Variety of Antimicrobial Peptides in the Bombina Maxima Toad and Evidence of Their Rapid Diversification

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Variety of antimicrobial peptides in the Bombina maxima toad and evidence of their rapid diversification

Wen-Hui Lee1, Yan Li2,3, Ren Lai1, Sha Li2,4, Yun Zhang1 and Wen Wang2

1 Department of Animal Toxinology, Kunming Institute of Zoology, The Chinese Academy of Sciences (CAS), Kunming, P. R. China 2 CAS-Max Planck Junior Scientist Group, Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, The Chinese Academy of Sciences (CAS), Kunming, P. R. China 3 Graduate School of the Chinese Academy of Sciences, Beijing, P. R. China 4 Department of Pathology, University of Chicago, Chicago, USA

Antimicrobial peptides secreted by the skin of many amphibians play an important role Received 30/8/04 in innate immunity. From two skin cDNA libraries of two individuals of the Chinese red Revised 4/2/05 belly toad (Bombina maxima), we identified 56 different antimicrobial peptide cDNA Accepted 14/2/05 sequences, each of which encodes a precursor peptide that can give rise to two kinds of [DOI 10.1002/eji.200425615] antimicrobial peptides, maximin and maximin H. Among these cDNA, we found that the mean number of nucleotide substitution per non-synonymous site in both the maximin and maximin H domains significantly exceed the mean number of nucleotide substitution per synonymous site, whereas the same pattern was not observed in other structural regions, such as the signal and propiece peptide regions, suggesting that these antimicrobial peptide genes have been experiencing rapid diversification driven by Darwinian selection. We cloned and sequenced seven genes amplified from skin or liver genomic DNA. These genes have three exons and share the same gene structure, in which both maximin and maximin H are encoded by the third exon. This suggests that alternative splicing and somatic recombination are less likely to play a role in creating Key words: the diversity of maximins and maximin Hs. The gene trees based on different domain Antimicrobial regions revealed that domain shuffling or gene conversion among these genes might peptides Rapid have happened frequently. diversification Bombina maxima

The first two authors contributed equally to this work. Introduction Correspondence: Wen Wang, CAS-Max Planck Junior Scientist Group, Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, The Chinese Academy of It has been widely recognized that antimicrobial Sciences (CAS), Kunming, Yunnan 650223, P. R. China peptides play an important role in protection from Fax: +86-871-5193137 invading microorganisms [1–4]. Antimicrobial peptides e-mail: [email protected] have successfully been isolated from plants [5], or invertebrates [6], vertebrates [7, 8], and humans [9, Yun Zhang, Department of Animal Toxinology, Kunming Institute of Zoology, The Chinese Academy of Sciences (CAS), 10]. In amphibians, antimicrobial peptides are usually Kunming, Yunnan 650223, P. R. China secreted by the dermal glands located in the outer layer Fax: +86-871-5191823 of the skin and they provide an immediate defense e-mail: [email protected] against harmful microorganisms [4, 11]. The Chinese red belly toad (Bombina maxima)isan Abbreviations: p : Proportion of non-synonymous difference N endemic amphibian in the mountainous regions of pS: Proportion of synonymous difference dN: Mean number of nucleotide substitution per non-synonymous site dS: Mean southwestern China [12]. The indigenous people report number of nucleotide substitution per synonymous site that the toad lives in very harsh environments such as UTR: Untranslated region

