The antimicrobial peptides of South American hylid frogs are derived from precursors, the preprodermaseptins, whose signal peptides and intervening sequences are remarkably conserved, bu
Trang 1Antimicrobial 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 enor-mous array of peptide antibiotics The hyperdivergence of modern antimicrobial peptides and the number of peptides per species result from repeated duplications of a 150-million-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 Keywords: antimicrobial peptides; frog skin; dermaseptin; hypermutation; gene family
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
anti-microbial 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, tri-dimensional 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, 2 Place Jussieu,
75251 Paris Cedex 05, France.
Fax: +33 1 44 27 59 94, Tel.: +33 1 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)
Trang 2in 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 annaeand Pachymedusa dacnicolor, respectively
[36] Analysis of cDNA clones of antimicrobial peptides
from South American hylids [32,34–37] and Asian, European
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 process-ing signal, Lys-Arg The pattern of conserved and variable regions in skin antimicrobial peptide precursors is therefore
Table 1 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.
Hylidae Phyllomedusinae Phyllomedusa bicolor Dermatoxin SLGSFLKGVGTTLASVGKVVSDQFGKLLQAGQa
Phylloxin GWMSKIASGIGTFLSGMQQa DRSB1 AMWKDVLKKIGTVALHAGKAALGAVADTISQa DRSB2 GLWSKIKEVGKEAAKAAAKAAGKAALGAVSEAVa DRSB3 ALWKNMLKGIGKLAGQAALGAVKTLVGAE DRSB4 ALWKDILKNVGKAAGKAVLNTVTDMVNQa DRSB6 ALWKDILKNAGKAALNEINQLVNQa
Agalychnis callydryas DRP-AC1 GLLSGILNTAGGLLGNLIGSLSNGES
DRP-AC2 GLLSGILNSAGGLLGNLIGSLSNGES DRP-AC3 SVLSTITDMAKAAGRAALNAITGLVNQGEQ Agalychnis annae DRP-AA11 SLGSFMKGVGKGLATVGKIVADQFGKLLEAGQG
DRP-AA2-5 GLVSGLLNTAGGLLGDLLGSLGSLSGGES DRP-AA3-1 SLWSKIKEMAATAGKAALNAVTGMVNQGEQ DRP-AA3-3 GMFTNMLKGIGKLAGQAALGAVKTLAGEQ DRP-AA3-4 GMWGSLLKGVATVVKHVLPHALSSQQS DRP-AA3-6 GMWSTIRNVGKSAAKAANLPAKAALGAISEAVGEQ Pachymedusa dacnicolor DRP-PD1-5 SLGSFMKGVGKGLATVGKIVADQFGKLLEAGKG
DRP-PD2-2 ALWKTLLKKVGKVAGKAVLNAVTNMANQNEQ DRP-PD3-3 GMWSKIKNAGKAAAKASKKAAGKAALGAVSEALGEQ DRP-PD3-6 GVVTDLLNTAGGLLGNLVGSLSGGER
DRP-PD3-7 LLGDLLGKTSKLVNDLTDTVGSIV Pelodryadinae Litoria caerulea Caerin 1.1 GLLSVLGSVAKHVLPHVVPVIAEHLa
Caerin 1.11 GLFSVLGSVAKHVVPRVVPVIAEHLa Caerin 1.12 GLFGILGSVAKHVLPHVVPVIAEHSa Caerin 1.13 GLLSVLGSLKLIVPHVVPLIAEHLa Caerin 1.14 SVLGKSVAKHLPHVVPVIAEKTa Caerin 1.15 GLFGLAKGSVAKPHVVPVISQLVa Ranidae Raninae Rana catesbeiana Ranalexin FLGGLIKIVPAMICAVTKKC
esculenta Brevinin-1 E FLPLLAGLAANFLPKIFCKTRKC
Brevinin-2 Ef GIMDTLKNLAKTAGKGALQSLVKMASCKLSGQC Esculentin 1B GIFSKLAGKKLKNLLISGLKNVGKEVGMDVVRTGIDI
AGCKIKGEC rugosa Gaegurin-4 GILDTLKQFAKGVGKDLVKGAAQGVLSTVSCKLAKTC
Gaegurin-5 DVEVEKRFLGALFKVASKVLPSVFCAITKKC temporaria Temporin B LLPIVGNLLKSLLa
Temporin H LSPNLLKSLLa Temporin G FFPVIGRILNGILa Brevinins ) 2Ta GILDTLKNLAKTAGKGILKSLVNTASCKLSGQC Brevinins ) 2Tb GILDTLKHLAKTAGKGALQSLLNHASCKLSGQC pipiens Ranatuerin-2P GLMDTVKNVAKNLAGHMLDKLKCKITGC
Ranatuerin-2 Pa GFLSTVVKLATNVAGTVIDTIKCKVTGGCRK
Trang 3the 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
immuno-globulins [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:
Phyllo-medusinae) We have determined the activity spectra of the
predicted antimicrobial peptides A combination of
phylo-genetic reconstruction, analysis of mutation rates and
geophysical models for the sequence of fragmentation of
Gondwana suggests that the hypervariability of
antimicro-bial peptides and the number of peptides per species reflect
the combination of speciation events, gene duplications,
targeted hypermutation and subsequent actions of
diversi-fying 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 La Ferme
Tropi-cale (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 ± 1C
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 94C for 240 s, 56C for 45 s, 72 C for 60 s and one cycle of
72C for 10 min 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 prod-ucts 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
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
for caerin 1.1, 5¢-CTAAGTGCTCAGCAATGACG-3¢ for caerin 1.11, 5¢-AGCATAACTGGAACGTGGG-3¢ for caerin 1.12, 5¢-CAGCAATAAGTGGAACAACG-3¢ for
