGAVKTLVGAE ALWKDILKNVGKAAGKAVLNTVTDMVNQa ALWKDILKNAGKAALNEINQLVNQa GLVTSLIKGAGKLLGGLFGSVTGGQS FLSLIPHIVSGVAALAKHLG GLLSGILNTAGGLLGNLIGSLSNGES GLLSGILNSAGGLLGNLIGSLSNGES SVLSTITDMAKAAGRAALNAITGLVNQGEQ SLGSFMKGVGKGLATVGKIVADQFGKLLEAGQG GLVSGLLNTAGGLLGDLLGSLGSLSGGES SLWSKIKEMAATAGKAALNAVTGMVNQGEQ GMFTNMLKGIGKLAGQAALGAVKTLAGEQ GMWGSLLKGVATVVKHVLPHALSSQQS GMWSTIRNVGKSAAKAANLPAKAALGAISEAVGEQ SLGSFMKGVGKGLATVGKIVADQFGKLLEAGKG ALWKTLLKKVGKVAGKAVLNAVTNMANQNEQ GMWSKIKNAGKAAAKASKKAAGKAALGAVSEALGEQ GVVTDLLNTAGGLLGNLVGSLSGGER LLGDLLGKTSKLVNDLTDTVGSIV GLLSVLGSVAKHVLPHVVPVIAEHLa GLFSVLGSVAKHVVPRVVPVIAEHLa GLFGILGSVAKHVLPHVVPVIAEHSa GLLSVLGSLKLIVPHVVPLIAEHLa SVLGKSVAKHLPHVVPVIAEKTa GLFGLAKGSVAKPHVVPVISQLVa FLGGLIKIVPAMICAVTKKC FLPLLAGLAANFLPKIFCKTRKC GIMDTLKNLAKTAGKGALQSLVKMASCKLSGQC GIFSKLAGKKLKNLLISGLKNVGKEVGMDVVRTGIDI AGCKIKGEC GILDTLKQFAKGVGKDLVKGAAQGVLSTVSCKLAKTC DVEVEKRFLGALFKVASKVLPSVFCAITKKC LLPIVGNLLKSLLa LSPNLLKSLLa FFPVIGRILNGILa GILDTLKNLAKTAGKGILKSLVNTASCKLSGQC GILDTLKHLAKTAGKGALQSLLNHASCKLSGQC GLMDTVKNVAKNLAGHMLDKLKCKITGC GFLSTVVKLATNVAGTVIDTIKCKVTGGCRK and North American ranins [24,29,38,39] has indicated that they are all derived from a single family of precursor polypeptides with unique features [39] Precursors belonging to this family, designated the preprodermaseptin, have an N-terminal preprosequence of approximately 50 residues that is remarkably well conserved both within and between species, while the C-terminal sequence corresponding to antimicrobial peptides varies markedly The conserved preproregion comprises a 22 residue signal peptide and an acidic propiece that ends in a typical prohormone processing signal, Lys-Arg The pattern of conserved and variable regions in skin antimicrobial peptide precursors is therefore Ó FEBS 2003 2070 D Vanhoye et al (Eur J Biochem 270) the opposite of that of conventional secreted peptides, suggesting that the conserved preproregion is important for the biology of the expressing cell The unexpected similarity between the preproregions of precursors that result in structurally diverse end-products suggests that the corresponding genes all came from a common ancestor The genes encoding dermaseptins B from Phyllomedusa bicolor and gaegurin-4 from Rana rugosa have been cloned [40,41] They have a two exon coding structure, the first contains codons for the 22-residue signal peptide and the first three residues of the acidic propiece and the second exon encodes the remainder of the acidic propiece plus the processing signal Lys-Arg and the antimicrobial peptide progenitor sequence As the conserved preproregion is encoded by the same gene as the mature peptide, it cannot have been added by post-transcriptional events The vast number of different peptides encoded by this gene family reflects an unprecedented degree of gene diversification similar to that of the gene families that mediate interactions between organisms, such as immunoglobulins [42,43] or venom-derived toxins [44,45] The amphibian antimicrobial peptides are thus ideal for studying the evolution of a large variable gene family We have used the remarkable degree of conservation in the preproregion of the preprodermaseptin transcripts to identify novel members of this family in Australian hylids belonging to the genus, Litoria (subfamily: Pelodryadinae) and in South American hylid frogs (subfamily: Phyllomedusinae) We have determined the activity spectra of the predicted antimicrobial peptides A combination of phylogenetic reconstruction, analysis of mutation rates and geophysical models for the sequence of fragmentation of Gondwana suggests that the hypervariability of antimicrobial peptides and the number of peptides per species reflect the combination of speciation events, gene duplications, targeted hypermutation and subsequent actions of diversifying selection directed by the coevolution of the cell membrane of microbes and/or adaptation to the particular microbial biota that these frogs encounter Materials and methods Frog species Specimens of Agalychnis callydryas, Pachymedusa dacnicolor and Litoria caerulea were obtained from ÔLa Ferme TropicaleÕ (Paris, France) All procedures involving frogs adhered to ARVO resolution of the use of animals in research and the guidelines of INSERM ethical committee on animal research Specimens of Phyllomedusa bicolor were housed in large wooden cages (120 · 90 · 90 cm), covered on three sides by plastic mosquito net as described previously [3] Phyllodendron, Potos and Dracena were used as perches, and water bowls were provided for nocturnal baths The frogs were fed crickets Relative humidity was maintained at 65% by a constantly operating humidifier The temperature was maintained at 25 ± °C cDNA cloning procedure One specimen of A callydryas and L caerulea were anesthetized by immersion in ice-water and sacrificed by pithing The skin was removed on dry ice and a sample of  180 mg of tissue was homogenized Poly(A+) RNAs were purified over an affinity oligo(dT) spin cellulose column supplied by Invitrogen (Micro-FastTrack kit) The cDNA was synthetized by RT-PCR, with 3¢ RACE (Invitrogen) using a 5¢-primer (5¢-GGCTTCCCTGAA GAAATCTC-3¢) corresponding to the nucleotide sequence encoding the conserved N-terminus of the preproregion