Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents

Biochimica et Biophysica Acta 1696 (2004) 1 – 14 www.bba-direct.com

Review

Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents J. Michael Conlon a,*, Jolanta Kolodziejek b, Norbert Nowotny b,c a

Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666 Al-Ain, United Arab Emirates b Clinical Virology Group, Institute of Virology, University of Veterinary Medicine, Vienna, Veterina¨rplatz 1, A-1210 Vienna, Austria c Department of Medical Microbiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666 Al-Ain, United Arab Emirates Received 24 June 2003; received in revised form 2 September 2003; accepted 5 September 2003

Abstract Granular glands in the skins of frogs of the genus Rana, a widely distributed group with over 250 species, synthesize and secrete a remarkably diverse array of peptides with antimicrobial activity that are believed to have arisen as a result of multiple gene duplication events. Almost without exception, these components are hydrophobic, cationic and form an amphipathic a-helix in a membrane-mimetic solvent. The peptides can be grouped into families on the basis of structural similarity. To date, brevinin-1, esculentin-1, esculentin-2, and temporin peptides have been found in ranid frogs of both Eurasian and North American origin; ranalexin, ranatuerin-1, ranatuerin-2 and palustrin peptides only in N. American frogs; and brevinin-2, tigerinin, japonicin, nigrocin and melittin-related peptides only in Eurasian frogs. It is generally assumed that this structurally diversity serves to protect the organism against a wide range of pathogens but convincing evidence in support of this hypothesis is still required. The possibility that ‘‘antimicrobial peptides’’ fulfill additional or alternative biological functions should not be rejected. The molecular heterogeneity of the peptide families, particularly brevinin-1, brevinin-2 and ranatuerin-2, may be exploited for the purposes of unequivocal identification of specimens and for an understanding of phylogenetic interrelationships between species. The broad-spectrum antibacterial and antifungal activities of certain peptides, for example esculentin-1, ranalexin-1 and ranatuerin, together with their relatively low hemolytic activity, make them candidates for development into therapeutically useful antiinfective agents. D 2003 Elsevier B.V. All rights reserved. Keywords: Antimicrobial peptide; Rana; Taxonomy; Phylogeny; Drug development

1. Introduction Frogs belonging to the genus Rana, often referred to as ‘‘true frogs’’, are included in the family Ranidae within the suborder Neobatrachia. These amphibians comprise more than 250 species that are distributed worldwide, except for the Polar Regions, southern South America and most of Australia. At least 36 species are found in North America [1]. The fossil record of the ranids is poor and so evolutionary relationships within the genus are not well understood. However, the ranid frogs are considered to be phylogenetically quite ancient with some species not having shared a common ancestor in over 100 million years [2]. Holarctic and Neotropical ranids are believed to have

* Corresponding author. Tel.: +971-3-7039484; fax: +971-3-767-2033. E-mail address: [email protected] (J.M. Conlon). 1570-9639/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2003.09.004

diverged around 50 million years ago when a land connection between eastern North America and western Eurasia was disrupted [3]. Morphological differences between species are often slight so that taxonomic classification of specimens can be difficult. Unambiguous identification of individuals is especially challenging in regions where several species coexist and produce hybrids so that there is clearly a need for a reliable and noninvasive molecular technique that can be used for this purpose. In common with other anuran species, particularly those belonging to the families Pipidae, Hylidae, Hyperoliidae, and Pseudidae (reviewed in Ref. [4]), the skin secretions of ranid frogs contain peptides with antimicrobial activity. These peptides are stored in granular glands, located mainly in the skin of the dorsal region, which are surrounded by myocytes and innervated by sympathetic fibers [5]. Adrenergic stimulation of myoctes causes compression of the peptide-containing serous cells and discharge of their con-

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tents by a holocrine-like mechanism. As a result, secretions contain not only antimicrobial peptides but also cytosolic components and intact polyadenylated mRNAs encoding the peptides [6]. The structural diversity of the antimicrobial peptides from ranid frogs is quite remarkable with virtually no single peptide from one species being found with an identical amino acid sequence in another. This review discusses the ways in which this molecular heterogeneity may be exploited as an aid to taxonomic and phylogenetic classification and in the design of peptide-based antimicrobial agents.

2. Peptidomic analysis of frog skin secretions In the laboratory (or in the field), skin secretions may be collected under noninvasive conditions that do not appear to cause major discomfort or long-term harm to the animal.

Mild electrical stimulation, typically 10-V DC applied in 1-s pulses at multiple sites on the dorsal region, is generally effective [7]. After stimulation, the secretions are collected by washing the skin surface thoroughly with water and acidified to inhibit the activity of endogenous peptidases. Alternatively, the animal may be injected bilaterally with norepinephrine (2 nmol/g body wt.) and placed in a buffered saline solution for 15 min [8]. The antimicrobial peptides may be isolated from the combined secretions and washings using Sep-Pak C-18 cartridges. Reverse-phase HPLC with on-line monitoring by electrospray mass spectrometry represents a powerful technique for the rapid identification of all the antimicrobial peptides present in the skin secretions of a particular species. The elution profiles are generally quite reproducible. Fig. 1 (panels A and B) shows the chromatography of samples of norepinephrine-stimulated skin secretions from a single specimen of the foothill yellow-legged frog, R.

