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An anionic antimicrobial peptide from toad Bombina maxima
BBRC Biochemical and Biophysical Research Communications 295 (2002) 796–799 www.academicpress.com
An anionic antimicrobial peptide from toad Bombina maximaq Ren Lai, Hen Liu, Wen Hui Lee, and Yun Zhang* Department of Animal Toxinology, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 East Jiao Chang Road, Kunming, Yunnan 650223, People’s Republic of China Received 10 June 2002
Abstract Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide, named maximin H5, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2 . Containing three aspartate residues and no basic amino acid residues, maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides, only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5, while the other bacterial and fungal strains tested were resistant to it. The presence of metal ions, like Zn2þ and Mg2þ , did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians. The results provide the first evidence that, together with cationic antimicrobial peptides, anionic antimicrobial peptides may also exist naturally as part of the innate defense system. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Anionic antimicrobial peptide; Cationic antimicrobial peptide; Amphibian; Bombina maxima; Maximin H; cDNA clone
In recent years, a large number of antimicrobial peptides have been discovered from animals and plants. These molecules, which are either constitutive or inducible, are recognized as important components of innate defense system [1–3]. Amphibians, being the first group of organisms forming a connecting link between land and water, are forced to adopt and survive in a variety of conditions laden with pathogenic microbes. Thereby, they are endowed with an excellent chemical defense system composed of pharmacological and antimicrobial peptides [4]. A considerable variety of antimicrobial peptides have been characterized from amphibians. Based on their sequence/tridimensional structure characteristics, the microbicidal peptides from amphibian skin can be grouped into three broad families [5–7]. The first one contains linear amphipathic helix forming peptides such q Abbreviations: BLPs, bombinin-like peptides; SAAPs, ovine pulmonary surfactant-associated anionic peptides; AAMPs, anionic antimicrobial peptides; CAMPs, cationic antimicrobial peptides; MIC, minimal inhibitory concentration. The nucleotide sequence data reported in this paper are available from GenBank database with Accession No. AF515614. * Corresponding author. Fax: +86-871-519-1823. E-mail address: [email protected] (Y. Zhang).
as magainins from African clawed frog Xenopus laevis and bombinin-like peptides (BLPs) from Bombina orientalis and B. variegata. The second family has four different groups of related peptides isolated from various species of the Ranidae family, which all contain two cysteine residues at the C-terminal part that form a disulfide bond. The third one includes temporins from Rana temporaria, which are only 10–13 amino acids long. In spite of great primary structure diversity, the majority of antimicrobial peptides documented are now characterized by a preponderance of cationic and hydrophobic amino acids, which are spatially organized in discrete sectors of the molecule. This amphipathic structure allows them to selectively interact with bacterial membranes, which usually comprise negatively charged phospholipids, by the aid of electrostatic interactions [1,8]. Ovine pulmonary surfactant-associated anionic peptides (SAAPs), being 7 amino acids long and comprising 5–7 aspartate residues, is the only example of anionic antimicrobial peptides (AAMPs), showing bactericidal activity on Pasteurella haemolytica in the presence of zinc [9]. In a previous study, two groups of antimicrobial peptides have been isolated from skin secretions of Chi-
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 7 6 2 - 3
R. Lai et al. / Biochemical and Biophysical Research Communications 295 (2002) 796–799
nese red belly toad B. maxima. Peptides in the first group, maximins 1–5, are structurally related to BLPs. Peptides in the second group, maximins H1–H4, are homologous with bombinin H peptides [10]. They are all cationic antimicrobial peptides (CAMPs). Here, we reported a gene encoded anionic peptide, named maximin H5, its cDNA cloning, synthesis, and antimicrobial activity.