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pools with microorganism-rich mud and that its skin is observation. While these studies shed insight into very “toxic”. In a preliminary test of biological activity, understanding of the evolution of antimicrobial peptides including antimicrobial, hemolytic and cytotoxic assays, in distinct species, data from closely related species or on the skin secretions of more than ten amphibian intraspecies antimicrobial peptide gene families might species, we found that the secretions of B. maxima have show whether alternative splicing and somatic recom- the strongest biological activity (Zhang, unpublished bination are involved in the creation of peptide diversity, observations). Our recent preliminary studies showed how these genes respond to different environments, and that the skin secretions of B. maxima contain many whether these genes are under unusually high selective different antimicrobial peptides [13, 14]. These anti- pressure. Here we describe of as many as 40 different microbial peptides could be grouped into two families, antimicrobial peptide genes expressed in a single B. maximin and maximin H, which are, respectively, maxima individual. Gene structure analysis did not equivalent to the bombinin and bombinin H identified provide evidence for alternative splicing and somatic in two other Bombina species, B. orientalis and B. recombination. Evolutionary analyses on these genes variegata [15–17]. The gene encoding bombinin and disclosed unusually high non-synonymous substitution bombinin H comprises two exons separated by an rates in the mature antimicrobial peptide regions of approximately 1-kb intron (Fig. 1A) [18, 19]. Exon 1 these genes after gene duplication. Structural and codes for the signal peptide, and exon 2 for bombinin, phylogenetic analyses suggest that domain shuffling bombinin H, and other structural peptides including a or gene conversion play an important role in the spacer and two acidic propieces; the latter have a evolution of this gene family. negative charge that serves to balance the positive charge in the mature antimicrobial peptides and thus prevent cytotoxicity prior to cleavage. We wanted to Results know (a) whether the antimicrobial peptides in B. maxima genes have a similar structure to that of Various antimicrobial peptides are found in bombinin and bombinin H, (b) how rich and diverse B. maxima the antimicrobial peptides are in B. maxima, and (c) how the genes encoding these peptides evolve. In an attempt to analyze the expression profile of the As for the evolution of antimicrobial peptides, it has skin of B. maxima, we randomly sequenced 793 cDNA been reported that, in animals (including amphibians), clones from the library constructed with mRNA antimicrobial peptides exhibit high levels of variation in extracted from the skin of one toad (specimen A). Of response to the selective pressure of quickly evolving these, 92 clones were found to be antimicrobial peptide pathogens [20, 21]. For example, Duda et al. [21] cDNA, on the basis of similarity to the maximin peptides, observed high non-synonymous substitutions among and were then fully sequenced. The sequences show antimicrobial peptides isolated from several distinct frog high level of similarity to the cDNA of bombinin and species (three hylid frogs and four ranid frogs), and bombinin H from B. orientalis and B. variegata [15, 16]. adaptive evolution was inferred to account for this Each of the cDNA codes for a precursor consisting of a

Fig. 1. Schematic gene structure of antimicrobial peptides from B. orientalis and B. maxima. Boxes with diag- onal lines are exons (E1, E2, E3), the dotted box is a putative promoter (P), and solid lines are introns. (A) Gene structure of bombinin peptides from B. orientalis. This gene contains two exons (E1 and E2) and a putative promoter in front of the exon E1 [18, 19]. (B) Gene structure of the maximin peptides from B. maxima. The corresponding region of the putative promoter proposed in the previously study [18, 19] is indicated in front of

the exon E2. Sd and Nd: the number of synonymous and non-synonymous differences compared with consen- sus sequence. *p<0.05 with Z-test.

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signal peptide, two acidic propiece peptides, an 8-amino 12 putative antimicrobial peptides are thus newly acid spacer, and two antimicrobial peptides, maximin identified. (27 amino acids) and maximin H (20 amino acids) To investigate variations in the maximin and (Fig. 1B). The 92 sequences can be grouped into 40 maximin H peptides between individuals, we screened independent cDNA sequences. In spite of the identity of a cDNA library constructed from the skin of B. maxima the coding regions of the mature antimicrobial peptides specimen B by a rapid PCR-based procedure, and among some clones (such as between clone 62 and clone sequenced 30 positive clones. Each of the 30 clones 488), these cDNA differ from each other in UTR or contains an insert encoding a maximin and maximin H structural regions. These 40 cDNA could encode 9 precursor, from which 16 independent cDNA were different mature maximin peptides (maximin 1–9), and obtained (Table 2). The cDNA structures are similar to 13 different maximin H peptides (maximin H1–13) those obtained in specimen A. These 16 cDNA encode 6 (Table 1). We have previously isolated maximin 1–5 and different maximin peptides, and 9 different maximin H maximin H1–5 from skin secretions [13, 14]. The other peptides. Maximin 2–5, and maximin H1, 3, 4, 5, 8, 11

Table 1. List of the 40 cDNA identified of the B. maxima specimen A and the number of clones showing peptide-specific cDNA sequences in the 92 sequenced clonesa)

a) Identical amino acids in the maximin and maximin H peptides are shown in bold.

