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FEBS 29802 FEBS Letters 579 (2005) 4443–4448

Maximin 9, a novel free thiol containing antimicrobial peptide with antimycoplasma activity from Bombina maximaq

Wen-Hui Lee, Jie Zhang, Ying-Xia Zhang, Yang Jin, Ren Lai, Yun Zhang* Department of Toxinology, Kunming Institute of Zoology, The Chinese Academy of Sciences, 32 East Jiao Chang Road, Kunming, Yunnan 650223, PR China

Received 30 May 2005; revised 20 June 2005; accepted 7 July 2005

Available online 21 July 2005

Edited by Judit Ovadi

ical defense system composed of pharmacological and antimi- Abstract skin is a rich resource of antimicrobial peptides, like maximins and maximin Hs from frog Bombina crobial peptides [4]. A considerable variety of antimicrobial maxima. Novel cDNA clones encoding a precursor protein, peptides have been characterized from . Based on which comprises a novel maximin peptide (maximin 9) and re- their sequence/three-dimensional structure characteristics, the ported maximin H3, were isolated from two constructed skin microbicidal peptides from amphibian skin can be grouped cDNA libraries of B. maxima. The predicted primary structure into three broad families [5–7]. The first one contains linear of maximin 9 is GIGRKFLGGVKTTFRCGVKDFASKHLY- amphipathic helix forming peptides such as magainins from NH2. A surprising substitution is at position 16, with a free cys- African clawed frog Xenopus laevis, bombinin-like peptides teine in maximin 9 rather than usual conserved glycine in other (BLPs) from Bombina orientalis and B. variegata. The second reported maximins. Maximin 9, the homodimer form and its family has four different groups of related peptides isolated Cys16 to Gly16 mutant were synthesized and their antimicrobial from various Ranidae species, which all contain two cysteine activities were evaluated. Unlike previously reported maximin 3, the tested bacterial and fungal strains were resistant to maxi- residues at the C-terminal part that form a disulfide bond. min 9, its homodimer and the Cys16 to Gly16 mutant (with The third one includes temporins from Rana temporaria, which MICs > 100 lM). On the other hand, interestingly, while eight are only 10–13 amino acid residues long. clinical Mollicutes strains were generally resistant to maximin These antimicrobial peptides have been shown to have a 9 homodimer and its Cys16 to Gly16 mutant, most of them are broad antimicrobial spectrum, able to kill most strains of bac- sensitive to maximin 9 at a peptide concentration of 30 lM, teria as well as some fungi, and in addition, many types of tu- especially in the presence of dithiothreitol. These results indicate mor cells. The detailed molecular mechanism of their that the presence of a reactive Cys residue in maximin 9 is antimicrobial activity and selectivity has not been clearly eluci- important for its antimycoplasma activity. The diversity of anti- dated. However, specificity for pathogenic cells is thought to microbial peptide cDNA structures encountered in B. maxima be due to the composition of the cell membranes. An increased skin cDNA libraries and the antimicrobial specificity differences of the peptides may reflect well the speciesÕ adaptation to the un- proportion of anionic phospholipids making the pathogen ique microbial environments. more susceptible and the presence of cholesterol making the 2005 Federation of European Biochemical Societies. Published membranes more resistant to the peptides are reviewed in [8]. by Elsevier B.V. All rights reserved. The Chinese red belly frog (B. maxima) is an endemic amphibian distributed in the mountainous area of south- Keywords: Amphibian; Antimicrobial peptide; cDNA western China. It has long been known by the indigenous sequence; Free thiol; Maximin; Bombina maxima people that the frog lives in very harsh environments like pools with microorganism-rich mud. Our recent studies showed that the skin secretions of B. maxima contain a large number of new antimicrobial peptides. Maximins and maxi- min Hs, which are equivalent to BLPs and bombinin Hs 1. Introduction identified in two other Bombina species, B. orientalis and B. variegata, respectively [9,10]. Particularly, maximin H5 In recent years, a large number of antimicrobial peptides represents the first example of anionic antimicrobial peptides have been characterized from and plants. These mole- from amphibians, providing evidences that together with cat- cules, which are either constitutive or inducible, are recognized ionic antimicrobial peptides, anionic antimicrobial peptides as important components of innate defense system [1–3]. also exist naturally as part of the innate defense system Amphibians, being the first group of organisms forming a con- [10]. Maximin S4, member of a novel group of antimicrobial necting link between land and water, are forced to adopt and peptides of 14–18 amino acid residues long, was found to survive in a variety of conditions laden with pathogenic have antimycoplasma activity but not anti-bacterial or microbes. Therefor, they are endowed with an excellent chem- anti-fungal activities [11]. Here, we reported identification, cDNA cloning, synthesis and antimicrobial activity of a novel maximin (maximin 9) q The nucleotide sequence data reported in this paper is available peptide, which is characterized by a free cysteine in the middle from GenBank database with accession number of AY652770. part of the molecule. With specificity targeting on myco- *Corresponding author. Fax: +86 871 5191823. plasma, maximin 9 might represent the first example of free E-mail address: [email protected] (Y. Zhang). thiol containing antimicrobial peptides from vertebrates.