Materials and methods cDNA cloning of antimicrobial peptides. As previously reported, several full-length clones with cDNA sequences encoding maximins plus maximin H peptides were obtained [10]. In an attempt to screen possible additional clones encoding novel antimicrobial peptides from constructed B. maxima skin cDNA library, a specific oligonucleotide primer Ma (50 -ATGAATTTTAAGTACATA-30 , in the sense direction, corresponding to residues 1–6 of the precursor protein of maximin 3 plus maximin H2), was designed. Primer Ma and a vector SP6 promoter primer (50 -CATACGATTTAGGTGACACTATAG-30 , in the antisense direction) located in 30 of the cloned insert were used in PCR screening of full-length antimicrobial peptide cDNAs as before [10]. All the oligonucleotide primers for PCR were prepared with a DNA synthesizer (Model 381A, Applied Biosystems). DNA sequencing was performed on an ABI PRISM 377 DNA sequencer, Applied Biosystems. Computer analysis of protein and nucleotide acid sequences was performed with the Clustal V sequence software package. Peptide synthesis. Amidated maximin H5 was synthesized by solid phase synthesis on an Applied Biosystems model 433A peptide synthesizer according to manufacturer’s standard protocols. After cleavage and side-chain deprotection, the crude synthetic peptide was purified on a Vydac 218TP510 C18 reverse-phase HPLC column (25 1 cm) eluted at a flowrate of 2 ml/min by a linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water. Identity of the peptide was confirmed by automated Edman degradation with a protein sequencer and mass spectrometry analysis. Fast atom bombardment mass spectrometry was carried out on an Autospec-3000 spectrometer, equipped with a high field magnet [11]. The synthetic peptide was then used for evaluating biological activities. Antimicrobial assays. Standard bacterial and fungal strains used in antimicrobial assays, Gram-positive bacterial strains Staphylococcus aureus (ATCC2592) and Bacillus megatherium, Gram-negative bacterial strains Escherichia coli (ATCC25922), Bacillus pyocyaneus (CMCCB10104), Bacillus dysenteriae, and Klebsiella pneumoniae, and fungal strains Candida albicans (ATCC2002) and Aspergillus flavus (IFFI4015), were obtained from Kunming Medical College. Antimicrobial activity was assayed as described [10]. Minimal inhibitory concentration (MIC) was determined in liquid LB medium by incubating the bacteria in LB broth with variable amounts of the sample tested. The MIC at which no visible growth occurred was recorded. To assay the possible synergetic effect of maximin H5 on maximin 3, maximin H5 and maximin 3 were co-applied to bacteria that are sensitive to maximin 3 and the MICs were then assayed. For assaying the possible influence of metal ions, different concentrations (20 lM– 1 mM) of ZnCl2 and MgCl2 were added to test their effects on antimicrobial activity of maxmin H5. The peptides were quantified by UV absorbance at 215 and 225 nm using the formula: concentration (mg/ml) ¼ ðA215 A225 Þ 0:144. Hemolytic assays. Hemolytic assay was tested with rabbit red cells in liquid medium as reported [12]. Serial dilutions of the peptides were used, and after incubation at 37 °C for 30 min, the cells were centrifuged and the absorbance in the supernatant was measured at 595 nm. Maximum hemolysis was determined by adding 1% Triton X-100 to a sample of cells.
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Results and discussion cDNA Cloning A cDNA library constructed from B. maxima skin was screened at high stringency, as described under ‘‘Materials and methods,’’ by an efficient and rapid PCR-based procedure. More than 60 positive clones, which contain an insert around 650 base pairs, were identified and isolated. Both strands of these clones were sequenced. In addition to previously identified clones that encode previously purified antimicrobial peptides maximins 1–5, and maximins H1–H4 [10], about 30 novel clones were found to encode new antimicrobial peptide precursor proteins. The encoded precursors comprise either a novel maximin with a novel maximin H peptide, or different combinations of these two kinds of peptides (data not shown). Surprisingly, clone 321 contains an insert that encodes a precursor protein comprising maximin 3 and a novel anionic peptide. The complete nucleotide and the deduced amino acid sequences of clone 321 are shown in Fig. 1. The overall
Fig. 1. Complete nucleotide and deduced amino acid sequences of the cDNA encoding maximin 3 plus maximin H5 (clone 321) and comparison with those of the cDNA encoding maximin 3 plus maximin H2 (clone 118) [10]. The nucleotides of the protein coding region are shown by uppercase letters and those of the noncoding region by lowercase letters. The deduced amino acid sequence is shown below. Gaps (-) have been introduced to optimize the sequence homology. Maximin H peptide sequences are boldfaced and italicized. The sequence of maximin 3 is boldfaced. Dots () indicate the same nucleotide and amino acid residues in both cDNAs.
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R. Lai et al. / Biochemical and Biophysical Research Communications 295 (2002) 796–799
Fig. 2. Primary structures of maximin H peptides. Identical residues among maximin H peptides are boldfaced. For comparison, the sequence of SAAP as reported in [9] is included. The sequences of maximins H1–H4 are from [10].