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Table 2. List of the 16 cDNA identified in the skin cDNA library in second individual of B. maxima and the number of clones showing peptide-specific cDNA sequences in the 30 sequenced clones.

from specimen B are the same as those in the specimen differed from each other by base variations. Unexpect- A, whereas maximin 10, 11, and maximin H14, 15 and 16 edly, all these genes comprise three exons. Exon 1 are putative new peptides with a few amino acid contains most of the 50 UTR sequence. Exon 2 contains differences from others. the remainder of the 50 UTR, the sequence encoding the signal peptide, and several residues of the acidic Gene structure of maximin and maximin propiece 1. Exon 3 encodes the remainder of acidic H peptides propiece 1, one copy of maximin, spacer peptide, acidic

To reveal the gene structure of these antimicrobial peptide-encoding cDNA, we first performed Southern hybridization of genomic DNA from B. maxima specimen A. The Southern hybridization patterns using three different restriction enzymes displayed many bands (Fig. 2A), suggesting that the antimicrobial peptide genes exist as a multiple copy gene family. PCR-based cloning of genomic DNA was subsequently carried out on DNA samples extracted from both specimens A (skin) and B (skin and liver). Two specific primers located in the 50 and 30 UTR regions that are conserved among all cloned antimicrobial peptide cDNA were used to amplify the antimicrobial peptide genes. The amplifications of three DNA samples resulted in one similar 2.4-kb band Fig. 2. (A) A Southern blot shows that antimicrobial peptide (Fig. 2B). We fully sequenced seven clones: two from the genes exist as a multiple copy gene family. The left lane shows band amplified from skin DNA from specimen A, two a ladder of molecular weight markers. The other three lanes show genomic DNA digested with Taq I, Xba I, and Hae III, from the skin of specimen B and three from the liver of respectively. (B) Results of genomic DNA PCR amplifications specimen B. All the genes sequenced had the same using primers located in the two UTR regions. Lane 1: specimen structure (Fig. 1B), although the seven sequences A skin, lane 2: specimen B skin, lane 3: specimen B liver.

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propiece 2, one copy of maximin H, and the 30 UTR (Fig. 1B). The similarity of matched regions among these genes and genes from B. orientalis (AJ298827) is over 85%.

Rapid diversification in the antimicrobial peptide domains

To look at the diversification patterns of this antimicrobial peptide family in B. maxima, we first analyzed Fig. 3. Amino acid structures of the maximin (A) and maximin sequence variations in the 40 cDNA from the first H (B). The a-helix region is marked as “h” under the sites, “c” individual (specimen A) because gene diversity is better indicates random coil regions. Amino acids marked with starts are the sites under positive selection, which have dN/dS ratios represented in this data set. Across the 40 cDNA (x) significantly larger than 1. sequences, we observed more non-synonymous substitutions than expected by neutrality. The ratios of the mean number of nucleotide substitution per non- ceeded synonymous substitutions in 103 pairs for synonymous site to that per synonymous site (dN/dS) maximin and 153 pairs for maximin H, whereas none for the maximin and maximin H regions are 1.774 and of the comparisons was significant for the remaining 3.567, respectively, both of which are significantly regions (Table 3). If we add the 16 cDNA obtained from higher than the neutral expectation in a test for the second individual (specimen B) into the analysis, the detecting selection (Z-test, Table 3). In contrast, the results remain significant (data not shown). ratios for structural regions 1 and 2 are only 0.708 and On the basis of the substitution patterns in the 0.394. These results suggest that the two maximin obtained 40 sequences and using the PAML software regions experienced strong Darwinian positive selec- [24], we narrowed down the sites that have been under tion. positive selection in the maximin and maximin H Because the sequences of the four tested regions, peptides. In Fig. 3, the sites under positive selection especially the mature peptide regions, are short, the use are marked with stars. of dN and dS may be misleading [22, 23]. We therefore also used the proportion of non-synonymous difference The distribution of sites under positive (pN) to the proportion of synonymous difference (pS)in selection in the secondary structures of the the statistical test (Table 3). The pN still significantly antimicrobial peptides exceed pS. Another way to avoid the problem of a small sample is to conduct a Fisher's exact test on sequence The active form of antimicrobial peptides is mainly a a- pairs. In the 780 possible comparisons, we observed that helix with two short random coils at the N and C termini the non-synonymous substitutions significantly ex- [17, 25, 26]. Like the structures found in other