Fig. 1. Reverse-phase HPLC on a semipreparative Vydac C-18 column of (A) a sample of R. boylii skin secretions, (B) a sample of R. boylii skin secretions collected from the same animal three months later, (C) a sample of R. sphenocephala skin secretions, and (D) a sample of R. sphenocephala skin secretions collected at the same time from a different animal. The masses of the antimicrobial peptides were determined by electrospray mass spectrometry. The dashed line shows the concentration of acetonitrile in the eluting solvent.

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synergistically [14]. An understanding of this molecular complexity is made more difficult by the fact that investigators have referred to peptides that clearly belong to the same family by different names. For example, gaegurin-5 and gaegurin-6 from the Korean frog, R. rugosa [15], ranatuerin-4 from the American bullfrog R. catesbeiana [16], and the histamine-releasing peptides pipinin-I, -II, and -III from the northern leopard frog R. pipiens [17] are clearly members of the brevinin-1 family (Section 3.1). In this review, we have adopted the terminology of Simmaco et al. [18] and have named peptides belonging to a particular family by the initial letter in capitals (or more than one letter in case of ambiguity) of the species to indicate their origin and lower case letters to designate isoforms, e.g. brevinin1Sa and brevinin-1Sb from R. sphenocephala.

boylii, taken on different occasions approximately three months apart [9]. Panels C and D in Fig. 1 show the chromatography of electrically stimulated skin secretions taken at the same time from two different specimens of southern leopard frog, R. sphenocephala [10]. The masses of the antimicrobial peptides, determined by electrospray mass spectrometry, associated with each peak are shown. It is apparent, therefore, that the elution profile and distribution of the molecular masses of the most abundant antimicrobial peptides in skin secretions constitute a characteristic ‘‘fingerprint’’ of a particular species or sub-species of ranid frog that may be used for an unequivocal taxonomic classification. Although furnishing less detailed or precise structural information than techniques utilizing HPLC and electrospray mass spectrometry, MALDI mass spectrometry of a unfractionated sample of skin secretions, after concentration on Sep-Pak cartridges if necessary, provides a rapid method for species identification. Analysis by MALDI generates a molecular mass ‘‘fingerprint’’ that is quite specific for a particular species or sub-species.

3.1. Brevinin-1 Brevinin-1 was first isolated from an extract of the skin of the Japanese pond frog R. brevipoda porsa [19] and members of the family were subsequently purified from a wide range of North American (Table 1) and Eurasian (Table 2) ranid species. Circular dichroism studies have demonstrated that brevinin-1 exists predominantly as a random coil in aqueous solution but adopts an extended a-helical conformation in a membrane-mimetic environment such as 50% trifluoroethanol [20]. As shown in Fig. 2, the amino acid sequence of brevinin-1 has been poorly conserved across species with only four invariant residues (Ala9, Cys18, Lys23, and Cys24). Although not invariant, Pro14 is present in most brevinin-1 peptides and NMR spectroscopy has shown that this residue produces a stable kink in the molecule [21]. It was speculated that this feature might be important in producing transmembrane pores that lead to bacterial cell lysis. Analysis of the known brevinin-1 peptides from North American ranids by the neighbor joining method generates an unrooted phylogenetic tree with three well-defined clades (Fig. 3). The multiple components from the closely related

3. Molecular diversity of the ranid antimicrobial peptides With the exception of the wood frog, R. sylvatica, from whose skin secretions only a single antimicrobial peptide (brevinin-1SY) was isolated [11], the ranid frogs synthesize and secrete multiple active components. For example, the skin secretions of the pickerel frog, R. palustris, contain at least 22 antimicrobial peptides [12] as well as several families of bradykinin-related peptides and peptides of unknown function [13]. On the basis of amino acid sequence similarity, antimicrobial peptides from ranid frogs may be divided into several families with multiple isoforms of a particular class of peptide frequently being identified in a single species. There is evidence that antimicrobial peptides belonging to different structural classes may act

Table 1 Distribution of the molecular forms of the antimicrobial peptides from the skins of N. American frogs Brevinin-1 R. R. R. R. R. R. R. R. R. R. R. R.

catesbeiana grylio clamitans pipiens berlandieri luteiventris boylii sphenocephala sylvatica tarahumara palustris aerolata

Esculentin-1

Esculentin-2

1

5 6 2 3 3 1 3 3 1

1 1 1

2 2

1

Temporin

Ranalexin

Ranatuerin-1

Ranatuerin-2

5 4 5 1

1 1 2

1 2 1

3 1

3 1 2 1 1 2 2

1 1

3 6 2

Palustrin-1

Palustrin-2

Palustrin-3

4

3 1

2 1

The values in the table show the number of peptides belonging to a particular family that are synthesized in that species.