structure of clone 321 cDNA is similar to those of the cDNAs encoding other maximins and maximin H peptides. Like previously reported clone 118 cDNA that encodes maximin 3 plus maximin H2, clone 321 cDNA was found to contain a coding region of 435 nucleotides. The encoded amino acid sequence corresponds to a polypeptide of 144 amino acids, which is composed of a signal peptide, an acidic peptide, a maximin 3 peptide, spacer octapeptide, another acidic peptide plus finally a C-terminal novel anionic peptide. As compared in Fig. 2, this novel peptide, possessing an identical N-terminal region, is a homolog of maximin H peptides. The novel anionic peptide was thus designated as maximin H5. Besides several conserved substitutions, maximin H5 is characterized by three remarkable aspartate residue replacements at positions 11, 14, and 15. In addition, two basic residues Lys18 –Lys19 in maximin H2 were replaced by hydrophobic residues Gly18 –Ile19 in maximin H5. Like in the case of maximin H2, the sequence of maximin H5 is preceded by a dibasic site (Lys–Arg), which forms the cleavage site for the releasing of mature maximin H5, and a C-terminal glycine provides the amide for the C-terminal leucineamide of maximin H5. Amphibians have naked skin and show a marked preference for damp habitats that teamup with potentially diverse pathogenic bacteria and fungi. Diverse antimicrobial peptides in B. maxima skin are necessary for its antipathogen potential and wider antipathogen spectrum. As compared in Fig. 1, sequence identity between cDNAs of clones 321 and 118 is more than 95% in the coding regions. Remarkably, the main differences were observed in the maximin H peptide coding sequences, in which 16 nucleotide mutations resulted in 12 amino acid substitutions. All 16 nucleotide mutations are necessary nonsynonymous mutations for encoding novel maximin H5 peptide, indicating a highly positive selection occurring in evolution processes. Recently, it has been revealed that the genes coding for antimicrobial peptides in B. orientalis are composed of two exons. Exon 1 codes for the signal peptide, while exton 2 contains the genetic information for one or two copies of a BLP (maximin-like) plus a bombinin H peptide (maximin H-like). Analysis of the structures of these genes suggests the occurrence of a gene duplication event, which leads to the generation of variability in this class of cytolytic peptides [13,14]. If the gene structures of
antimicrobial peptides in B. maxima were similar to those of B. orientalis, intra-exon shuffling or fragment exchange [15] might also occur in exon 2, which could be an additional way the toad adapted for rapid formation of novel functional genes. A detailed analysis of the gene structures of antimicrobial peptides in Bombina species will certainly help to explain this interesting phenomenon and its biological implication. Antimicrobial activity According to the primary structure of maximin H5 as deduced from its cDNA sequence, the peptide was chemically synthesized in the C-terminal amidated form by a peptide synthesizer and used in antimicrobial assays. Different from other maximins and maximin H peptides that possess strong antimicrobial activity and broad antimicrobial spectrum [10], maximin H5 had a limited antimicrobial spectrum and relative lower antimicrobial potency on tested bacterial and fungal strains (Table 1). Only S. aureus was found to be sensitive to maximin H5, with an MIC of 80 lM, while the other bacterial and fungal strains were resistant to it. No synergetic effect of maximin H5 with maximin 3 was observed when the peptides were co-applied to bacterial culture media. Different from SAAPs, which are zincdependent anionic peptides with bactericidal activity for Pasteurella haemolytica [9], Zn2þ and Mg2þ had no effects on the antimicrobial activity of maximin H5. As expected from the lack of positive charge, maximin H5 did not induce hemolysis of rabbit red cells in a concentration up to 80 lM. Several bombinin H peptides isolated from skin secretions of B. variegate and B. orientalis are known to contain a D -amino acid in the second position, in addition to their corresponding all L -isomers, resulting in distinctive antimicrobial activities [16]. Whether in natural maximin H5 such a post-translational modification occurs remains to be clarified.
Table 1 Antimicrobial activity of maximin H5 MIC (lM) Microorganisms
Maximin H5
Staphylococcus aureus ATCC2592 Bacillus megatherium Bacillus pyocyaneus CMCCB10104 Bacillus dysenteriae Escherichia coli ATCC25922 Klebsiella pneumoniae Candida albicans ATCC2002 Aspergillus flavus IFFI4015
80 – – – – – – –
The data represent mean values ( 25%) of three independent experiments performed in duplicates. (–) no detectable activity.
R. Lai et al. / Biochemical and Biophysical Research Communications 295 (2002) 796–799
Antimicrobial host defense peptides are widely distributed in animals and plants, and are among the most ancient host defense factors. Most of these peptides have cationic properties [1–3] that allow both electrostatic and hydrophobic interactions with the bacterial cytoplasmic membrane, which usually comprises negatively charged phospholipids. However, bacterial pathogens, like S. aureus and Salmonella enterica, have found multiple ways to limit the effectiveness of CAMPs. The countermeasures of bacterial pathogens include reducing the net negative charge of the bacterial cell envelope through covalent modification of anionic molecules (like teichoic acids, phospholipids, and lipid A), expelling CAMPs through energy-dependent pumps, altering membrane fluidity and cleaving CAMPs with proteases [17–20]. Accordingly, the existence of AAMPs, like maximin H5, in B. maxima skin, should contribute importantly to its innate immunity defense system. Coevolution of host–pathogen and/or predator–prey, as have been observed in major histocompatibility complex class I [21], merozoite surface antigen-1 [22], and primate ribonuclease genes [23], may provide evolution forces for the toad to achieve the relevant anionic antimicrobial factors, reflecting the species’ adaptation to the unique microbial environments. Maximin H5 represents the first example of AAMPs from amphibians. Even though their antimicrobial spectrum and action mechanisms have not yet been well established, the results provide evidences that together with CAMPs, AAMPs also exist naturally as part of the innate defense system.