Table 3. Summary of statistics on substitution patterns among maximin and maximin H antimicrobial peptides of the first individual of B. maximaa)

a) All: the number of total pair-wise comparisons. *p<0.05.

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antimicrobial peptides, the simulation result showed tree to tree (Fig. 5). For example, the four trees based on that both the secondary structures of maximin and the four peptide regions for the cDNA clones 401, 158, maximin H consist of a a-helix in the middle and random 569, 88, 62, 129, 96, and 488 position these genes at coils at the N and C termini. Subsequently, we mapped very different branches, suggesting that the four peptide all the sites that have been under positive selection onto regions have evolved independently. the predicted secondary structures of maximin and maximin H (Fig. 3A, B). Interestingly, almost all the selected (variable) sites in the maximins are located Discussion along the hydrophobic side of the a-helix, whereas three very conservative lysines (positions 11, 15, 19) are In this study, we were able to identify as many as 40 located on the other side. In contrast, in the maximin H, different antimicrobial peptide cDNA in one B. maxima almost all sites under positive selection are located along individual (specimen A). In addition, 16 distinct the hydrophilic side. antimicrobial peptide cDNA were obtained in the second individual (specimen B) from a rough screening. Both History of the gene family individuals were randomly selected from two animal batches collected at different times at the same location, We noticed that in different genes, a certain maximin and showed a similar antimicrobial peptide expression sequence could link with different maximin H se- profile with a rich diversity of both mRNA and peptides. quences, and, on the other hand, a certain maximin H The major maximin and maximin H peptides, like sequence could also link with different maximins maximin 2, 3, 4, 5, and maximin H1, 3, 4, 5, 8, 11 are (Fig. 4). For example, maximin 3 was observed to be found in both individuals. The presence or absence of linked with five different downstream maximin H some peptide members, like maximin 1, between two (maximin H2, 3, 5, 9, and 11), and maximin H3 to be individuals might be caused by either limitation of cDNA linked with three different upstream maximins (max- data from the specimen B or polymorphism between imin 3, 4, and 9). To resolve evolutionary relationships each other. The rich diversity of the peptides identified among these genes, we constructed neighbor-joining from toad mRNA is consistent with the biochemical data. gene trees for the maximin, maximin H, structural Maximin 1–5 and maximin H1–5 have been purified region 1, and structural region 2, respectively. The from the skin secretions of B. maxima [13, 14]. When the results showed that the clustering relationships among antimicrobial peptide fractions from pooled skin secre- these cDNA clones disclosed by the four trees vary from tions of 20 B. maxima individuals (including specimen A) were analyzed by MALDI-TOF mass spectrometry, the molecules corresponding to maximin 1–8, 10 and maximin H4–10, 12 were well defined (Lee et al., unpublished observations), providing evidence that these peptides are effectively produced at the surface of the skin. To test whether there is positive Darwinian selection underlying the rich diversity of antimicrobial peptides in B. maxima, we analyzed the obtained cDNA in three ways: (1) overall synonymous and non-synonymous substitution patterns, (2) substitution patterns between gene pairs, and (3) particular sites possibly subject to selection. The results strongly suggest that the antimicrobial peptides in B. maxima have been subject to positive selection. First, at the level of overall compar-