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Table 2 Distribution of the molecular forms of the antimicrobial peptides isolated from the skins of Eurasian ranid frogs

R. R. R. R. R. R. R. R. R.

esculenta temporaria brevipoda porsa ornativentris tagoi rugosa nigromaculata japonica tigerina

Brevinin-1

Brevinin-2

Esculentin-1

Esculentin-2

Temporin

4 1 1

12

3

2

3 10

1

4 1

1

2

1 2 5 1

Japonicin-1

Japonicin-2

Tigerinin

Nigrocin-2

MRP

2 1 1

1 4

The values in the table show the number of peptides belonging to a particular family that are synthesized in that species. MRP, melittin-related peptide.

leopard frogs R. pipiens [22] and R. berlandieri [23] segregate together in different clades, suggesting that these peptides arose from relatively recent gene duplication events within the lineages after the species diverged from each other. Similarly, the peptides from R. boylii [9] and the Columbia spotted frog R. luteiventris [22] segregate together in a single clade which supports the accepted close phylogenetic relationship between these species [23,24]. On the basis of both morphological and molecular criteria, R. sphenocephala is generally classified as being a close relative of R. pipens [23]. Consistent with this, brevinin-1Sc is found in the R. pipiens clade but brevinin-1Sa and -1Sb are found in the R. berlandieri clade, suggesting that the gene may have duplicated in the putative common ancestor of the species. The placement of the wood frog R. sylvatica is controversial with the species being classified with both the eastern North American and Neotropical Rana [3] and with the western North American (R. boylii group) and Palearctic (R. temporaria) Rana [25]. An analysis based upon the brevinin-1 sequences does not support a very close phylogenetic relationship with R. boylii although both species appear on the same branch of the tree. Unexpectedly, R. sylvatica appears as the sister group of the bullfrog R. catesbeiana and it is of interest to note that a phylogenetic tree based upon the amino acid sequence of pancreatic polypeptide also supports this placement [26]. Brevinin-1 peptides exhibit high potency against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi but they are associated with very strong hemolytic activity [9,18,22]. In some cases, e.g. brevinin-1E from R. esculenta, HC50 values (the concentration producing 50% hemolysis) are less than 1 AM [18] severely limiting their potential for therapeutic use. A synthetic replicate of brevinin-1 from R. brevipoda porsa showed antiviral activity by protecting African Green Monkey kidney cells against infection by both herpes simplex virus type 1 and type 2 [27]. Structure – activity studies with brevinin-1 indicate that the disulfide bridge is not necessary for high antimicrobial potency but the linear acetamidomethylcysteinyl analog had appreciably less hemolytic activity that the native peptide [20]. The antiviral activity of brevinin-1 is also retained after reduction and carboxamidomethylation [27]. Similarly,

transposing the C-terminal cyclic heptapeptide domain to the central region of the peptide reduced hemolytic activity without affecting its antibacterial properties [28]. 3.2. Brevinin-2 Brevinin-2, first isolated from an extract of the skin of the Japanese pond frog R. brevipoda porsa [19], has a wide distribution in those species of Asian and European ranid frogs examined to-date (Table 2) but has yet to be identified in a North American species. As shown in Fig. 2, the primary structure of brevinin-2 has been very poorly conserved between species as well as between individual members of the family within a single species. Only four amino acid residues (Lys7, Cys27, Lys28, and Cys33) are invariant in the peptide. Three peptides of the brevinin-2 family were isolated from specimens of R. rugosa collected in Korea that were termed gaegurin 1 –3 [15] whereas two structurally similar, but not identical, peptides that were termed rugosins A and B were isolated from the same species collected in Japan [29]. Assuming that one group of workers had not incorrectly classified the animals, it would appear that different strains of R. rugosa inhabiting different regions synthesize a different array of antimicrobial peptides. The common edible frog R. esculenta Linnaeus 1758 is considered to have a hybrid origin arising from the marsh frog, R. ridibunda Pallas 1771, and the pool frog, R. lessonae Camerano 1882, and so is best regarded as a complex rather than a discrete species [30]. Analysis of electrically stimulated skin secretions from a single specimen of R. esculenta led to the identification of seven members of the brevinin-2 family [18] but a further three members of the family (brevinin-2Ei, brevinin-2Ej and brevinin-2Ek) were identified in a pooled extract of skins from multiple specimens collected in the wild [31] and two additional members (brevinin-2Eg and brevinin-2Eh) were isolated from a pooled extract of gastric tissue from specimens also collected in the wild [32]. As shown in Fig. 4, cladistic analysis of the amino acid sequences of the known brevinin-2 peptides provides insight into phylogenetic relationships among Eurasian ranids. The peptides segregate into three well-defined clades. With the exception of brevinin-2Ei, the brevinins from R. esculenta

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Fig. 2. The primary structures of the known peptides belonging to the most widely distributed families of antimicrobial peptides present in the skin secretions of ranid frogs. The amino acid sequence of the first member of the family to be identified is shown and the amino acid substitutions found in the known isoforms are arranged in order of decreasing hydrophobicity according to [80]. The species from which the peptides have been isolated are shown in Tables 1 and 2. (*) denotes deletion of a residue. The sequence of temporin is a consensus sequence derived by Wade et al. [48].