0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.07.010 4444 W.-H. Lee et al. / FEBS Letters 579 (2005) 4443–4448

2. Materials and methods 2.4. Hemolytic assays Hemolytic assay was tested with human erythrocytes in liquid med- 2.1. Construction and sequencing of a cDNA library ium as reported [9]. Serial dilutions of the peptides with or without Adult specimens of Chinese red belly frog B. maxima were collected in DTT (2 mM, final concentration) were used, and after incubation at Chuxiong County, Yunnan Province, South West of PR China. mRNAs 37 C for 30 min, the cells were centrifuged and the absorbance of were prepared from the total RNA extracted from the skin of a single the supernatant was measured at 595 nm. Maximum hemolysis was individual by oligo(dT) cellulose chromatography with a mRNA purifi- determined by adding 1% Triton X-100 to the tested cells. cation kit (Promega). A directional cDNA library was constructed with plasmid cloning kit (SuperScript Plasmid System, GIBCO/BRL) with some modifications as described previously [9]. Briefly, linkers contain- 3. Results and discussion ing a NotI restriction site and a SalI site were added to size-selected cDNAs. These cDNAs were cloned into the pSPORT1 vector (GIB- CO/BRL) by insertion at the NotI and SalI site of pSPORT1 vector arms 3.1. cDNA cloning and used to transform Escherichia coli HB101 competent cells, produc- Seven hundred and ninety-three clones, which contain an 4 ing a library of about 2.8 · 10 independent colonies. insert larger than 500 bp from a cDNA library constructed In attempt to analyze the expression profile of the frog skin, a strat- egy of random sequencing of the cloned cDNAs was used [12]. Briefly, from B. maxima individual A skin, were randomly picked the library was titered and bacteria were plated out in LB plates con- out and sequenced. Total 92 antimicrobial peptide clones, with taining 100 lg/ml ampicillin. Single colonies were picked out and ana- an insert around 650-bp, were identified and isolated by ran- lyzed by PCR to confirm the presence of an insert sequence and its size dom sequencing. Meanwhile, by PCR-based high stringency (>500 bp), under the following conditions: 2 min at 94 C, followed by screening method [15], thirty B. maxima antimicrobial peptide 30 cycles of 10 s at 92 C, 30 s at 50 C, 40 s at 72 C with a Peltier Ther- clones were screened out and sequenced from another con- mal Cycler (model PTC-200, M.J. Research, USA). A vector T7 primer (50-GTAATACGACTCACTATAGGGCGA-30, in the sense direc- structed individual B skin cDNA library. Both strands of these 0 tion) and a vector SP6 promoter primer (5 -CATACGATTTAGGT- clones were sequenced. In addition to previously identified 0 0 0 GACACTATAG-3 , in the antisense direction) located in 5 and 3 of clones that encode previously purified antimicrobial peptides the cloned insert, respectively, were used in PCR analysis. All the oligo- nucleotide primers for PCR were prepared with a DNA synthesizer maximins 1–5, and maximins H1–H5 [9,10], approximate 30 (Model 381A, Applied Biosystems). For obtained antimicrobial peptide novel clones were found to encode new antimicrobial peptide full-length cDNAs, both strands of the clones were sequenced on an precursor proteins. The encoded precursors comprise either a Applied Biosystems DNA sequencer, model ABI PRISM 377. Com- novel maximin with a novel maximin H peptide, or different puter analysis of protein sequences and nucleotide acid sequences were performed with the Clustal V sequence software package. combinations of these two kinds of peptides. Surprisingly, both clone 252 and clone 91 contain an insert that encodes a precursor protein comprising a novel antimicrobial peptide 2.2. Peptide synthesis Amidated maximin 9, Cys16-Gly16 mutant of maximin 9 and homodi- (designated as maximin 9) and previously reported maximin 0 mer of maximin 9 were synthesized by solid phase synthesis on an H3. The open reading frame and 3 -untranslated region of Applied Biosystems model 433A peptide synthesizer according to the clone 252 and clone 91 are identical, but clone 91 have fewer manufacturerÕs