ison, all the ratios of dN/dS and pN/pS for the maximin and maximin H regions are significantly larger than 1 in the Z-tests (p<0.05). In contrast, these values for the two structural regions are both smaller than 1 (Table 3). Second, with Fisher's exact test for pair-wise compar- Fig. 4. Linkage patterns observed between maximin and isons, the non-synonymous substitutions significantly maximin H regions in the 40 cDNA derived from the specimen exceed synonymous ones in a large number of pairs for A. Notably, maximin 3 is linked with five different downstream maximins H (maximin H2, 3, 5, 9, and 11), whereas maximin H3 maximin (103) and maximin H (153), respectively. is linked with three different upstream maximins (maximin 3, However, none of the pairs between the structural 4, and 9). propieces is significant. Finally, analysis on substitution

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Fig. 5. Gene trees of different regions of the antimicrobial peptide genes constructed with the neighbor-joining (NJ) method with cDNA from specimen A. (A) Maximin region, (B) maximin H region, (C) structural region 1, (D) structural region 2 (see Fig. 1 for structural region 1 and 2). The numbers along the right side indicate the codes of the cDNA clones (see Table 1).

patterns using maximum likelihood method in the PAML this study, we found that the sites subject to positive suggests that in maximins and maximin Hs, there are selection in maximin and maximin H are distributed on indeed a number of sites affected by positive selection different sides of the a-helix of the secondary structures. (Fig. 3). All this is consistent with the conclusion that The amino acid distribution difference may account for amino acid changes in the maximin and maximin H the difference in selectivity to pathogens observed domains have been subject to strong positive selection. between these two families of antimicrobial peptides The distribution of the sites affected by positive [13, 14]. It may also imply that these two peptides adopt selection may tell us something about the process of very different mechanisms to interact with membranes adaptive evolution and pathogen selectivity of the of microorganisms and hence in fighting microorgan- antimicrobial peptides. Generally, when antimicrobial isms: the distribution of variable sites in maximins may peptides fight microorganisms they first have to attach be able to diversify their pathogen selectivity, while the to the acidic bacterial membranes with their cationic distribution pattern in maximin Hs may change their residues. After having attached to bacteria, either by attachment to the membranes. carpet-like or barrel-stave mechanisms, they perturb It has been reported that the genes encoding the bacterial membranes [27, 28]. During this process, the bombinin and bombinin H in another Bombina species, hydrophilic and hydrophobic parts of antimicrobial B. orientalis, (Fig.1A) comprise two exons separated by a peptides could play important roles in the specific 1–2-kb intron [18, 19]: exon 1 codes for the signal recognition of different targets on the surface or in the peptide, and exon 2 for a bombinin, a bombinin H, and core of lipid bilayer of microbial membranes [27, 28]. In other structural peptides including two acidic propieces