form a single group suggest that they arose from multiple gene duplications within that lineage. Brevinin-2T from R. temporaria [5,33] is embedded in a clade comprising the brevinins from the Asian frogs R. rugosa and R. ornativentris that is quite distinct from the R. esculenta clade. Although R. esculenta and R. temporaria are both predominantly of European origin, phylogenetic analysis of the

nucleotide sequences of ribosomal DNAs indicates that the R. esculenta complex is an outgroup of other Holarctic and Neotropical Rana families [2], which is consistent with the placement in Fig. 4. Nigrocin-1 from R. nigromaculata (Section 3.12) and brevinin-2 from R. brevipoda porsa form a mini-clade consistent with the known sister group relationship of these Japanese pond frogs [34].

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Fig. 3. An unrooted phylogenetic tree based upon the amino acid sequences of brevinin-1 peptides isolated from North American ranid frogs. Analysis was performed with the PHYLIP (version 3,57c) package. Genetic distances between each pair of sequences were calculated using the PROTDIST program based on the Dayhof Pam matrix. From those distance matrices the phylogenetic trees were generated by the neighbor-joining method of the NEIGHBOR program and the best tree was displayed by the program DRAWTREE.

The brevinin-2 peptides from R. esculenta [31,32] and R. ornativentris [35] tested in the authors’ laboratory showed high potency against Escherichia coli (minimum inhibitory concentration, MIC < 10 AM) but were also active against Staphylococcus aureus and against the fungus Candida albicans. The hemolytic activities of peptides of the brevinin-2 family are much less (>10-fold) than those of the brevinin-1 family [18]. The physiological significance, if any, of the synthesis of brevinin-2 peptides in gastric tissue from R. esculenta is unknown but, in view of the rather low concentrations of peptides present [32], a major role in protecting the gastrointestinal tract from pathogens seems unlikely.

The native peptide exhibits very high potency (MIC < 1 AM) against a range of human pathogens such as E. coli, S. aureus, Pseudomonas aeruginosa and C. albicans [18]. A recombinant linear analog containing the substitutions

3.3. Esculentin-1 The 46-amino-acid-residue peptide esculentin-1 was first isolated from the skin of European frogs belonging to the R. esculenta complex [18] but the peptide has subsequently been identified in skin secretions from the closely related North American species, R. palustris [12] and R. areolata (crawfish frog) [36] (Tables 1 and 2). Compared with the brevinins, the primary structure of esculentin-1 has been relatively well conserved among the seven known members of the family (Fig. 2). Most substitutions involve replacement of an amino acid by one of similar polarity. The biological properties of esculentin-1 make it a promising candidate for drug development (Section 5).

Fig. 4. An unrooted phylogenetic tree based upon the amino acid sequences of brevinin-2 peptides isolated from Eurasian ranid frogs. The tree was constructed as described in the legend to Fig. 3.

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Met28 ! Leu, Cys40 ! Ser and Cys46 ! Ser and bearing an additional C-terminal Met residue retained high antimicrobial potency and also lacked hemolytic activity [37]. In a study that has clear commercial implications, DNA encoding [Leu28]esculentin-1 under the control of an appropriate promoter was introduced in the genome of the tobacco plant, Nicotiana tabacum [38]. The antimicrobial peptide, in its correctly processed form, could be isolated from the leaves of the transgenic plants, which demonstrated enhanced resistance to bacterial and phytopathogens and to insects. 3.4. Esculentin-2 Esculentin-2 was first isolated from the skin of frogs belonging to the R. esculenta complex [18] and, like esculentin-1, has a restricted distribution amongst species (Tables 1 and 2). Gaegurin-4 [15] and rugosin C [29] from the Asian frog, R. rugosa, are also members of the esculentin-2 family. The primary structure of the peptide has been very poorly conserved with only eight amino acid residues invariant amongst the eight members of the family that have been isolated to date (Fig. 2). Esculentin-2 peptides show broad-spectrum antimicrobial activity with high potency against E. coli and S. aureus (MIC < 10 AM) and moderate potency against C. albicans (MIC in the range 30– 50 AM) [18,22]. 3.5. Ranalexin Ranalexin was first isolated from an extract of whole R. catesbeiana tadpoles [39] and its distribution is limited to the closely related North American species R. grylio (pig frog) [40] and R. clamitans (green frog) [41] (Table 1). The primary structure of the peptide has been relatively well conserved across the three species with only conservative substitutions of hydrophobic amino acids (Fig. 2). The organization of the cyclic heptapeptide ring is reminiscent of that of the antibiotic polymyxin, first isolated from the bacteria Bacillus polymyxa, in which the lysine residues are replaced by 2,4-diaminobutyric acid. The disulfide bond does not, however, strongly affect either the conformation or the antimicrobial activity of the peptide [42]. Both the reduced and oxidized forms of ranalexin are mainly unstructured in water but adopt an a-helical conformation in the central region of the molecule in 30% trifluoroethanol. The activity of ranalexin against a wide range of clinical isolates of bacteria has been investigated. The peptide was most active against Gram-positive strains such as methicillin-resistant S. aureus, Staphylococcus epidermidis and Streptococcus pneumoniae but was inactive against some clinically important Gram-negative strains such as P. aeruginosa and Proteus mirabilis [43]. The activity of ranalexin alone against the intestinal parasite Cryptosporidium parvum is relatively low but its effectiveness is considerably enhanced when used in combination with conventional