standard protocols. After cleavage and side-chain depro- nucleotides at 50-untranslated region compared with clone tection, the crude synthetic peptide was purified on a Vydac 218TP510 252. The complete nucleotide sequence and the deduced amino C18 reverse phase-HPLC column (25 · 1 cm) eluted at a flow rate of 2 ml/min by a linear gradient of acetonitrile in 0.1% trifluoroacetic acid acid sequence of clone 252 are shown in Fig. 1. The overall in water. Identity of the peptide was confirmed by automated Edman structure of clone 252 cDNA is similar to that of the cDNAs degradation with a protein sequencer and mass spectrometry analysis. encoding other maximins and maximin Hs. Like previously re- Fast atom bombardment mass spectrometry was carried out on an Auto- ported clone 96 cDNA that encodes maximin 4 plus maximin spec-3000 spectrometer, equipped with a high field magnet [9]. The syn- H3, clone 252 cDNA was found to contain a coding region of thetic peptides were then used for evaluating biological activities. The peptides were quantified by UV absorbance at 215 and 225 nm using 435 nucleotides. The encoded amino acid sequence corre- the formula: concentration (mg/ml) = (A215–A225) · 0.144. sponds to a polypeptide of 144 amino acid residues, which is composed of a signal peptide, an acidic peptide, a novel homo- 2.3. Antimicrobial assays logue of maximin peptides, spacer octapeptide, another acidic Microbial strains used in antimicrobial assays, Gram-positive bacte- peptide plus finally a C-terminal maximin H3 peptide. Com- rial strains Staphylococcus aureus (ATCC2592), Gram-negative bacte- paring with maximins 1–5, the deduced amino acid sequence rial strains E. coli (ATCC25922), Bacillus pyocyaneus (CMCCB10104), of the acidic peptide followed signal peptide is: RSXXX- fungal strains Candida albicans (ATCC2002), were obtained from Kunming Medical College. Antimicrobial activity was assayed as de- XXQSLSQRDVLEEESLREIR, the putative C-terminal of scribed previously [9]. Minimal inhibitory concentration (MIC) was maximin 9 is followed by GKR, in which the glycine provides determined in liquid LB medium at pH 7.0 by incubating the bacteria the amide for the C-terminal tyrosineamide of the peptide and in LB broth with variable amounts of the sample tested. The MIC at the dibasic site (Lys-Arg) forms the cleavage site for the releas- which no growth occurred, as measured at absorbance of 600 nm, was recorded. The eight strains of Mollicutes, including two clinic ing of the mature peptide. Thus, amino acid sequence of the strains of Mycoplasma hominis and six clinic strains of Ureaplasma ure- deduced mature maximin 9 is GIGRKFLGGVKTTFRC- alyticum, were obtained from The First PeopleÕs Hospital of Yunnan GVKDFASKHLY-NH2. As compared in Fig. 2, this novel Province. The mollicutes were cultured under anaerobic conditions peptide is a homologue of maximin peptides (sequence identi- at 37 C for 48 h in appropriate liquid media (starting pH 6.0) as de- ties around 26–48% with maximins 1–11). The conserved resi- scribed [13,14]. Inhibitory effects were determined by culturing the cells in 96-well microtitration plates in the presence of 2-fold serial dilutions dues are found at positions 2–3, 11, 19 and 22 compared with of the peptides and following the color change of phenol red resulting other reported maximins 1–11 originated from the same from acidification of the culture medium during growth. The starting sources. Surprisingly, different from all maximins so far identi- 5 cell concentration was 10 colony-forming units (CFU/ml). To analyze fied, maximin 9 is characterized by a single unique cysteine res- the effect of a reducing agent on the activity of maximin 9, 5 mM (final concentration) dithiothreitol (DTT, Sigma) was added to the growth idue at positions 16. In addition, several significant medium. Organisms were considered to be resistant when their growth replacements are observed among maximin 9 and maximins was not prevented by fixed peptide concentrations of 30 lM. 1–11. Maximin 9 differs by amino acid substitutions with W.-H. Lee et al. / FEBS Letters 579 (2005) 4443–4448 4445