of dermaseptin precursors [37] and a primer specific to the 3¢adaptator under the following conditions: 35 cycles of 94 °C for 240 s, 56 °C for 45 s, 72 °C for 60 s and one cycle of 72 °C for 10 The PCR product was cloned in the pGEMt-easy vector system (Promega) using standard procedures [46] and used to transform competent JM 109 E coli After overnight incubation, the white positive colonies were screened both with T7 (5¢-ATTATGC TGAGTGATACCCGCT-3¢) and SP6 (5¢-ATTTAGGTG ACACTATAGAATAC-3¢) primers Amplification products of the expected sizes (400–500 base pairs) were sequenced by the dideoxy chain terminator method We determined the sequence for the 5¢-end of the preprocaerins with the cDNA as a template in RACE PCR with a sense primer specific of 5¢-adaptator and an antisense specific primer as follows: 5¢-GGATGCTCAACTCTT TATTGACC-3¢ for caerin 1.1, 5¢-ATGACTTTATCCT AAGGC-3¢ for caerin 1.11, 5¢-CTGAGTGAACAGCTA TAACTG-3¢ for caerin 1.12, 5¢-GACTTTATCCTAA GTGTTCAGC-3¢ for caerin 1.13, 5¢-TGTGGAAGGTG TTTACTAATGG-3¢ for caerin 1.14, and 5¢-GAAGT ACGTGCTTAGCAACGG-3¢ for caerin 1.15, and for nested PCR: 5¢-ATAACTGGAACAACGTGTGG-3¢ for caerin 1.1, 5¢-CTAAGTGCTCAGCAATGACG-3¢ for caerin 1.11, 5¢-AGCATAACTGGAACGTGGG-3¢ for caerin 1.12, 5¢-CAGCAATAAGTGGAACAACG-3¢ for caerin 1.13, 5¢-GTGTTTAGCAACGGATTTACC-3¢ for caerin 1.14 and 5¢-AGCAACGGATCCTAGGA CAC-3¢ for caerin 1.15 The temperature cycle used for the RACE PCRs was: 94 °C for 240 s, 35 cycles at 94 °C for 40 s, 56 °C for 45 s, 72 °C for 60 s, and a final extension step of 72 °C for 10 The PCR products were cloned and sequenced as above A similar approach was used to clone PBN1 and PBN2 cDNAs from P bicolor, and DRPAC1, and cDNAs from A callydryas Solid phase peptide synthesis Caerin 1.11, dermaseptin B2 and PBN2 were synthesized using solid-phase FastMoc chemistry procedures on an Applied Biosystems 433 A automated Peptide Synthesizer (Applera, France) Fmoc-protected amino acids and resins were from Senn Chemicals (Switzerland) and solvents from Sds (France) The carboxylic acid terminal peptides were prepared on a 4-benzyloxybenzyl alcohol resin (Wang PS resin) substituted at 1.18 mmolỈg)1 Carboxamidated peptides were prepared on a 4-methylbenzhydrylamin Polystyren resin (Rink Amide MBHA PS resin) substituted at 0.81 mmolỈg)1 Synthesis was carried out using a doublecoupling protocol: Fmoc amino acids (10 molar excess) were coupled for 30–60 with 2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate 1-hydroxybenzotriazol in a solution of N,N-dimethylformamide and diisopropylethylamine as activating agents with the Ó FEBS 2003 Evolution of amphibian antimicrobial peptides (Eur J Biochem 270) 2071 addition of N-methylpyrrolidone Capping with acetic anhydride was performed at the end of each cycle Temporary N-Fmoc protecting groups were removed by 20% piperidine in N-methyl-2-pyrrolidone Side chains were protected with tert-butyloxycarbonyl (tBoc) for lysine and tryptophane; O-tert-butyl ester (OtBu) for glutamic acid and aspartic acid; trityl (Trt) for histidine, threonine, glutamine and asparagine; O-tert-butyl ether (tBu) for serine and 2,2,4,6,7-pentamethyldihydrobenzofurane-5sulfonyl (Pbf) for arginine Cleavage of the peptidyl resin and side chain deprotection were carried out in a mixture composed of 95% trifluoroacetic acid, 2.5% triisopropylsilane and 2.5% water for h at room temperature The resulting mixture was filtered to remove the resin and the crude peptides were precipitated with ether at )20 °C They were recovered by centrifugation at 5000 g for 15 at °C, washed three times with cold ether, dried under a stream of nitrogen, dissolved in 10% acetic acid and lyophilized The lyophilized crude peptides were purified by reverse-phase HPLC on a Nucleosil C18 column (5 lm, 10 · 250 mm) eluted at mLỈmin)1 with a 0–60% linear gradient of acetonitrile in 0.07% trifluoroacetic acid/water over 30 The homogeneity of the synthetic peptides was assessed by MALDI-TOF mass spectrometry (Voyager DE RP, Perseptive Biosystems) and analytical HPLC as described previously [32] Antimicrobial Assays Gram-positive eubacteria (Aerococcus viridans, Bacillus megaterium, Staphylococcus aureus and Staphylococcus haemolyticus), Gram-negative eubacteria (E coli B, Salmonella typhimurium, Salmonella enteritidis, Enterobacter cloacae, Klebsiella pneumoniae) and Saccaromyces cerevisiae were cultured as described previously [34,35] The minimal inhibitory concentrations (MICs) of peptides were determined in 96-well microtitration plates by growing the bacteria in the presence of twofold serial dilutions of peptide Aliquots (10 lL) of each serial dilution were incubated with 100 lL of a suspension of a midlogarithmic phase culture of bacteria at a starting A630 value of 0.001 in Poor-Broth nutrient medium (1% bactotryptone, 0.5% NaCl, w/v) or yeast/peptone/glucose for S cerevisiae Inhibition of growth was assayed by measuring the A630 value after 16 h at 37 °C for bacteria growth and at 30 °C for yeast The minimal inhibitory concentration (MICs) was defined as the lowest concentration of peptide that inhibited the growth of 99% of the cells Bacteria were incubated for h with different concentrations of peptides and plated on solid