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antibiotics such as lasalocid and azithromycin [44]. Similarly, studies with an in vivo rat model indicate that a combination of ranalexin and cefazolin is effective in preventing infection of Dacron vascular prosthetic grafts by S. epidermidis [45]. 3.6. Ranatuerin-1 As shown in Table 1, ranatuerin-1 peptides, like the ranalexins, have a limited species distribution occurring only in the skins of three closely related North American bullfrogs R. catesbeiana [16], R. grylio [40] and R. clamitans [41]. Circular dichroism studies have shown that ranatuerin-1, in common with most frog skin antimicrobial peptides, exists as a random coil in aqueous solution but adopts a conformation with a-helical character in 50% trifluoroethanol. The primary structure of the peptide has been relatively well conserved between the four known members of the family with only conservative substitutions of amino acids. Consistent with data obtained with ranalexin [42], replacement of the cysteine residues in ranatuerin-1 by serine has only minor effects on conformation and biological activity but deletion of the cyclic heptapeptide gives an inactive compound. The predicted conformation of the peptide comprises three structural domains: a-helix (residues 1 –8), h-sheet (residues 11 –16) and h-turn (residues 20 – 25). Disruption of the h-sheet by replacement of the glycine residues at positions 10, 13 and 15 by lysine gave analogs with greatly reduced antimicrobial potency (unpublished data). The antimicrobial and hemolytic activities of ranatuerin-1 and its analogs are discussed in Section 5. 3.7. Ranatuerin-2 The ranatuerin-2 family of peptides, first identified in the skin of the bullfrog R. catesbeiana [16], is widely distributed in ranid frogs of North American origin (Table 1) but, as yet, has not been isolated from any Eurasian species. A C-terminal cyclic hexapeptide domain, rather than the more common heptapeptide, characterizes the peptide. Its primary structure has been poorly conserved with several residue deletions and only five amino acids (Gly1, Ala15, Lys22, Cys23, and Cys28) invariant. This variation in amino acid sequence is matched by a correspondingly wide range of antimicrobial potencies. Reported MIC values against E. coli range from 2 AM (ranatuerin-2Cb) to 30 AM (ranatuerin-2ARa) and against S. aureus from 2 AM (ranatuerin2B) to >200 AM (ranatuerin-2ARa). Activity against C. albicans is generally low with ranatuerin-2B being the most active (MIC = 35 AM). The hemolytic activities (HC50 values) of ranatuerin-2 peptides against human erythrocytes are in the range of 35 AM (ranatuerin-2Ga) to >200 AM (ranatuerin-2BYb). As shown in Fig. 5, an unrooted phylogenetic tree, based upon the known sequences of ranatuerin-2 peptides from North American ranids, identifies three well-defined clades.

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Fig. 5. An unrooted phylogenetic tree based upon the amino acid sequences of ranatuerin-2 peptides isolated from North American ranid frogs. The tree was constructed as described in the legend to Fig. 3.

With the exception of ranatuerin-2PLc, the multiple peptides isolated from R. palustris segregate together with ranatuerin-2ARa from the sister taxon R. areolata. Ranatuerin2PLc and ranatuerin-2ARb are localized to a different clade, suggesting that a gene duplication event may have occurred in the putative common ancestor of these species. Consistent with the placement in Fig. 3, the ranatuerins from R. boylii and R. luteventris segregate within the same clade. However, the peptides from the closely related bullfrogs, R. catesbeiana, R. grylio and R. clamitans, do not form a single assemblage nor do the three isoforms isolated from R. catesbeiana occur in the same clade. This suggests that ranatuerin-2 may represent a less reliable phylogentic marker than either brevinin-1 (Fig. 3) or brevinin-2 (Fig. 4). 3.8. Temporin Members of the temporin family, comprising between 10 and 14 amino acid residues, are among the smallest antimicrobial peptides to be found in nature and all known components are C-terminally a-amidated. Temporins were first identified in the skins of the European frogs R. esculenta [46] and R. temporaria [47] but subsequent work has demonstrated that the family is widely distributed in ranid frogs of both N. American and Eurasian origin (Tables 1 and 2). More than 30 members of the family have been identified and the consensus sequence (FLPLIASLLSKLL.NH2) shown in Fig. 2 has been derived by Wade et al. [48] from an analysis of the known sequences. The primary structures of the temporins are highly variable with no residue invariant. Most peptides