Acknowledgments This work was supported by Grants of ‘‘STZ98-3-01’’, ‘‘Western Light Project,’’ and ‘‘Tenth Five Plan’’ pre-research project from Chinese Academy of Sciences; and grants from National Natural Science Foundation (30170195) and Yunnan Science and Technology Commission (2001C0061M).
References [1] R.M. Epand, H.J. Vogel, Diversity of antimicrobial peptides and their mechanisms of action, Biochim. Biophys. Acta 1462 (1999) 11–28. [2] H.G. Boman, Innate immunity and the normal microflora, Immunol. Rev. 173 (2000) 5–16. [3] M. Zasloff, Antimicrobial peptides of multicellular organisms, Nature 415 (2002) 389–395.
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[4] B.T. Clarke, The natural history of amphibian skin secretions, their normal functioning and potential medical applications, Biol. Rev. 72 (1997) 365–370. [5] D. Barra, M. Simmaco, Amphibian skin: a promising resource for antimicrobial peptides, Trends Biotechnol. 13 (1995) 205–209. [6] P. Nicolas, A. Mor, Peptides as weapons against microorganisms in the chemical defense system of vertebrates, Annu. Rev. Microbiol. 49 (1995) 277–304. [7] M. Simmaco, G. Mignogna, D. Barra, Antimicrobial peptides from amphibian skin: what do they tell us, Biopolymers 47 (1998) 435–450. [8] K. Matsuzaki, Why and how are peptide–lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes, Biochim. Biophys. Acta 1462 (1999) 1–10. [9] K.A. Brogden, A.J. de Lucca, J. Bland, S. Elliott, Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica, Proc. Natl. Acad. Sci. USA 93 (1996) 412–416. [10] R. Lai, Y.T. Zheng, J.H. Shen, G.J. Liu, H. Liu, W.H. Lee, S.Z. Tang, Y. Zhang, Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima, Peptides 23 (2002) 427– 435. [11] R. Lai, H. Liu, W.H. Lee, Y. Zhang, A novel bradykinin-related peptide from skin secretions of toad Bombina maxima and its precursor containing six identical copies of the final product, Biochem. Biophys. Res. Commun. 286 (2001) 259–263. [12] G.S. Bignami, A rapid and sensitive hemolysis neutralization assay for palytoxin, Toxicon 31 (1993) 817–820. [13] R. Miele, D. Ponti, H.G. Boman, D. Barra, M. Simmaco, Molecular cloning of a bombinin gene from Bombina orientalis: detection of NF-jB and NF-IL6 binding sites in its promoter, FEBS Lett. 431 (1998) 23–28. [14] R. Miele, M. Borro, D. Fiocco, D. Barra, M. Simmaco, Sequence of a gene from Bombina orientalis coding for the antimicrobial peptide BLP-7, Peptides 21 (2000) 1681–1686. [15] W. Gilbert, D.J. de Souza, M. Long, Origin of genes, Proc. Natl. Acad. Sci. USA 94 (1997) 7698–7703. [16] M.L. Mangoni, N. Grovale, A. Giorgi, G. Mignogna, M. Simmaco, D. Barra, Structure–function relationships in bombinins H, antimicrobial peptides from Bombina skin secretions, Peptides 21 (2000) 1673–1679. [17] J. Harder, J. Bartels, E. Christophers, J.M. Schroder, A peptide antibiotic from human skin, Nature 387 (1997) 861. [18] R.K. Ernst, T. Guina, S.I. Miller, How intracellular bacteria survive: surface modifications that promote resistance to host innate immune responses, J. Infect. Dis. 179 (1999) S326–S330. [19] A. Peschel, L.V. Collins, Staphylococcal resistance to antimicrobial peptides of mammalian and bacterial origin, Peptides 22 (2001) 1651–1659. [20] A. Peschel, How do bacteria resist human antimicrobial peptides? Trends Microbiol. 10 (2002) 179–186. [21] A.L. Hughes, M. Nei, Pattern of nucleotide substitution at major histocompatibility complex class I loci reveals overdominant selection, Nature 335 (1988) 167–170. [22] A.L. Hughes, Positive selection and interallelic recombination at the merozoite surface antigen-1 (MSA-1) locus of Plasmodium falciparum, Mol. Biol. Evol. 9 (1992) 381–393. [23] J. Zhang, H.F. Rosenberg, M. Nei, Positive Darwinian selection after gene duplication in primate ribonuclease genes, Proc. Natl. Acad. Sci. USA 95 (1998) 3708–3713.