culture medium containing 1% noble agar to distinguish between bacteriostatic and bactericidal effects The plates were subsequently incubated and examined daily for the formation of colonies All assays were performed in triplicate plus positive-controls without peptide and negative-controls with 0.7% formaldehyde Sequence analysis The nucleotide sequences of cDNAs encoding 18 known dermaseptin B, phylloxins, dermatoxins, and dermaseptinrelated peptides from the hylids A annae, P dacnicolor and P bicolor were obtained from GenBank, in addition to the cDNAs encoding PBN1, PBN2, DRP-AC 1, and and caerins identified in this study (Table 1) The nucleotide sequences of the cDNAs encoding 13 known brevinins, esculentins, gaegurins, ranalexins, temporins and ranatuerin from the ranins, Rana catesbeiana, R esculenta, R rugosa, R temporaria and R pipiens were also obtained from GenBank (Table 1) We aligned the nucleotide sequences of the antimicrobial peptide transcripts with CLUSTAL X [47] and by eye We first aligned the predicted amino acid sequences of the different domains of the peptides and nucleotide sequences of the 5¢- and 3¢-untranslated regions (UTR) separately with CLUSTAL X Then we added the nucleotide sequences of the different regions and finally adjusted the alignment manually Molecular phylograms from the alignments were determined with NeighborJoining from Kimura-two-parameters distances [48] using PAUP [49] Levels of support for branches were estimated with bootstrapping methods (1000 replicates) also with PAUP Sequence groups were denoted based on the existence of distinct clades and similarity of predicted amino acid sequences We interpreted the origins of the gene families from the topologies of the phylogram; we assume that the sequences represent distinct loci in the species sampled We estimated the proportion of synonymous substitutions per synonymous sites (Ds) from the beginning of the preproregion to the last codon before the stop codon with method I of Ina [50] in South American hylids, Australian hylids and ranins to determine if different gene regions are subject to different mutation rates For comparaison, Jukes–Cantor distances [51] were estimated for the 5¢- and 3¢-UTR using the same program The transversion/transition rate ratios (Tv/Ts) were estimated by pairwise comparison of the sequences from South American hylids, Australian hylids and ranins transcripts by counting with JADIS the proportion of sites with transitional and transversional differences between two sequences Tv and Ts were calculated independently within the signal, propiece and mature domains Results cDNA cloning of preprodermaseptins from Australian and South American hylids The dermal secretions of Australian tree frogs of the genus Litoria (family Hylidae; subfamily: Pelodryadinae) all contain broad-spectrum antimicrobial peptides, the caerins, whose structures are very different from those of South American hylid antimicrobial peptides [52–54] Four caerin subfamilies, caerins 1–4, have been identified to date, each comprising several distinct peptides The most widespread of these is caerin 1.1, which has the sequence, GLLSVLGSVAKHVLPHVVPVIAEHL-amide (Table 1) Nothing was known about the gene encoding these peptides 3¢-RACE analysis of skin mRNA from L caerulea using a primer based on the conserved coding region of preprodermaseptins revealed six different cDNAs (Fig 1) One of these cDNAs encoded caerin 1.1, while the remaining five cDNAs coded for new members of the caerin family The five predicted peptides, tentatively designated caerins 1.11–1.15, were 22–25 residues long and their amino acid sequence was 58–88% identical to caerin 1.1 They are more distantly related to the members of the other caerin 2072 D Vanhoye et al (Eur J Biochem 270) Ó FEBS 2003 Fig Nucleic acid and deduced amino acid sequences of cDNAs encoding caerins from the skin of Litoria caerulea (A) Nucleic acid and predicted amino acid sequence of the cDNA encoding caerin 1.1 The predicted amino acid sequence of preprocaerin 1.1 is given in capital letters under the nucleotide sequence The amino acid sequence of mature caerin 1.1 is given in bold letters A solid line is drawn under the amino acid sequence of the signal peptide Nucleotides are numbered positively from the 5¢- to 3¢-ends of the cDNA Amino acids are numbered starting with position in the open reading frame *Stop codon The glycine residue at the end of the peptide progenitor sequence is involved in the formation of the C-terminal amide of caerin 1.1 (52, 53) (B) The deduced amino acid sequences of cDNAs encoding caerins 1.1., 1.11, 1.12, 1.13, 1.14 and 1.15 A solid line is drawn under the amino acid sequences of the signal peptides Predicted amino acid sequences of mature caerins are given in bold letters families Each precursor polypeptide had an extra-glycine residue at the carboxyl terminus of its progenitor sequence, indicating that C-terminal amidation is involved in production of the final peptide The N-terminal regions of the caerin precursors encompassing the signal peptides all contained 22 residues and were superimposable with only four exceptions (82% identical) The acidic propieces contained 27 residues and the amino