for caerin 1.14 and 5¢-AGCAACGGATCCTAGGA CAC-3¢ for caerin 1.15 The temperature cycle used for the RACE PCRs was: 94C for 240 s, 35 cycles at 94 C for
40 s, 56C for 45 s, 72 C for 60 s, and a final extension step of 72C for 10 min The PCR products were cloned and sequenced as above A similar approach was used to clone PBN1 and PBN2 cDNAs from P bicolor, and DRP-AC1, 2 and 3 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 mmolÆg)1 Carboxamidated pep-tides were prepared on a 4-methylbenzhydrylamin Polysty-ren resin (Rink Amide MBHA PSresin) substituted at 0.81 mmolÆg)1 Synthesis was carried out using a double-coupling protocol: Fmoc amino acids (10 molar excess) were coupled for 30–60 min with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate 1-hydro-xybenzotriazol in a solution of N,N-dimethylformamide and diisopropylethylamine as activating agents with the
Trang 4addition 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-5-sulfonyl (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%
triisopropylsi-lane and 2.5% water for 2 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 min at
4C, 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 4 mLÆmin)1with a 0–60% linear
gradient of acetonitrile in 0.07% trifluoroacetic acid/water
over 30 min 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,
Salmon-ella typhimurium, SalmonSalmon-ella enteritidis, Enterobacter
cloa-cae, Klebsiella pneumoniae) and Saccaromyces cerevisiae
were cultured as described previously [34,35] The minimal
inhibitory concentrations (MICs) of peptides were
deter-mined 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 A630value 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 37C 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
2 h with different concentrations of peptides and plated on
solid culture medium containing 1% noble agar to
distin-guish 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
nega-tive-controls with 0.7% formaldehyde
Sequence analysis
The nucleotide sequences of cDNAs encoding 18 known
dermaseptin B, phylloxins, dermatoxins, and
dermaseptin-related peptides from the hylids A annae, P dacnicolor and
P bicolorwere obtained from GenBank, in addition to the
cDNAs encoding PBN1, PBN2, DRP-AC 1, 2 and 3 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 withCLUSTAL 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 Neighbor-Joining from Kimura-two-parameters distances [48] using PAUP [49] Levels of support for branches were estimated with bootstrapping methods (1000 replicates) also withPAUP 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 prepro-region 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 JADISthe propor-tion of sites with transipropor-tional and transversional differences between two sequences Tv and Ts were calculated inde-pendently 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 wide-spread 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 caeru-leausing 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 remain-ing five cDNAs coded for new members of the caerin 1 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
Trang 5families Each precursor polypeptide had an extra-glycine
residue at the carboxyl terminus of its progenitor sequence,
indicating that C-terminal amidation is involved in
pro-duction 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
corres-ponding 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
preproderma-septin family
A similar approach was used to identify new
prepro-dermaseptin-related cDNA sequences in the skins of South
American hylid frogs (Fig 2) Two of these sequences from
P bicolorencoded novel putative peptides we have called
PBN1 and PBN2 PBN1 (FLSLIPHIVSGVAALAKHL)
and PBN2 (GLVTSLIKGAGKLLGGLFGSVTGGQS)
do 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, 2 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 bicolorantimicrobial peptides As shown previously, the common structural feature of linear cationic antimicrobial peptides such as caerin 1.1 and dermaseptins Sand 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 concentra-tions 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
Fig 1 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 1 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.