An anionic antimicrobial peptide from toad Bombina maximaq Ren Lai, Hen Liu, Wen Hui Lee, and Yun Zhang* Department of Animal Toxinology, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 East Jiao Chang Road, Kunming, Yunnan 650223, People’s Republic of China Received 10 June 2002
Abstract Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide, named maximin H5, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2 . Containing three aspartate residues and no basic amino acid residues, maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides, only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5, while the other bacterial and fungal strains tested were resistant to it. The presence of metal ions, like Zn2þ and Mg2þ , did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians. The results provide the first evidence that, together with cationic antimicrobial peptides, anionic antimicrobial peptides may also exist naturally as part of the innate defense system. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Anionic antimicrobial peptide; Cationic antimicrobial peptide; Amphibian; Bombina maxima; Maximin H; cDNA clone
In recent years, a large number of antimicrobial peptides have been discovered from animals and plants. These molecules, which are either constitutive or inducible, are recognized as important components of innate defense system [1–3]. Amphibians, being the first group of organisms forming a connecting link between land and water, are forced to adopt and survive in a variety of conditions laden with pathogenic microbes. Thereby, they are endowed with an excellent chemical defense system composed of pharmacological and antimicrobial peptides [4]. A considerable variety of antimicrobial peptides have been characterized from amphibians. Based on their sequence/tridimensional structure characteristics, the microbicidal peptides from amphibian skin can be grouped into three broad families [5–7]. The first one contains linear amphipathic helix forming peptides such q Abbreviations: BLPs, bombinin-like peptides; SAAPs, ovine pulmonary surfactant-associated anionic peptides; AAMPs, anionic antimicrobial peptides; CAMPs, cationic antimicrobial peptides; MIC, minimal inhibitory concentration. The nucleotide sequence data reported in this paper are available from GenBank database with Accession No. AF515614. * Corresponding author. Fax: +86-871-519-1823. E-mail address: [email protected] (Y. Zhang).
as magainins from African clawed frog Xenopus laevis and bombinin-like peptides (BLPs) from Bombina orientalis and B. variegata. The second family has four different groups of related peptides isolated from various species of the Ranidae family, which all contain two cysteine residues at the C-terminal part that form a disulfide bond. The third one includes temporins from Rana temporaria, which are only 10–13 amino acids long. In spite of great primary structure diversity, the majority of antimicrobial peptides documented are now characterized by a preponderance of cationic and hydrophobic amino acids, which are spatially organized in discrete sectors of the molecule. This amphipathic structure allows them to selectively interact with bacterial membranes, which usually comprise negatively charged phospholipids, by the aid of electrostatic interactions [1,8]. Ovine pulmonary surfactant-associated anionic peptides (SAAPs), being 7 amino acids long and comprising 5–7 aspartate residues, is the only example of anionic antimicrobial peptides (AAMPs), showing bactericidal activity on Pasteurella haemolytica in the presence of zinc [9]. In a previous study, two groups of antimicrobial peptides have been isolated from skin secretions of Chi-
0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 6 - 2 9 1 X ( 0 2 ) 0 0 7 6 2 - 3
R. Lai et al. / Biochemical and Biophysical Research Communications 295 (2002) 796–799
nese red belly toad B. maxima. Peptides in the first group, maximins 1–5, are structurally related to BLPs. Peptides in the second group, maximins H1–H4, are homologous with bombinin H peptides [10]. They are all cationic antimicrobial peptides (CAMPs). Here, we reported a gene encoded anionic peptide, named maximin H5, its cDNA cloning, synthesis, and antimicrobial activity.