acid sequences were 92.5% identical Lastly, the 5¢- and 3¢-UTR of the corresponding cDNAs were 84% and 77% identical, respectively A comparison of the amino acid sequences of the preproregions of the six caerin precursors with those of preprodermaseptins from South American hylid frogs revealed that the signal peptides (95% identitical) and the acidic propieces (96% identical) were highly conserved (Fig 2) This similarity also extended to the 5¢- and 3¢untranslated regions of the respective mRNAs (not shown) Caerin 1.1 and related peptides from Australian hylids are thus an unexpected addition to the structurally diverse peptides encoded by genes belonging to the preprodermaseptin family A similar approach was used to identify new preprodermaseptin-related cDNA sequences in the skins of South American hylid frogs (Fig 2) Two of these sequences from P bicolor encoded novel putative peptides we have called PBN1 and PBN2 PBN1 (FLSLIPHIVSGVAALAKHL) and PBN2 (GLVTSLIKGAGKLLGGLFGSVTGGQS) not resemble any antimicrobial peptides identified to date in the skin secretions of P bicolor (Table 1) The other three sequences from A callidryas encoded peptides, called DRP-AC 1, and 3, that are structurally related, but not identical to DRP-AAs from A annae Secondary structure and antimicrobial activities of predicted peptides We selected caerin 1.11 and PBN2 to evaluate whether within-species differences between antimicrobial peptides reflect functional differentiation Caerin 1.11 and caerin 1.1 differ only by three amino acid substitutions In contrast, the sequence of PBN2 is very different from that of other P bicolor antimicrobial peptides As shown previously, the common structural feature of linear cationic antimicrobial peptides such as caerin 1.1 and dermaseptins S and B is the adoption of a stable amphipathic a-helix upon binding to the membrane surface [55,56] The predicted secondary structures of caerin 1.11 and PBN2 suggest that they can assume an amphipathic a-helical structure, and therefore be antimicrobial peptides (Fig 3) According to Segrest et al [57], both peptides belong to the class L group of helices that are highly positively charged with a narrow polar face and a highly hydrophobic apolar face The average charged polar face subtended a mean radial angle of  200° for both caerin 1.11 and PBN2 The circular dichroism spectra of synthetic PBN2 and caerin 1.11 (not shown) had strong minima at 200 nm in aqueous solution, reflecting the great degree of unordered structure Adding micellar concentrations of SDS to the aqueous solution greatly altered the dichroic spectra of both peptides Ellipticity decreased at 208 and 222 nm and increased at 192 nm, indicating the stabilization of the a-helical structure (30% helix content for caerin 1.11 and 42% for PBN2) in the membrane-mimetic environment The antibacterial and cytotoxic activities of synthetic caerin 1.11 and PBN2 were tested (Table 2) The Ó FEBS 2003 Evolution of amphibian antimicrobial peptides (Eur J Biochem 270) 2073 Fig Conserved preproregions and hypervariable antimicrobial domains of preprodermaseptins (A) Diagram of preprodermaseptin cDNAs The coding region, including the signal peptide, the acidic propiece and the antimicrobial progenitor sequence is drawn as a rectangle (B) Alignment of the predicted amino acid sequences (single-letter code) of preprodermaseptin cDNAs obtained from hylid and ranin frogs, including the signal peptide, the acidic propiece and the antimicrobial progenitor sequence The predicted hydrophobic signal peptide includes the first 22 amino acid residues, while the acidic propiece comprises 16–27 residues Gaps (–) have been introduced to maximize sequence similarities Identical (black background) and similar (shaded background) amino acid residues are highlighted Among the hylid sequences, DRS, dermaseptin B from P bicolor, DRP, dermaseptin-related peptide (appended with AA, AC or PD to indicate that the sequences were identified from A annae, A callidryas and P dacnicolor, respectively) Among the ranin sequences, temporins B, H and G and brevinins 2Ta and 2Tb are from Rana temporaria, brevinins 1E and 2Ef and esculentin 1B from R esculenta, ranalexin from R catesbeiana, gaegurins and from R rugosa, and ranatuerin-2P and Pa from R pipiens cDNAs encoding PBN1 and PBN2 (GenBank accession numbers: AY218784 and AY218783), DRP-AC 1, and (accession numbers: AY218775, AY218776 and AY218777) and caerins (accession numbers: AY218778-82 and AY218785) were identified in this study corresponding values for dermaseptin B2 from P bicolor are shown for comparison Although their primary structures are very different, PBN2 and dermaseptin B2 showed overlapping antimicrobial spectra They had broadspectrum antibacterial activities, inhibiting the growth of Gram-positive bacteria, Gram-negative bacteria and yeast with minimal inhibitory concentrations in the lM range The dose–response profiles showed sharp curves in which 0–100% inhibition was generated within a 1–2-fold peptide dilution (not shown) Bacteria incubated overnight with 25 lM PBN2 or dermaseptin B2 produced no colony forming units, indicating that the peptides are bactericidal A combination of PBN2 and dermaseptin B2 was also