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Antimicrobial peptides from diverse families isolated from the skin of the Asian frog, Rana grahami
peptides 27 (2006) 2111–2117
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Antimicrobial peptides from diverse families isolated from the skin of the Asian frog, Rana grahami J. Michael Conlon a,*, Nadia Al-Ghaferi a, Bency Abraham a, Hu Jiansheng b, Pascal Cosette c,d, Je´roˆme Leprince c,e, Thierry Jouenne c,d, Hubert Vaudry c,e a
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates b School of Life Science, Yunnan University, Kunming, Yunnan Province, China c European Institute for Peptide Research, CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France d CNRS UMR-6522, CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France e INSERM U-413, CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France
article info
abstract
Article history:
Seven peptides with antimicrobial activity were isolated in pure form from an extract of the
Received 15 February 2006
skin of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. The peptides were
Received in revised form
identified as belonging to the nigrocin-2 (three peptides), brevinin-1 (one peptide), brevi-
4 March 2006
nin-2
Accepted 7 March 2006
(GLFGKILGVGKKVLCGLSGMC) containing three lysine residues, represented the peptide
Published on line 18 April 2006
with highest potency against microorganisms (MIC = 3 mM against Escherichia coli, 12.5 mM
Keywords:
activity against human erythrocytes (LD50 = 40 mM). In contrast, nigrocin-2GRa (GLLSGIL-
Antimicrobial peptides
GAGKHIVCGLSGLC) and nigrocin-2GRc (GLLSGILGAGKNIVCGLSGLC), with only a single
Frog skin
lysine residue, showed weak antimicrobial and hemolytic activity. Phylogenetic relation-
Brevinin
ships among Eurasian ranid frogs are less well understood than those of North American
Esculentin
ranids but the primary structures of the R. grahami antimicrobial peptides suggest a close
(three
peptides),
and
esculentin-1
(one
peptide)
families.
Nigrocin-2GRb
against Staphylococcus aureus and 50 mM against Candida albicans) and the greatest hemolytic
relationship of this species with the Japanese pond frogs R. nigromaculata and R. porosa
Nigrocin
brevipoda. # 2006 Elsevier Inc. All rights reserved.
1.
Introduction
Molecular techniques of phylogenetic analysis, particularly the comparison of nucleotide sequences of orthologous genes, are becoming increasingly important for an understanding of the evolutionary history of amphibia [13]. The elucidation of phylogenetic relationships among frogs belonging to the genus Rana (Neobatrachia, Ranidae), often referred to as ‘‘true frogs’’, poses a particularly challenging problem for the taxonomist. Ranid frogs represent a diverse group comprising
more than 250 species that are distributed worldwide, except for the Polar Regions, southern South America and most of Australia [12]. Morphological differences between species are often slight and the fossil record is poor so that ‘‘classical’’ techniques based upon morphological analyses alone are generally unable to provide unambiguous resolution of issues relating to phylogenetic placement. Peptides with broad-spectrum antibacterial and antifungal activities are synthesized in the skins of most, but not all, species of ranid frogs and represent a component of the
* Corresponding author. Tel.: +791 3 7137484; fax: +791 3 7672033. E-mail address: [email protected] (J.M. Conlon). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.03.002
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peptides 27 (2006) 2111–2117
animal’s system of innate immunity that defends the animal against invasion by pathogenic microorganisms [6,23,25]. On the basis of common structural features, these antimicrobial peptides may be grouped together in families that share a common evolutionary origin but the variation in amino acid sequences of homologous peptides is considerable [4,6]. Virtually no single peptide from one species is found with an identical amino acid sequence in another and there is an extremely wide variation in antimicrobial potency and specificity for different microorganisms. Consequently, determination of the primary structures of these peptides can be used to complement morphological and other types of molecular analysis to provide valuable insight into phylogenetic relationships. The New World is home to about one-quarter of known species of ranid frogs and they are regarded as belonging to a monophyletic group [14]. As a result of a series of comprehensive analyses based upon comparisons of nucleotide sequences from the mitochondrial genome, their evolutionary history is becoming much better understood. In particular, the molecular systematics of the Rana catesbiana species group (Aquarana) [2], Rana boylii group (Amerana) [17], Rana pipiens complex (Pantherana) [14], and the Mexican leopard frogs of the Rana berlandieri group [32] have been studied in detail. The molecular systematics of the Eurasian ranids has not been as extensively investigated. Phylogenetic relationships based upon mitochondrial gene sequences have been assessed for the Japanese brown frogs (R. dybowskii, R. japonica, R. okinavana, R. ornativentris, R. pirica, R. sakuraii, R. tagoi, and R. tsushimensis) [29], Japanese pond frogs (R. nigromaculata, R. porosa porosa, and R. p. brevipoda) [28], and for selected pond frogs distributed in Eurasia [27]. Previous work from the author’s laboratory has focussed upon a systemic study of the primary structures and biological properties of antimicrobial peptides from Japanese brown frogs [5,7–9,15,16]. We now extend this work to investigate the structures and properties of the antimicrobial peptides present in an extract of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. This species, also known as the diskless-fingered odorous frog, is found in evergreen forests in mountainous regions of southern Shanxi, Sichuan, Guizhou, and Yunnan, China and in Vietnam. According to the classification system of Dubois [11], R. grahami is placed in the Hylarana group.