Trang 6corresponding values for dermaseptin B2 from P bicolor
are shown for comparison Although their primary
struc-tures are very different, PBN2 and dermaseptin B2 showed
overlapping antimicrobial spectra They had
broad-spectrum antibacterial activities, inhibiting the growth of
Gram-positive bacteria, Gram-negative bacteria and yeast
with minimal inhibitory concentrations in the lMrange 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 sepa-rately (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 within-species differences between members of the caerin 1 family indicates that the peptides are functionally differentiated
Fig 2 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 4 and 5 from R rugosa, and ranatuerin-2P and 2 Pa from R pipiens cDNAs encoding PBN1 and PBN2 (GenBank accession numbers: AY218784 and AY218783), DRP-AC
1, 2 and 3 (accession numbers: AY218775, AY218776 and AY218777) and caerins (accession numbers: AY218778-82 and AY218785) were identified in this study.
Trang 7The 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
param-eters, namely hydrophobicity, hydrophobic moment,
bulky-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 pre-prodermaseptin 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 antimicro-bial 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 3 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.
Table 2 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.
Microorganism
Peptide minimal inhibitory concentration (l M )
Caerin 1.11
DRS
DRS B2 + 0.25 l M
PBN2 Escherichia coli B 25 1.5 1.5 0.4 Salmonella typhimurium Ra 3.1 3.1 0.8 Salmonella enteritidis R 3.1 1.5 1.5 Enterobacter cloacae 50 3.1 12.5 3.1 Klebsiella pneumoniae 25 1.5 0.8 0.2 Aerococcus viridans ND ND 3.1 ND Bacillus megaterium R 0.4 0.8 0.4 Staphylococcus aureus R 12.5 3.1 0.8 Staphylococcus
haemolyticus
Saccaromyces cerevisiae ND 5.5 3.1 0.8
a MIC > 100 l M
Fig 4 Molecular phylogeny of preprodermaseptins (A) Neighbor-joining tree constructed from Kimura two parameters distances com-puted 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.
Trang 8values 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
Trang 9sequences 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
evolution-ary 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
diversi-fied 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 do 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
substitu-tions 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¢-untrans-lated regions were calcu3¢-untrans-lated 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 6 Genes were segregated
in clearly defined branches according to the classical
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,
Fig 5 Accelerated mutation of the antimicrobial peptide domain of preprodermaseptins (A) Average (± SD) pairwise Jukes–Cantor dis-tances (5¢- and 3¢-untranslated regions) and proportions of synony-mous substitutions per synonysynony-mous site (D) among the signal peptide, acidic propiece and antimicrobial peptide progenitor domains esti-mated 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 deter-mined by Maor-Shoshani et al [62] for DNA polymerases Pol III and Pol V are shown for comparison.
Trang 10error-prone, DNA polymerases similar to DNA polymerase
V from E coli [44,60,61] Molecular signatures of the
SOS-inducible 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 vitromeasured 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 hypermuta-tions and the subsequent achypermuta-tions of diversifying selection have all contributed to the evolution and diversification of this large family of hypervariable genes
Fig 6 Unrooted neighbor-joining trees constructed using Kimura two parameters distances computed from comparison of specific regions of prepro-dermaseptin cDNA sequences.