dramatically synergistic, so that the mixture sometimes had 15-times greater antibiotic activity than the peptides separately (Table 2) Although their primary structures are very similar, caerin 1.1 and caerin 1.11 differred unexpectedly in their capacity to inhibit the growth of various bacteria For instance, whereas caerin 1.11 effectively inhibited E coli (MIC, 25 lM), caerin 1.1 was inactive [54] Conversely, caerin 1.1 effectively inhibited the proliferation of S aureus [54], while caerin 1.11 had no effect Clearly, the withinspecies differences between members of the caerin family indicates that the peptides are functionally differentiated Ó FEBS 2003 2074 D Vanhoye et al (Eur J Biochem 270) Table Inhibition of yeast and bacterial growth in vitro by caerin 1.11, PBN2 and dermaseptin B2 The MIC is the minimal dose producing 100% inhibition of growth after incubation for 24 h in culture medium ND, not determined Peptide minimal inhibitory concentration (lM) Microorganism Escherichia coli B Salmonella typhimurium Salmonella enteritidis Enterobacter cloacae Klebsiella pneumoniae Aerococcus viridans Bacillus megaterium Staphylococcus aureus Staphylococcus haemolyticus Saccaromyces cerevisiae a Caerin 1.11 DRS B2 PBN2 DRS B2 + 0.25 lM PBN2 25 Ra R 50 25 ND R R 50 1.5 3.1 3.1 3.1 1.5 ND 0.4 12.5 6.2 1.5 3.1 1.5 12.5 0.8 3.1 0.8 3.1 0.8 0.4 0.8 1.5 3.1 0.2 ND 0.4 0.8 0.8 ND 5.5 3.1 0.8 MIC > 100 lM ness of the apolar face, net charge and conformational flexibility play a crucial role in modulating the biological potency of linear helical peptide antibiotics Molecular phylogeny of preprodermaseptins We examined the evolutionary relationships between preprodermaseptin cDNAs by constructing phylogenetic trees from alignments of DNA and predicted protein sequences (Fig 2) The phylogenetic reconstruction shown in Fig 4A indicates that the 10 frog species from which the 42 preprodermaseptin sequences were obtained fall into two distinct clusters The nucleotide sequences of the antimicrobial peptides from the South American and Australian hylids cluster separately from those from the ranins The cluster of hylid sequences is not very well resolved, but there were several distinct clades: one clade of 12 dermaseptins B and dermaseptin-related peptides was supported by a bootstrap Fig Helical wheel plots (73) of (A) caerin 1.11 and (B) PBN2 Apolar residues are in bold letters The amino acid sequence of each peptide is shown beneath the corresponding wheel plot The predicted helical domains are underlined The data presented here showed that despite very different primary structures, caerin 1.11 and PBN2 belong to the class of cationic, amphipathic a-helical antimicrobial peptides which interact with and disrupt cell membrane CD and antimicrobial assays showed that the helical contents of PBN2 and caerin 1.11 are not correlated with antibacterial potency These observations suggest that additional parameters, namely hydrophobicity, hydrophobic moment, bulky- Fig Molecular phylogeny of preprodermaseptins (A) Neighborjoining tree constructed from Kimura two parameters distances computed from comparison of entire preprodermaseptin cDNA sequences (including 3¢- and 5¢-untranslated regions) obtained from hylid and ranid frogs Bootstrap values from 1000 replicates greater than 50% are indicated on branches The distance scale is drawn below the tree Maximum parsimony, maximum likelihood and LogDet analyses yielded the same ordinal phylogeny Phylogram is midpoint rooted (B) Paleogeographic reconstruction of the fragmentation of Gondwana during the late Jurassic/early Cretaceous period [64] Land areas are shaded Ancestors of Australian hylids are believed to have crossed Antartica to Australia from South America  150–130 Ma Ancestors of Asian, European and North American ranins evolved on isolated India between 150 and 65 Ma and colonized the Laurasian land mass after India collided with Asia Abbreviations; AF, Africa; IND, India; AUS, Australia; SA, South America; ANT, Antartica Reconstruction map from http://www.odsn.de/odsn/services Ó FEBS 2003 Evolution of amphibian antimicrobial peptides (Eur J Biochem 270) 2075 values of 81% (hylid clade 1) while a second clade of 17 dermaseptin-related peptides included the caerins from Litoria (hylid clade 2) Within hylid clade 2, the sequences from Litoria clustered together monophyletically and were more closely related to PBN1 from P bicolor The average Kimura 2-parameter pairwise distances were 0.34 for the 2076 D Vanhoye et al (Eur J Biochem 270) Ó FEBS 2003 sequences from South American hylids, 0.32 for those from ranins, and 0.08 for the sequences from L caerulea The average pairwise distances were much greater between each cluster of sequences (0.70 between South American hylids and ranins, and 0.72 between ranins and L caerulea) Thus, despite considerable variations between the sequences of the mature antimicrobial peptides, phylogenetic reconstruction using the complete sequence of the preprodermaseptin transcript produced a tree topology that agreed with the traditional classification of neobatrachian frogs [19,58] The divergence of the antimicrobial peptides and their evolutionary relationships would never have been apparent without the