contain a single basic residue (Lys or Arg) but temporin-C, -D, and -E from R. temporaria [47], temporin-1Od from R. ornativentris [34], and temporin-1Ja from R. japonica [49] lack this feature. In general, the temporins are active only against Gram-positive bacteria such as S. aureus and Enterococcus faecium with MIC values ranging from 1 AM to >100 AM [47,50]. The exception is temporin L (FVQWFSKFLGRIL.NH2) from R. temporaria which bears a net positive charge of + 3 and is active against clinically relevant Gram-negative strains such as E. coli and P. aeruginosa and against the fungus C. albicans [51]. This peptide is strongly hemolytic and also cytolytic towards several human tumor cell lines. Structure – activity studies with temporin A (FLPLIGRVLSGIL.NH2) have indicated that a hydrophobic Nterminal residue, a positively charged amino acid at position 7, and bulky hydrophobic residues at positions 5 and 12 are important determinants of antibacterial activity. Replacement of the isoleucine residues by leucine resulted in an increase in antimicrobial potency [50]. A positively charged amino acid is not necessary for activity as illustrated by the fact that the MIC of temporin-1Od (FLPLLASLFSGLF.NH2) against S. aureus is 13 AM [34]. The all-D enantiomer of temporin A is equipotent with the native peptide against bacteria, indicating that effects on the cell membrane are mediated through non-chiral interactions [50]. Studies involving the use of liposomes of different composition have suggested that the temporins produce bacterial cell death by formation of transmembrane pores rather than causing a detergent-like disruption of the cell membrane into peptidecoated vesicles [52].

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Temporin-1Gb (SILPTIVSFLSKFL.NH2) and temporin1Gd (FILPLIASFLSK FL.NH2), isolated from R. grylio, elicited concentration-dependent relaxations of preconstricted vascular rings from the rat thoracic aorta with EC50 values of approximately 2 AM but the physiological significance, if any, of the observation is unclear [40]. 3.9. Palustrin family Analysis of electrically stimulated skin secretions from the pickerel frog, R. palustris led to the identification of three families of antimicrobial peptides, termed palustrin-1, -2, and -3, that bore little similarity to previously characterized peptides from ranid frogs (Fig. 6) [12]. As shown in Table 1, palustrin-1 has not been identified in any species other than R. palustris but palustrin-2 and a peptide structurally related to palustrin-3, but containing a cyclic heptapeptide rather than a hexapeptide ring, were isolated from the skin secretion of the sister group species R. areolata [36]. Palustrin-3 contains regions of sequence similarity to esculentin-1 but, unlike the latter peptide, is active only against E. coli and not against S. aureus. Circular dichroism spectra demonstrate that palustrin-3ARa exists as a random coil conformation in 10 mM sodium phosphate buffer solution pH 7.0, but in 50% trifluoroethanol the peptide is associated with appreciable a-helicity (unpublished data).

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identified only in the skin secretions of the Indian frog, R. tigerina (Fig. 6) [53]. Circular dichroism studies in 5 mM HEPES buffer indicate that the tigerinins exist as a mixed population of unordered and h-turn conformations. The tigerinins contain a nine-membered disulfide-bridged ring and replacement of the cysteine residues by either leucine or a-aminoisobutyric acid results in a drastic loss in antimicrobial activity. It was speculated that linearization of the peptide prevented the h-turn formation that is necessary for membrane permeabilization. Increasing cationicity by substitution of a threonine by a lysine residue increased antimicrobial potency and, conversely, decreasing cationicity by replacing the C-terminal CONH2 group by COOH resulted in loss of activity [54]. 3.11. Japonicin-1 and -2 The skin of the Japanese brown frog R. japonica produces two peptides that show little amino acid sequence similarity to previously characterized antimicrobial peptides isolated from ranid frogs (Fig. 6) [49]. Japonicin-2 contains a disulfide-bridged cyclic octapeptide region and adopts a a-helical conformation in 50% trifluoroethanol. The peptide is active against both E. coli and S. aureus. Japonicin-1 contains the more common cyclic heptapeptide domain that shows some structural similarity to the corresponding region of brevinin-2. The peptide is active only against E. coli.

3.10. Tigerinin 3.12. Nigrocin-2 The tigerinins comprise a family of small (11 – 12 amino acids), C-terminally a-amidated peptides with broad spectrum antimicrobial activity that, at this time, have been

Two peptides with antimicrobial activity, termed nigrocin-1 and -2, were isolated from the skin of the Korean

Fig. 6. The primary structures of antimicrobial peptides with a restricted distribution in ranid frogs. The palustrins were isolated from R. palustris [12] and R. areolata [33], the tigerinins from R. tigerina [51], japonicin-1 and -2 from R. japonica [47] and nigrocin-2 from R. nigromaculata [53]. Amino acid substitutions found in the known isoforms are arranged in order of decreasing hydrophobicity according to Ref. [80]. (*) denotes deletion of a residue.