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and a spacer. Our results suggest that the gene structure Materials and methods of antimicrobial peptides from B. maxima (Fig. 1B) is different from the reported one. On the basis of the high Construction and sequencing of skin cDNA libraries level of similarity between these two genomic se- Adult specimens of the B. maxima were collected in Chuxiong quences, we suspect that the first exon was missed in County, Yunnan Province of China. Two batches were collected the previous study probably due to incomplete cDNA in the summer of 1999 and 2002, respectively. From each sequence. We compared the upstream sequences in front batch, one individual was randomly selected designated as of the signal peptide region between the genes from the specimen A (collected in 1999) and specimen B (collected in two species, and high similarity was found in this region, 2002). mRNA was prepared from the total RNA extracted from including those putative promoter recognition sites for the skin of a single individual of B. maxima by oligo(dT) nuclear factors previously proposed for the genes of B. cellulose chromatography with an mRNA purification kit orientalis [18, 19]. These sites could either have been (Promega). Initiated with about 3 lg mRNA, a directional falsely identified, or different genes have different cDNA library was constructed with a plasmid cloning kit TM promoters. Further analysis on the B. orientalis genes (SuperScript Plasmid System, Gibco/BRL) following the will help to clarify this uncertainty. manufacturer's instructions. Briefly, linkers containing a Not I As with other species of genus Bombina (B. orientalis restriction site and a Sal I site were added to size-selected cDNA. These cDNA were cloned into the pSPORT1 vector and B. variegata, 2n=24), the karyotype of B. maxima is (Gibco/BRL) by insertion between the Not I and Sal I site of diploid (2n=28) [29, 30]. Therefore, we would only pSPORT1 vector arms and were then used to transform E. coli have observed, at most, two copies in one individual if HB101 competent cells. Two libraries with about 2.8104 and the diversity had simply resulted from allelism. How- 3.5104 independent colonies were obtained from the skin of ever, the rich diversity of cDNA, the variations in the specimen A and B, respectively. seven cloned genomic DNA sequences, and the Southern In the case of specimen A, the cloned cDNA was randomly hybridization patterns we observed in this study suggest sequenced. Those clones containing typical 50 ends of full- that the antimicrobial peptide genes belong to a multi- length maximin peptide cDNA were selected and both strands gene family. Gene duplication has been thought to be an of the clones were then sequenced. In the case of specimen B, a important mechanism in creating new genes [31, 32], PCR-based method for high stringency screening of cDNA and we have recently demonstrated that many genes can libraries was used for screening and isolating the antimicrobial peptide clones as described [36]. Two specific oligonucleotide be generated by gene duplications in a very short primers located in highly conserved regions of antimicrobial evolutionary time [33]. It is conceivable that gene 0 0 peptide coding cDNA, M1 (5 -AGATGAATTTTAAGTACATA-3 , duplication is an efficient way to create these maximin in the sense direction), corresponding to residues 1–6 of the genes. It is interesting that all these genes have similar signal peptide, and M2R (50-TGAAAGCAAAGTTTTTGGTT-30, structures, regardless of the source of the cDNA: liver, in the anti-sense direction), in the 30 UTR, were used in PCR skin, or from different individuals. This result suggests reactions under the following conditions: 2 min at 94C, that alternative splicing and somatic recombination are followed by 30 cycles of 10 s at 92C, 30 s at 50C, 40 s at less likely to account for the diversity of the anti- 72C. All the oligonucleotide primers for PCR were prepared microbial peptides. with a DNA synthesizer (Model 381A, Applied Biosystems). The shuffling/linkage patterns between different DNA sequencing was performed on an Applied Biosystems maximins and maximin H in precursors suggest that DNA sequencer, model ABI PRISM 377. different regions in these antimicrobial peptide genes Southern hybridization and PCR-based cloning of may have different evolutionary history. The inconstant genomic DNA phylogenetic relationships among the genes in the four gene trees (Fig. 5) suggest that domain shuffling or gene Total DNA was extracted from the liver of one B. maxima conversion among these genes has frequently happened individual. The DNA was digested with Taq I, Xba I, and Hae III, as discovered in the MHC loci [34, 35]. Moreover, the respectively. Subsequent Southern blotting was carried out

synonymous substitution rate (dS, assumed to be the following the standard procedure [37]. The nylon membrane neutral rate) in the maximin region is about 0.134, but with the transferred digested DNA was hybridized with a this values for maximin H and the two structural regions digoxigin-labeled (Roche Inc.) probe, which was made by are 0.047, 0.036 and 0.080, respectively (Table 3). This using one of the cDNA clone as template. 0 0 pattern suggests that the extant maximin sequences Primers located in both the 5 and 3 UTR conserved regions were used to amplify the genomic sequence to determine gene have diverged much earlier than those of maximin H and structures. The resulting products were cloned using TOP- the structural regions, probably due to domain shuffling cloning kit (Invitrogen). The inserts were then sequenced or gene conversion among the different regions in these using the vector primers and subsequently designed walking genes. primers.

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