strong conservation of the precursor preproregion The molecular phylogram shows that the genes encoding the antimicrobial peptides in Hylidae and Ranidae arose from a common ancestral locus that subsequently diversified by several rounds of duplication and subsequent divergence of loci Most of the duplication events predated the radiations of ranins and of South American hylids and occurred before cladogenesis in a species ancestral to all these species, i.e., the sequences not cluster according to species and are more closely related between than within species In contrast, the phylogenetic tree suggests that gene duplications in L caerulea occured after the divergence of South American and Australian hylids Accelerated mutation of the antimicrobial peptide domain of preprodermaseptins The strikingly greater variability in the antimicrobial peptide progenitor sequences of the precursors compared to the highly conserved preproregions may indicate different mutation rates in the corresponding regions of the genes We tested this hypothesis by measuring the rates of nucleotide synonymous (silent) substitutions in the three domains (signal peptide, acidic propiece and antimicrobial peptide progenitor sequence) of the translated regions of the preprodermaseptin sequences in South American and Australian hylids and in ranins As synonymous substitutions are apparently neutral, the fixation rates can be considered to be proportional to the mutation rates and the number of synonymous substitutions per synonymous sites, Ds, to be an adequate representation of the mutation rate The nucleotide substitution rates in the 5¢- and 3¢-untranslated regions were calculated using the Jukes–Cantor one parameter model to be consistent with the Ds estimation The average Ds in the mature antimicrobial domains were more than two to five times greater than Ds estimated from the signal peptide domain or the Jukes–Cantor distance estimated from the untranslated regions (Fig 5A) and the substitution rates for the mature peptide domain were certainly underestimated because of multiple substitutions per site (saturation) in the mature region and because additions and deletions were ignored in the calculation In all cases, the length of the signal peptide was constant while many additions and deletions were needed for optimal alignment of the antimicrobial peptide progenitor sequences (Fig 2) The different mutation rates of the different regions in the transcripts of the preprodermaseptins is also indicated by the unrooted trees shown in Fig Genes were segregated in clearly defined branches according to the classical Fig Accelerated mutation of the antimicrobial peptide domain of preprodermaseptins (A) Average (± SD) pairwise Jukes–Cantor distances (5¢- and 3¢-untranslated regions) and proportions of synonymous substitutions per synonymous site (D) among the signal peptide, acidic propiece and antimicrobial peptide progenitor domains estimated from the nucleotide sequences of preprodermaseptins from South American and Australian hylids and from ranins (Fig 4A) (B) Average (± SD) ratios of nucleotide transversions to transitions (Tv/ Ts) calculated for the signal peptide, acidic propiece and antimicrobial peptide progenitor domains of preprodermaseptins from South American and Australian hylids and from ranins Tv/Ts ratios determined by Maor-Shoshani et al [62] for DNA polymerases Pol III and Pol V are shown for comparison phylogeny of species when using either the 5¢-UTR, the signal peptide sequences or the acidic propiece sequences In contrast, transcripts started to diverge directly from the origin with mixed branches when using the antimicrobial progenitor sequences Synonymous substitutions should accumulate at similar rates in different regions of a gene Deviations from this behavior may be due to difference in codon usage, or differences in mutation rate across the gene As no codon bias was detected, our data suggest that a mechanism is operating that leads to very different mutation rates in adjacent regions of these small genes Molecular signatures of mutagenic polymerases targeted to the antimicrobial peptide domain Whereas diversifying (positive) selection contributes to the accelerated evolution of antimicrobial peptides [59], recent studies have suggested that hypervariability in specific gene regions may result from the actions of targeted mutagenic, Ó FEBS 2003 Evolution of amphibian antimicrobial peptides (Eur J Biochem 270) 2077 Fig Unrooted neighbor-joining trees constructed using Kimura two parameters distances computed from comparison of specific regions of preprodermaseptin cDNA sequences error-prone, DNA polymerases similar to DNA polymerase V from E coli [44,60,61] Molecular signatures of the SOSinducible polymerase V are low processivity and a strong bias for transversions over transitions [62] We examined the transversion/transition (Tv/Ts) ratios in alignments of preprodermaseptin transcripts from hylid and ranid frogs The Tv/Ts ratios in the different regions of the transcripts were clearly different (Fig 5B) It increased from the signal peptide to the acidic propiece, to the antimicrobial peptide progenitor sequence, with a twofold bias for Tv over Ts in the progenitor