Materials and methods cDNA cloning of antimicrobial peptides. As previously reported, several full-length clones with cDNA sequences encoding maximins plus maximin H peptides were obtained [10]. In an attempt to screen possible additional clones encoding novel antimicrobial peptides from constructed B. maxima skin cDNA library, a specific oligonucleotide primer Ma (50 -ATGAATTTTAAGTACATA-30 , in the sense direction, corresponding to residues 1–6 of the precursor protein of maximin 3 plus maximin H2), was designed. Primer Ma and a vector SP6 promoter primer (50 -CATACGATTTAGGTGACACTATAG-30 , in the antisense direction) located in 30 of the cloned insert were used in PCR screening of full-length antimicrobial peptide cDNAs as before [10]. All the oligonucleotide primers for PCR were prepared with a DNA synthesizer (Model 381A, Applied Biosystems). DNA sequencing was performed on an ABI PRISM 377 DNA sequencer, Applied Biosystems. Computer analysis of protein and nucleotide acid sequences was performed with the Clustal V sequence software package. Peptide synthesis. Amidated maximin H5 was synthesized by solid phase synthesis on an Applied Biosystems model 433A peptide synthesizer according to manufacturer’s standard protocols. After cleavage and side-chain deprotection, the crude synthetic peptide was purified on a Vydac 218TP510 C18 reverse-phase HPLC column (25 1 cm) eluted at a flowrate of 2 ml/min by a linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water. Identity of the peptide was confirmed by automated Edman degradation with a protein sequencer and mass spectrometry analysis. Fast atom bombardment mass spectrometry was carried out on an Autospec-3000 spectrometer, equipped with a high field magnet [11]. The synthetic peptide was then used for evaluating biological activities. Antimicrobial assays. Standard bacterial and fungal strains used in antimicrobial assays, Gram-positive bacterial strains Staphylococcus aureus (ATCC2592) and Bacillus megatherium, Gram-negative bacterial strains Escherichia coli (ATCC25922), Bacillus pyocyaneus (CMCCB10104), Bacillus dysenteriae, and Klebsiella pneumoniae, and fungal strains Candida albicans (ATCC2002) and Aspergillus flavus (IFFI4015), were obtained from Kunming Medical College. Antimicrobial activity was assayed as described [10]. Minimal inhibitory concentration (MIC) was determined in liquid LB medium by incubating the bacteria in LB broth with variable amounts of the sample tested. The MIC at which no visible growth occurred was recorded. To assay the possible synergetic effect of maximin H5 on maximin 3, maximin H5 and maximin 3 were co-applied to bacteria that are sensitive to maximin 3 and the MICs were then assayed. For assaying the possible influence of metal ions, different concentrations (20 lM– 1 mM) of ZnCl2 and MgCl2 were added to test their effects on antimicrobial activity of maxmin H5. The peptides were quantified by UV absorbance at 215 and 225 nm using the formula: concentration (mg/ml) ¼ ðA215 A225 Þ 0:144. Hemolytic assays. Hemolytic assay was tested with rabbit red cells in liquid medium as reported [12]. Serial dilutions of the peptides were used, and after incubation at 37 °C for 30 min, the cells were centrifuged and the absorbance in the supernatant was measured at 595 nm. Maximum hemolysis was determined by adding 1% Triton X-100 to a sample of cells.
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Results and discussion cDNA Cloning A cDNA library constructed from B. maxima skin was screened at high stringency, as described under ‘‘Materials and methods,’’ by an efficient and rapid PCR-based procedure. More than 60 positive clones, which contain an insert around 650 base pairs, were identified and isolated. Both strands of these clones were sequenced. In addition to previously identified clones that encode previously purified antimicrobial peptides maximins 1–5, and maximins H1–H4 [10], about 30 novel clones were found to encode new antimicrobial peptide precursor proteins. The encoded precursors comprise either a novel maximin with a novel maximin H peptide, or different combinations of these two kinds of peptides (data not shown). Surprisingly, clone 321 contains an insert that encodes a precursor protein comprising maximin 3 and a novel anionic peptide. The complete nucleotide and the deduced amino acid sequences of clone 321 are shown in Fig. 1. The overall
Fig. 1. Complete nucleotide and deduced amino acid sequences of the cDNA encoding maximin 3 plus maximin H5 (clone 321) and comparison with those of the cDNA encoding maximin 3 plus maximin H2 (clone 118) [10]. The nucleotides of the protein coding region are shown by uppercase letters and those of the noncoding region by lowercase letters. The deduced amino acid sequence is shown below. Gaps (-) have been introduced to optimize the sequence homology. Maximin H peptide sequences are boldfaced and italicized. The sequence of maximin 3 is boldfaced. Dots () indicate the same nucleotide and amino acid residues in both cDNAs.
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R. Lai et al. / Biochemical and Biophysical Research Communications 295 (2002) 796–799
Fig. 2. Primary structures of maximin H peptides. Identical residues among maximin H peptides are boldfaced. For comparison, the sequence of SAAP as reported in [9] is included. The sequences of maximins H1–H4 are from [10].