Fig. 1. Complete nucleotide sequence and deduced amino acid sequence of the cDNA encoding maximin 9 plus maximin H3 (clone 252), and comparison with those of the cDNA encoding maximin 4 plus maximin H3 (clone 96) [9]. The nucleotides of protein coding region are shown by uppercase letters and those of noncoding region by lowercase letters. The deduced amino acid sequence is shown below. Gaps (–) have been introduced to optimize the sequence homology. Maximin H3 peptide sequences are boldfaced and italicized. The sequences of maximin 9 and maximin 4 are boldfaced. Dots () indicate the same nucleotide residue and amino acid residue in both cDNAs. two phenylalanines at positions 6 and 21. An aromatic residue evolving pathogens [16,17]. As compared in Fig. 1, sequence Tyr/Phe at position 25 in maximins 1–11 is replaced by His25 in identity between cDNAs of clones 252 and 96 is 89.4% in the maximin 9. The well conserved hydrophobic residue at posi- coding regions. Remarkably, the main difference was observed tion 4 among maximins 1–11 is replaced by a basic Arg4 in in the maximin peptide coding sequences, in which 30 nucleo- maximin 9 (Fig. 2). No other cysteine-containing antimicrobial tide mutations resulted in 19 amino acid residue substitutions. peptide precursor was found from the two constructed skin All nucleotide mutations are necessary nonsynonymous muta- cDNA libraries of B. maxima in this study. tions for encoding novel maximin 9 peptide. Among all the It has been reported that in animals, including amphibians, antimicrobial peptides obtained, we found that the mean num- antimicrobial peptides exhibit high levels of variation caused ber of nucleotide substitution per nonsynonymous site in both by selection pressure responding to a diversity of quickly the maximin and maximin H domains significantly exceed the 4446 W.-H. Lee et al. / FEBS Letters 579 (2005) 4443–4448

the growth of B. pyocyaneus and C. albicans in the assay sys- tems. In the presence of DTT, increase of the sensitivities of bacteria S. aureus and E. coli to both maximin 9 and its Cys16-Gly16 mutant might be either caused by the influence of DTT on the growth of the strains or by the enhancing effect of DTT on the sensitivities of the strains to the peptides. Sim- ilar to maximins 1–5, maximin 9, the homodimer and its Cys16- Gly16 mutant did not induce hemolysis of human red cells in the presence or absence of DTT under the tested concentration up to 100 lM. Mycoplasma belong to the class of Mollicutes. These ‘‘soft skin’’ bacteria emerged from the Gram-positive branch of Fig. 2. Primary structures of maximin peptides. Identical residues the eubacterial phylogenetic tree by genome size reduction to among maximin peptides are boldfaced. Significant amino acid such an extent that several mycoplasma species are the smallest substitutions are shaded. The sequences of maximins 1–11 are from [9,10,18]. and the simplest self-replicating organisms [19,20]. Since these bacteria have no cell wall, unlike typical bacteria, their plasma membrane contains cholesterol to stabilize the membrane. The mean number of nucleotide substitution per synonymous site, sensitivities of eight clinical strains of Mollicutes (two of M. whereas the same pattern was not observed in other structural hominis and six of U. urealyticum) to maximin 9, the homodi- regions, such as the signal and propiece peptide regions, sug- mer and its Cys16-Gly16 mutant were tested (see Table 2). gesting that these antimicrobial peptides have been experienc- Interestingly, in our assay conditions, four out of eight clinical ing rapid diversification driven by Darwinian selection [18]. strains were found to be sensitive to maximin 9 with varied sensitivities. Furthermore, the activity was found to be depen- 3.2. Antimicrobial activity dent on a reducing environment. Since two strains of M. hom- According to the deduced primary structure of maximin 9 inis, which were resistant to maximin 9 in the absence of DTT, from its cDNA sequences, the peptide was chemically synthe- were found to become sensitive to the peptide after addition of sized in a C-terminal amidated form by peptide synthesizer and DTT. It should be noted that the oxidation of free thiol by air used in antimicrobial assays. To better evaluate the contribu- is limited in our assay conditions as the test samples and the tion of Cys16 in the antimicrobial activity of the peptide, a culture media were covered with mineral oil to maintain anaer- maximin 9 Cys16-Gly16 mutant was synthesized based on the obic conditions. In addition, in a parallel experiment, the pres- well conserved Gly16 in maximins 1–11. Homodimer of maxi- ence of DTT did not influence the growth of tested min 9 was also synthesized in a C-terminal amidated form and mycoplasma strains. In the cases of maximin 9 homodimer itÕs antimicrobial activity was tested. and its Cys16-Gly16 mutant, the majority of the strains tested The antibacterial activity of maximin 9, its Cys16-Gly16 mu- were resistant to them. This is in accordance with the results tant and homodimer were tested on different bacterial and fun- of magainins, a representative of these linear amphipathic anti- gal strains. Unlike previously reported maximin 3 that microbial peptides from amphibians. When tested the antibi- possesses potent antimicrobial activity and broad antimicro- otic activity of several amphipathic peptides from different bial spectrum on tested strains with MICs within 1–20 lM origins on mycoplasma strains, it was proven that the assayed [9], the tested bacterial and fungal strains were resistant to mycoplasma strains, like M. gallisepticum, M. genitalium are maximin 9, its homodimer and the Cys16-Gly16 mutant (with fully resistant to the magainins, even when used at concentra- MICs > 100 lM) (Table 1). In the presence of DTT, S. aureus tions up to 100 lM [14]. Different from the case of maximin 9, and E. coli were found to be more sensitive to maximin 9 with the presence of DTT did not increase the sensitivities of the observed MICs changed to 100 and 75 lM, respectively. On mycoplasma strains to the Cys16-Gly16 mutant. the other hand, the decreases of MIC values of the Cys16- The experimental results indicate that the presence of a reac- Gly16 mutant on S. aureus and E. coli were also observed. tive Cys residue in maximin 9 do have some effects on its DTT (a reducing agent) alone (2 mM, final concentration) antimycoplasma properties. This is probably due to the inter- influenced the growth of the tested bacteria S. aureus and action of free Cys residues with thiol groups on the outside of E. coli, showing a 20–30% inhibition, while it did not affect bacterial cells, as suggested for the antimicrobial peptides nisin