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pond frog R. nigromaculata [55]. Nigrocin-1 is a member of the brevinin-2 family but nigrocin-2 (Fig. 6) shows little structural similarity to other peptides from ranid frogs. In 50% trifluoroethanol solution, nigrocin 2 adopts an amphipathic a-helical conformation spanning residues (3-18). Nigrocin-1 and -2 showed comparable potencies against a range of Gram-positive and Gram-negative bacteria and against C. albicans, but neither peptide was appreciably hemolytic. 3.13. Melittin-related peptides The 26-amino-acid peptide melittin is the main peptide component of the venom of the honeybee, first isolated from Apis mellifera and subsequently from the related species, Apis florea and Apis cerana [56]. Melittin exhibits potent hemolytic and cytolytic actions against mammalian cells and a broad spectrum of antibacterial, antifungal, antiviral, and antiprotozoal properties (reviewed in Ref. [57]). A melittin-related peptide (MRP), displaying 78% sequence identity to melittin from A. florea, was isolated along with a temporin from an extract of the skin of the Japanese frog, Tago’s brown frog R. tagoi [57], and a peptide with lower sequence identity with melittin was isolated from the skin of the European frog R. temporaria [5] (Fig. 7). MRP showed potent broad-spectrum antibacterial and candidacidal activity and was 13-fold less hemolytic than melittin against human erythrocytes. In contrast to the reported anti-viral activity of melittin, MRP, at noncytotoxic concentrations ( V 8 AM), did not protect HeLa cells from cell death produced by infection with human rhinovirus type 2 or herpes simplex virus type 1 [57]. 3.14. Caerulein precursor-related fragments Three structurally related peptides with no sequence similarity with antimicrobial peptides isolated from other species of ranid frogs, which potently and selectively inhibit the growth of E. coli (MIC < 5 AM), were identified in a pooled extract of skins from approximately 100 specimens belonging to the R. esculenta complex [31]. These peptides show limited amino acid sequence similarity to the homologous exon gene products that encode the N-terminal flanking peptides of preprocaerulein, preproxenopsin and preprolevitide [58] and so have been termed caerulein

Fig. 7. A comparison of the primary structures of the melittin-related peptides from the skins of R. tagoi and R. temporaria with melittin from the venom of the honeybees Apis florea and Apis mellifera.

Fig. 8. A comparison of the primary structures of caerulein precursorrelated fragments (CPRF-E) isolated from frogs of the R. esculenta complex with the consensus sequences of the caerulein precursor fragments (CPF), xenopsin precursor fragments (XPF) and levitide precursor fragments (LPF) identified in the skin of Xenopus laevis. Peptide XT-7 was isolated from the skin of Xenopus tropicalis [81]. The shaded residues indicate sequence identity with one or more CPRF-E peptides.

precursor-related fragments (CPRF-Ea, CPRF-Eb and CPRF-Ec) (Fig. 8).

4. Evolutionary relationships The primary structure of the biosynthetic precursors of the ranid antimicrobial peptides may be deduced from the nucleotide sequences of cloned cDNAs in the case of brevinin-1 from R. esculenta [18], R. rugosa (gaegurin-5) [59], and R. pipiens [6], ranatuerin-2P from R. pipiens [6], esculentin-2 from R. esculenta [18] and R. rugosa (gaegurin-4) [59], esculentin-1 from R. esculenta [18], ranalexin from R. catesbeiana [39] and temporin from R. temporaria [47]. The structural organization of these precursors is quite similar, comprising a signal peptide sequence, an N-terminal spacer peptide region containing several aspartic and glutamic acid residues, and the antimicrobial peptide at the Cterminus of the precursor. Despite the fact that the primary structures of antimicrobial peptides from the various families show very little similarity to each other, the amino acid sequences of the signal peptide and acidic pro-region have been remarkably well conserved in the different precursors. Frogs belonging to the Phyllomedusinae subfamily of the family Hylidae also produce in their skins antimicrobial peptides (dermaseptins, phylloxin, and dermatoxin) that have no structural similarity with the ranid peptides, but the signal peptide and pro-region of the precursors of these peptides are structurally quite similar to the corresponding regions of the precursors of the ranid antimicrobial peptides. This observation has led to the speculation that all these antimicrobial peptides arose from a common gene in the ancestor of the Ranidae and Hylidae before their proposed divergence in the Mesozoic [60,61]. The molecular diversity in present-day species is a consequence of multiple duplications of this ancestral gene during radiation of the species, and within individual species. Selective pressure has acted to conserve the N-terminal proregion whereas the C-terminal functional region has evolved rapidly in response to