sequence This value was similar to that predicted for random substitions and corresponded to the in vitro measured transversion bias of DNA Pol V reported by Maor-Shoshani et al [62] These results suggest that a targeted mutagenic process involving a DNA Pol V-like enzyme has operated in hylids and ranins within the peptide progenitor sequence of antimicrobial peptide loci, but not in the signal peptide and acidic propiece domains Thus speciation events, gene duplications, targeted hypermutations and the subsequent actions of diversifying selection have all contributed to the evolution and diversification of this large family of hypervariable genes 2078 D Vanhoye et al (Eur J Biochem 270) Discussion Given the low dispersal abilities of amphibians over salty environments, tectonic movments and changes in sea-level have been of importance in shaping the distribution of lineages of ranid and hylid frogs As emphasized by Savage [63], the historical biogeography of neobatrachian frogs is associated mainly with Gondwanaland Thus, the current biogeographic distributions of ranids and hylids together with tectonic events during the fragmentation of Gondwana can be used to provide a temporal framework for the origins and evolution of the preprodermaseptin genes The supercontinent Gondwana began to break up about 150 Ma when India/Madagascar separated from Africa (Fig 4B) [64] India drifted north-eastward during the ensuing 100 Myr, and finally collided with Asia 65–56 Ma Antartica/Australia became disjoined from South America around 150 Ma They were reconnected by a narrow land bridge between 140 and 130 Ma, after which there was an archipelago until 110 Ma Australia separated from Antartica about 60 Ma Africa and South America were substantially separated around 125 Ma and became completely detached before 100 Ma The many endemic and very diverse species of ranid frogs in Africa, Madagascar and India [19,65], is usually interpreted as indicating that they originated on these land masses before Africa and India/Madagascar separated Furthermore, thorough analyses of the phylogenetic relationships of ranids based on mitochondrial and nuclear DNA sequences [65–67] has demonstrated that the speciation events giving rise to the Indian ranid lineages took place  130–150 Ma when India had separated from Africa Indian Raninae, including the genus Rana, originated and began to diversify on the drifting Indian block between 130 and 65 Ma, spread from India after its collision with Asia 65–56 Ma and radiated into more than 200 species in Asia, Europe and North America [19,67] Biogeographic and molecular evidence indicates that South America is the site from which the Hylidae radiated [19,65] Among the four subfamilies of hylids, the Phyllomedusinae, the Hemipractinae and the Hylinae are predominantly South American, while the Pelodryadinae, including the genus Litoria, are restricted to Australia and New Guinea These data suggest that ancestors of the Australian hylids originated in South America and probably reached Australia via the connection with Antartica and South America [66] Evidence for the dispersal of land vertebrates from South America to Australia via Antartica also comes from fossils and molecular data on marsupials [68] Thus, the paleogeographic reconstruction shown in Fig 4B suggests that the immediate ancestor of Australian Pelodryadinae must have been present in Australia before Australia and Antartica separated 60 Ma, and in Antartica well before the last known connection between Antartica and South America 150–130 Ma Once South America and Antartica were separated, the hylid fauna in Australia and South America diversified independently This historical reconstruction shows that the gene family encoding antimicrobial peptides from South American and Australian hylids and from Indian, Eurasian and North American ranins arose before the isolation of India and South America from Africa in a pan-Gondwanan common ancestor of these species Given Ó FEBS 2003 that Archeobatrachia (Ôarchaic frogsÕ) and Neobatrachia diverged about 200 Ma [69], and that preprodermaseptins have not been detected in archaic frogs, the present data indicates maximum and minimum origination dates of approximately 200–150 Ma Our study provides evidence that several different mechanisms appear to have contributed to the diversity of modern antimicrobial peptide loci since the Jurassic period These include several gene duplications, most of them predating the divergence of species, and diversifying (positive) selection We have shown previously that members of the preprodermaseptin gene family have been subject to positive selection within the acidic propiece and the antimicrobial peptide domain of hylids and only within the antimicrobial peptide domain of ranins [59] The third mechanism is an accelerated rate of nucleotide substitution that is focused on the antimicrobial peptide domain in preprodermaseptin genes This mechanism, that has yet to be proved, may involve a mutagenic DNA polymerase with a strong transversion bias, such as DNA Pol V from E coli which can generate multiple changes in the sequence in a single step Antimicrobial peptides are synthesized in the multinucleated cells