Table 1 Antimicrobial activity of maximin 9 Microorganisms MIC (lM) Maximin 9 Maximin 9 Maximin 9 Maximin 9 Mutant C16G plus DTT homodimer mutant C16G plus DTT Staphylococcus aureus ATCC2592 R 100 R R 75 Escherichia coli ATCC25922 150 75 >150 150 100 Bacillus pyocyaneus CMCCB10104 R R R R R Candida albicans ATCC2002 R R R R R The data represent mean values of three independent experiments performed in duplicates. R, resistance (growth of the organisms unaffected by peptide concentrations up to 100 lM). MIC > 150 lM: a slowing down of the growth of the organisms was observed at concentrations >100 lM, but the MIC could not be obtained when the peptide dosages used up to 150 lM. W.-H. Lee et al. / FEBS Letters 579 (2005) 4443–4448 4447

Table 2 Antimycoplasma activity of maximin 9 on assayed clinical mycoplasma strains Microorganisms Sensitivities to maximin 9, its derivatives and antibiotics Maximin 9 Maximin 9 Maximin 9 Maximin 9 Mutant C16G ERY LML OFL ROX AZI plus DTT homodimer mutant C16G plus DTT M. hominis Strain 1 R ++ R R R R R R R R Strain 2 R ++ R R R + R + ++ ++

U. urealyticum Strain 1 ++ + R R R R + + ++ + Strain 2 R R R + + + + + + ++ Strain 3 ++ ++ R R R R + + + + Strain 4 ++ ++ R + + + R R ++ ++ Strain 5 + R R R R + R + ++ ++ Strain 6 R ++ + R R R + ++ + + The data represent three parallel experiments. R, resistance (growth of the organisms unaffected in the presence of the peptides or antibiotics); ++, growth of the organisms was totally inhibited; +, a slowing down of the growth of the organisms was observed. ERY, erythromycin; LML, lomefloxacin; OFL, ofloxacin; ROX, roxithromycin; AZI, azithromycin. The peptide and the antibiotic concentrations used in the experiments are fixed at 30 lM and 10 lg/ml, respectively.