structure of clone 321 cDNA is similar to those of the cDNAs encoding other maximins and maximin H peptides. Like previously reported clone 118 cDNA that encodes maximin 3 plus maximin H2, clone 321 cDNA was found to contain a coding region of 435 nucleotides. The encoded amino acid sequence corresponds to a polypeptide of 144 amino acids, which is composed of a signal peptide, an acidic peptide, a maximin 3 peptide, spacer octapeptide, another acidic peptide plus finally a C-terminal novel anionic peptide. As compared in Fig. 2, this novel peptide, possessing an identical N-terminal region, is a homolog of maximin H peptides. The novel anionic peptide was thus designated as maximin H5. Besides several conserved substitutions, maximin H5 is characterized by three remarkable aspartate residue replacements at positions 11, 14, and 15. In addition, two basic residues Lys18 –Lys19 in maximin H2 were replaced by hydrophobic residues Gly18 –Ile19 in maximin H5. Like in the case of maximin H2, the sequence of maximin H5 is preceded by a dibasic site (Lys–Arg), which forms the cleavage site for the releasing of mature maximin H5, and a C-terminal glycine provides the amide for the C-terminal leucineamide of maximin H5. Amphibians have naked skin and show a marked preference for damp habitats that teamup with potentially diverse pathogenic bacteria and fungi. Diverse antimicrobial peptides in B. maxima skin are necessary for its antipathogen potential and wider antipathogen spectrum. As compared in Fig. 1, sequence identity between cDNAs of clones 321 and 118 is more than 95% in the coding regions. Remarkably, the main differences were observed in the maximin H peptide coding sequences, in which 16 nucleotide mutations resulted in 12 amino acid substitutions. All 16 nucleotide mutations are necessary nonsynonymous mutations for encoding novel maximin H5 peptide, indicating a highly positive selection occurring in evolution processes. Recently, it has been revealed that the genes coding for antimicrobial peptides in B. orientalis are composed of two exons. Exon 1 codes for the signal peptide, while exton 2 contains the genetic information for one or two copies of a BLP (maximin-like) plus a bombinin H peptide (maximin H-like). Analysis of the structures of these genes suggests the occurrence of a gene duplication event, which leads to the generation of variability in this class of cytolytic peptides [13,14]. If the gene structures of
antimicrobial peptides in B. maxima were similar to those of B. orientalis, intra-exon shuffling or fragment exchange [15] might also occur in exon 2, which could be an additional way the toad adapted for rapid formation of novel functional genes. A detailed analysis of the gene structures of antimicrobial peptides in Bombina species will certainly help to explain this interesting phenomenon and its biological implication. Antimicrobial activity According to the primary structure of maximin H5 as deduced from its cDNA sequence, the peptide was chemically synthesized in the C-terminal amidated form by a peptide synthesizer and used in antimicrobial assays. Different from other maximins and maximin H peptides that possess strong antimicrobial activity and broad antimicrobial spectrum [10], maximin H5 had a limited antimicrobial spectrum and relative lower antimicrobial potency on tested bacterial and fungal strains (Table 1). Only S. aureus was found to be sensitive to maximin H5, with an MIC of 80 lM, while the other bacterial and fungal strains were resistant to it. No synergetic effect of maximin H5 with maximin 3 was observed when the peptides were co-applied to bacterial culture media. Different from SAAPs, which are zincdependent anionic peptides with bactericidal activity for Pasteurella haemolytica [9], Zn2þ and Mg2þ had no effects on the antimicrobial activity of maximin H5. As expected from the lack of positive charge, maximin H5 did not induce hemolysis of rabbit red cells in a concentration up to 80 lM. Several bombinin H peptides isolated from skin secretions of B. variegate and B. orientalis are known to contain a D -amino acid in the second position, in addition to their corresponding all L -isomers, resulting in distinctive antimicrobial activities [16]. Whether in natural maximin H5 such a post-translational modification occurs remains to be clarified.
Table 1 Antimicrobial activity of maximin H5 MIC (lM) Microorganisms
Maximin H5
Staphylococcus aureus ATCC2592 Bacillus megatherium Bacillus pyocyaneus CMCCB10104 Bacillus dysenteriae Escherichia coli ATCC25922 Klebsiella pneumoniae Candida albicans ATCC2002 Aspergillus flavus IFFI4015
80 – – – – – – –
The data represent mean values ( 25%) of three independent experiments performed in duplicates. (–) no detectable activity.
R. Lai et al. / Biochemical and Biophysical Research Communications 295 (2002) 796–799
Antimicrobial host defense peptides are widely distributed in animals and plants, and are among the most ancient host defense factors. Most of these peptides have cationic properties [1–3] that allow both electrostatic and hydrophobic interactions with the bacterial cytoplasmic membrane, which usually comprises negatively charged phospholipids. However, bacterial pathogens, like S. aureus and Salmonella enterica, have found multiple ways to limit the effectiveness of CAMPs. The countermeasures of bacterial pathogens include reducing the net negative charge of the bacterial cell envelope through covalent modification of anionic molecules (like teichoic acids, phospholipids, and lipid A), expelling CAMPs through energy-dependent pumps, altering membrane fluidity and cleaving CAMPs with proteases [17–20]. Accordingly, the existence of AAMPs, like maximin H5, in B. maxima skin, should contribute importantly to its innate immunity defense system. Coevolution of host–pathogen and/or predator–prey, as have been observed in major histocompatibility complex class I [21], merozoite surface antigen-1 [22], and primate ribonuclease genes [23], may provide evolution forces for the toad to achieve the relevant anionic antimicrobial factors, reflecting the species’ adaptation to the unique microbial environments. Maximin H5 represents the first example of AAMPs from amphibians. Even though their antimicrobial spectrum and action mechanisms have not yet been well established, the results provide evidences that together with CAMPs, AAMPs also exist naturally as part of the innate defense system.