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exposure to different pathogens [60]. It has been speculated that a mutagenic, error-prone DNA polymerase similar to E. coli Pol V may be involved in this accelerated rate of molecular evolution [61]. At this time, there has been no biological activity reported for the N-terminal flanking peptide. The validity of this hypothesis is dependent upon the frequently made assumption that the hypervariability of the frog skin antimicrobial peptides is a consequence of their biological role in protecting the organism against invasion by a wide range of microbial species [62,63]. Evidence that the peptides produced in the skins of ranid frogs really do fulfill this function is not overwhelming. Studies with model peptides have suggested that any peptide that satisfies the criteria of appropriate hydrophobicity and cationicity and can adopt an amphipathic a-helical conformation will show at least some antimicrobial activity [64]. Hence, one is entitled to question whether the biological role of ‘‘antimicrobial peptides’’ described in this article really is to protect against microorganisms and whether synthesis confers a significant advantage to the animal. Evidence in favor of a protective role is provided by the observation that many of the frog skin antimicrobial peptides isolated from ranid frogs are active in vitro against the pathogens to which the frog may be exposed in the wild. Peptides belonging to the brevinin-1, ranatuerin-1, ranatuerin-2, ranalexin, esculentin-1, esculentin-2, palustrin-3 and temporin families are active against Batrachochytrium dendrobatidis, the chytrid fungus associated with global amphibian declines [65 – 67]. Similarly, esculentin-2P and ranatuerin-2P, isolated from the skin of R. pipiens, have been shown to be active against frog virus 3, a pathogenic iridovirus infecting anurans [68]. It has been reported that R. esculenta specimens kept in a sterile environment produce only very low amounts of antimicrobial peptides but synthesis is induced in response to exposure to natural flora, such as the yeast Candida guiller-mondii [69]. Similarly, indirect evidence for induction of synthesis of brevinin-1SY in R. sylvatica in response to thermally induced bacterial challenge has been obtained [11]. On the other hand, the range of a species such as R. sylvatica overlaps with those of tree frogs such as Hyla versicolor and Pseudacris triseriata and with toads such as Bufo americanus and Scaphiopus holbrooki. All attempts by the authors to detect antimicrobial peptide in the skins of any species of Hyla, Pseudacris, Bufo, and Scaphiopus have been unsuccessful as have attempts to induce synthesis in specimens from these species by exposing them to E. coli and S. aureus (unpublished data). Similarly, species belonging to other genera in the family Ranidae, for example the African bullfrog Pyxicephalus adspersus [70] and the treefrog Rhacophorus schlegeli (unpublished data), do not appear to produce antimicrobial peptides in their skin secretions. In this light, it is unclear just how a great an advantage the synthesis of antimicrobial peptides confers on ranid frogs when several species that are presumably subject

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to very similar environmental challenges appear to survive perfectly well without them. The possibility that ‘‘antimicrobial peptides’’ in ranid frogs may have a biological additional to, or even instead of, defense of the animal should not be ignored.

5. Towards the development of peptide-based therapeutic agents The emergence in recent years of numerous strains of pathogenic microorganisms that have developed resistance to a wide range of formerly efficacious antibiotics has been a major cause of concern not only to the medical profession but also to the general public. A number of recent reviews have addressed the feasibility of using naturally occurring antimicrobial peptides, including those from frog skin, in clinical practice [71 – 73]. On the positive side, because of their relatively nonspecific mechanism of action, the development of resistance to the peptides occurs at rates that are orders of magnitude lower than those observed for conventional antibiotics [74]. The major obstacle to the development of therapeutically useful anti-infectives based upon the Rana skin peptides is their toxicity, particularly if they are to be administered systemically. The relative ability of a peptide to lyse bacterial and mammalian cells is the result of a complex interrelationship of factors involving conformation, charge, hydrophobicity and amphipathicity [75]. Several studies have demonstrated a direct correlation between degree of amphipathicity and hemolytic activity in peptides that form an a-helix [76 – 78]. It has been suggested that increasing the hydrophobic moment of an antimicrobial peptide has a relatively modest effect on the ability to permeabilize the negatively charged cell membrane of microorganisms but a marked effect on the more zwitterionic phospholipid membrane of the erythrocyte [77]. The cationic residues in an antimicrobial peptide are considered to be important in the initial binding to the negatively charged phospholipids in the cell membranes of microorganisms [77,79] and, up to a certain limit, there is a good correlation between peptide cationicity and antimicrobial potency in a range of systems. A promising approach, therefore, to the development of analogs of the Rana skin peptides with increased antimicrobial potency but decreased hemolytic activity is to synthesize peptides with increased cationicity but reduced amphipathicity. For example, ranatuerin-1 shows broad-spectrum antimicrobial activity against a range of human pathogens (e.g. MIC values: E. coli 13 AM; S. aureus 25 AM; P. aeruginosa 20 AM; C. albicans 50 AM) but is moderately hemolytic towards human erythrocytes (HC50 between 120 and 180 AM). Increasing cationicity by the substitution Asn8 ! Lys gave an analog with sixfold increased potency against E. coli and twofold increased potency against both S. aureus and C. albicans with only a very small increase in hemolytic activity. However, increasing hydrophobicity and a-helicity

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by the substitution Asn22 ! Ala increased antimicrobial against these three microorganisms twofold but hemolytic activity increased threefold (unpublished data). Toxicity, the possibility of provoking an immune response, together with the relatively short half-life of the peptides in the circulation suggests that future therapeutic applications of ranatuerin-1 are more likely to involve topical rather than systemic administration.

Acknowledgements Experimental work described in this review was supported by an Interdisciplinary Grant (03/12-8-03-01) and a Faculty Support Grant (NP/03/01) from the United Arab Emirates University.

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