Z mutants S5C and M17C, and lactococcin B, which are pro- mer nor maximin 9 homodimer was identified by MALDI- duced by certain Lactococcus lactis strains [21,22]. Lactococcin TOF mass analysis from partially purified antimicrobial frac- B contains naturally one Cys residue and two mutants of nisin tions in our previous experiments [11]. Evidences revealed that Z (S5C and M17C) contain engineered one Cys residue in each bathing B. orientalis with bacteria induced a significant molecule. It was observed that they exhibit antimicrobial activ- increase in the biosynthesis of antibacterial peptides, and the ity when the Cys residue is in the reduced state. Alternatively, expression of the antibacterial peptides in B. orientalis was reg- the existence of a reactive Cys residue in maximin 9 might also ulated by NF-jB and NF-IL6 [28,29]. Moreover, the synthesis facilitate its attack on Mollicutes strains by using cholesterol as of antimicrobial peptides in skin glands of R. esculenta is also a target, as those observed for a widespread group of mem- demonstrated to be induced by the presence of microorganisms brane-damaging thiol-activated toxins, active against mamma- but not under sterile conditions, suggesting that this phenom- lian cells. These toxins all contain a single Cys residue, which enon might be common in amphibians [30]. We have recently facilitates their binding to cholesterol-containing membrane demonstrated that the putative recognition sites of NF-jB [23]. and NF-IL6 in B. maxima skin and liver genomic antimicro- Homodimeric and heterodimeric antimicrobial peptides bial peptide DNAs are identical to those of B. orientalis [18]. linked with one disulfide bond were reported from different Thus, the expression and regulation of antimicrobial peptides species. Dicynthaurin purified from hemocytes of Halocynthia in B. maxima should be similar to that of B. orientalis. aurantium and 11-kDa polypeptide from Guinea pig neutro- Whether the expression of maximin 9 will be enhanced by phils are homodimers [24,25]. Halocidin and distinctin are het- mycoplasma bathing need to be further investigated. erodimers (composed of two similar monomers) naturally The existence of maximin 9 coding cDNAs in two individual existed in hemocytes of H. aurantium and skin secretions of B. maxima skin cDNA libraries should have important contri- tree-frog Phyllomedusa distincta, respectively [26,27]. Haloci- bution in the innate immunity defense system of the frog. din is composed of two peptides of 18 amino acid residues Coevolution of host-pathogen, as have been observed in major and 15 amino acid residues long. Experimental results indi- histocompatibility complex class I [31] and merozoite surface cated that besides purified heterodimer, the 18-amino acid res- antigen-1 [32], may provide evolution forces for the frog to idues monomer and its homodimer also show potent achieve the relevant antimicrobial factors. The diversity of antimicrobial activities on tested strains of S. aureus and Pseu- antimicrobial peptide cDNA structures encountered in B. domonas aeruginosa, and the activities are even stronger than maxima skin cDNA libraries and the antimicrobial specificity those of purified heterodimeric halocidin [26], suggesting the differences of the peptides may reflect well the speciesÕ adapta- natural existence of its monomer and homodimer. Differently, tion to the unique microbial environments. in the case of maximin 9, both monomer and homodimer showed very poor antimicrobial activities on tested bacterial Acknowledgments: We thank two anonymous reviewers for their help- and fungal strains. The significant difference between maximin ful comments on the manuscript. This work was supported by ‘‘Wes- tern Light’’ Projects to Dr. Lee W.H and Dr. Zhang Y from The 9 and its homodimer observed at present study is that maximin Chinese Academy of Sciences; and Grants from National Natural Sci- 9, but not its homodimer, possesses antimycoplasma activity ence Foundation (30170195, 30200044, 30470380) and Yunnan Science that is enhanced in a reducing environment. Thus, maximin and Technology Commission (2003C0066M). 9 might represent a novel class of antimycoplasma peptide, which is free thiol dependent. Amphibians have naked skin and show a marked preference References for damp habitats that teem with potentially diverse patho- genic bacteria and fungi. Diverse antimicrobial peptides in B. [1] Epand, R.M. and Vogel, H.J. (1999) Diversity of antimicrobial peptides and their mechanisms of action. Biochim. Biophys. Acta maxima skin are necessary for its antipathogen potential and 1462, 11–28. wider antipathogen spectrum. Unfortunately, from skin secre- [2] Boman, H.G. (2000) Innate immunity and the normal microflora. tions of 20 individuals of B. maxima, neither maximin 9 mono- Immunol. Rev. 173, 5–16. 4448 W.-H. Lee et al. / FEBS Letters 579 (2005) 4443–4448

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