Acknowledgments This work was supported by Grants of ‘‘STZ98-3-01’’, ‘‘Western Light Project,’’ and ‘‘Tenth Five Plan’’ pre-research project from Chinese Academy of Sciences; and grants from National Natural Science Foundation (30170195) and Yunnan Science and Technology Commission (2001C0061M).
References [1] R.M. Epand, H.J. Vogel, Diversity of antimicrobial peptides and their mechanisms of action, Biochim. Biophys. Acta 1462 (1999) 11–28. [2] H.G. Boman, Innate immunity and the normal microflora, Immunol. Rev. 173 (2000) 5–16. [3] M. Zasloff, Antimicrobial peptides of multicellular organisms, Nature 415 (2002) 389–395.
799
[4] B.T. Clarke, The natural history of amphibian skin secretions, their normal functioning and potential medical applications, Biol. Rev. 72 (1997) 365–370. [5] D. Barra, M. Simmaco, Amphibian skin: a promising resource for antimicrobial peptides, Trends Biotechnol. 13 (1995) 205–209. [6] P. Nicolas, A. Mor, Peptides as weapons against microorganisms in the chemical defense system of vertebrates, Annu. Rev. Microbiol. 49 (1995) 277–304. [7] M. Simmaco, G. Mignogna, D. Barra, Antimicrobial peptides from amphibian skin: what do they tell us, Biopolymers 47 (1998) 435–450. [8] K. Matsuzaki, Why and how are peptide–lipid interactions utilized for self-defense? Magainins and tachyplesins as archetypes, Biochim. Biophys. Acta 1462 (1999) 1–10. [9] K.A. Brogden, A.J. de Lucca, J. Bland, S. Elliott, Isolation of an ovine pulmonary surfactant-associated anionic peptide bactericidal for Pasteurella haemolytica, Proc. Natl. Acad. Sci. USA 93 (1996) 412–416. [10] R. Lai, Y.T. Zheng, J.H. Shen, G.J. Liu, H. Liu, W.H. Lee, S.Z. Tang, Y. Zhang, Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima, Peptides 23 (2002) 427– 435. [11] R. Lai, H. Liu, W.H. Lee, Y. Zhang, A novel bradykinin-related peptide from skin secretions of toad Bombina maxima and its precursor containing six identical copies of the final product, Biochem. Biophys. Res. Commun. 286 (2001) 259–263. [12] G.S. Bignami, A rapid and sensitive hemolysis neutralization assay for palytoxin, Toxicon 31 (1993) 817–820. [13] R. Miele, D. Ponti, H.G. Boman, D. Barra, M. Simmaco, Molecular cloning of a bombinin gene from Bombina orientalis: detection of NF-jB and NF-IL6 binding sites in its promoter, FEBS Lett. 431 (1998) 23–28. [14] R. Miele, M. Borro, D. Fiocco, D. Barra, M. Simmaco, Sequence of a gene from Bombina orientalis coding for the antimicrobial peptide BLP-7, Peptides 21 (2000) 1681–1686. [15] W. Gilbert, D.J. de Souza, M. Long, Origin of genes, Proc. Natl. Acad. Sci. USA 94 (1997) 7698–7703. [16] M.L. Mangoni, N. Grovale, A. Giorgi, G. Mignogna, M. Simmaco, D. Barra, Structure–function relationships in bombinins H, antimicrobial peptides from Bombina skin secretions, Peptides 21 (2000) 1673–1679. [17] J. Harder, J. Bartels, E. Christophers, J.M. Schroder, A peptide antibiotic from human skin, Nature 387 (1997) 861. [18] R.K. Ernst, T. Guina, S.I. Miller, How intracellular bacteria survive: surface modifications that promote resistance to host innate immune responses, J. Infect. Dis. 179 (1999) S326–S330. [19] A. Peschel, L.V. Collins, Staphylococcal resistance to antimicrobial peptides of mammalian and bacterial origin, Peptides 22 (2001) 1651–1659. [20] A. Peschel, How do bacteria resist human antimicrobial peptides? Trends Microbiol. 10 (2002) 179–186. [21] A.L. Hughes, M. Nei, Pattern of nucleotide substitution at major histocompatibility complex class I loci reveals overdominant selection, Nature 335 (1988) 167–170. [22] A.L. Hughes, Positive selection and interallelic recombination at the merozoite surface antigen-1 (MSA-1) locus of Plasmodium falciparum, Mol. Biol. Evol. 9 (1992) 381–393. [23] J. Zhang, H.F. Rosenberg, M. Nei, Positive Darwinian selection after gene duplication in primate ribonuclease genes, Proc. Natl. Acad. Sci. USA 95 (1998) 3708–3713.