of the dermatous glands of the skin and large amounts are stored in the secretory granules of these glands [70,71] The glands may release their content onto the skin surface by a holocrine mechanism involving the rupture of the plasma membrane and the extrusion of the granules through a duct opening to the surface Skin antimicrobial peptides not participate in the general metabolism and physiology of the frog producing them As no deleterious effects are expected when a new peptide variant rapidly emerges by multiple changes in a current sequence, the new sequence may bypass the actions of neutral and negative (purifying) selections Thus, the combination of targeted hypermutations to generate great variation plus the subsequent action of positive selection may explain both the hypervariation and large number of antimicrobial peptides per species Although the organization of eukaryotic signal peptides is evolutionary conserved, the sequences are not similar The evolutionary pressure that results in the conservation of the signal peptide and, albeit to a lesser extent, the acidic propiece in preprodermaseptins is especially striking in view of the extreme variations in the contiguous antimicrobial peptide domain and the very ancient history of the gene family This suggests that these conserved elements have important functions Conticello et al have suggested that conserved elements in an otherwise hypermutable DNA sequence might be protected from mutagenesis by specifically bound macromolecules that serve to prime DNA Pol V-like polymerase in the vicinity [60] By stopping normal DNA replication, DNA-bound macromolecules may then create single-strand gaps in the replicating strand, thus recruiting mutagenic polymerase as part of the damage response to the lesion This hypothetical scenario is attractive in that it provides a plausible explanation for the conservation of the signal peptide sequences for millions of years although it lies in the precursors of very different antimicrobial peptides produced by very distantly related species of frog Finally, the underlying biology of preprodermaseptin diversification within the hylid and ranin frogs invites some Ó FEBS 2003 Evolution of amphibian antimicrobial peptides (Eur J Biochem 270) 2079 speculation Each ranin or hylid frog species produces its own set of preprodermaseptin-derived antimicrobial peptides Some of these peptides differ by only a few amino acid substitutions or deletions and have similar biochemical characteristics Other peptides have widely different sequences and physicochemical properties The presence in frog skin of numerous antimicrobial peptides, acting separately or in concert, may have a selective survival value in habitats laden with microorganisms As shown in this and other studies [52–55,70], a comparison of antimicrobial peptides issued from Phyllomedusa or from Litoria revealed that most of them kill a broad spectrum of microorganisms but differ widely in their capacity to kill the various agents Even peptides with very similar structures, such as caerin 1.1 and caerin 1.11 or dermaseptins S [9], target specific microorganisms Without direct functional characterization, it would have been difficult to predict the different spectra of these peptides Moreover, these peptides act in synergy, with the mixture having up to a 10- to 100-fold greater antibiotic activity than the peptides separately (Table 2) [72] Hence, the hypervariability of skin antimicrobial peptides and the proposed mechanisms of diversification could be part of a strategy for providing frogs with a maximum protection against a wide range of infectious microorganisms Also, these antimicrobial peptides with such diverse structures and spectrum of action can be viewed as the successful evolution of a multidrug defence system, which minimizes the chance of microorganisms developing resistance to individual peptides Comparisons of species has shown that different frog species have sets of homologous but different peptides that have diversified in a species-specific manner South American and Australian hylids, as well as Indian, European, Asian and North American ranins, have different patterns of distribution with respect to geography, climate, vegetation and habitats (aquatic, semiaquatic, terrestrial, arboreal, torrential, rocky), some of them showing very unusual and extreme adaptations [19] The diversification of antimicrobial peptides between species could thus be part of an optimum evolutionary strategy developed during the radiations of these frog species when microbial predators changed very rapidly with shifts to new ecological niches Focal hypermutation of the C-terminal antimicrobial-coding region of preprodermaseptin genes might have evolved as a way of increasing genetic diversity and so accelerating the adaptation of frogs to noxious microbial fauna Acknowledgements This work was partly supported by the CNRS and the ÔProgramme de Recherche Fondamentale en Microbiologie 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[64] Land areas are shaded Ancestors of Australian hylids are believed to have crossed Antartica to Australia from South America  150–130 Ma Ancestors of Asian, European and North American ranins