2.
Materials and methods
2.1.
Tissue collection and extraction
Adult male specimens of the Yunnanfu Kunming frog Rana grahami (n = 3; length 6.6–7.3 cm, body weight 28–31 g) were collected under permit in summer, 2005 in Guandu county, Kunming, Yunnan province, China. The animals were anesthetized by immersion in ice–water and sacrificed by pithing. Skin was immediately removed and freeze-dried for shipment to UAE. University. The dried tissue (1.41 g) was extracted by homogenization in ethanol/0.7 M HCl (3:1 v/v; 100 mL) at 0 8C using a Waring blender. The homogenate was
stirred for 2 h at 0 8C and centrifuged (4000 g for 30 min at 4 8C). Ethanol was removed from the supernatant under reduced pressure and, after further centrifugation (4000 g for 30 min at 4 8C), the extract was pumped onto 4 Sep-Pak C18 cartridges (Waters Associates) connected in series at a flow rate of 2 mL/min. Bound material was eluted with acetonitrile/ water/trifluoroacetic acid (70.0:29.9:0.1, v/v/v) and freezedried.
2.2.
Antimicrobial and hemolytic assays
Purification of the peptides was monitored by incubating lyophilized aliquots of chromatographic effluent in Mueller– Hinton broth (50 mL) with an inoculum (50 mL of 106 colony forming units per milliliter) from a log-phase culture of reference strains Staphylococcus aureus (NCTC 8325) and Escherichia coli (ATCC 25922) in 96-well microtiter cell-culture plates for 18 h at 37 8C in a humidified atmosphere of air. Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 h at 35 8C using an inoculum (50 mL) of 5 104 colony forming units per milliliter. After incubation, the absorbance at 630 nm of each well was determined using a microtiter plate reader. In order to monitor the validity and reproducibility of the assays, incubations with bacteria were carried out in parallel with increasing concentrations of bacitracin and incubations with C. albicans in parallel with amphotericin B. Minimum inhibitory concentration (MIC) was measured by standard microdilution methods [21,22] and was taken as the lowest concentration of peptide where no visible growth was observed. Peptides in the concentration range 15–500 mM were incubated with washed human erythrocytes (2 107 cells) from a healthy donor in Dulbecco’s phosphate-buffered saline, pH 7.4 (100 mL) for 1 h at 37 8C. After centrifugation (12,000 g for 15 s), the absorbance at 450 nm of the supernatant was measured. A parallel incubation in the presence of 1% (v/v) Tween-20 was carried out to determine the absorbance associated with 100% hemolysis. The LC50 value was taken as the mean concentration of peptide producing 50% hemolysis in three independent experiments.
2.3.
Peptide purification
The skin extract, after partial purification on Sep-Pak cartridges, was redissolved in 0.1% (v/v) trifluoroacetic acid/ water (2 mL) and injected onto a (2.2 cm 25 cm) Vydac 218TP1022 (C-18) reverse-phase HPLC column (Separations Group) equilibrated with 0.1% (v/v) trifluoroacetic acid/water at a flow rate of 6.0 mL/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min and to 63% (v/v) over 60 min using linear gradients. Absorbance was monitored at 214 and 280 nm, and fractions (1 min) were collected. The abilities of freeze-dried aliquots (50 mL) of the fractions to inhibit the growth of S. aureus and E. coli were determined as described in the previous section. Fractions with antimicrobial activity were successively chromatographed on a (1 cm 25 cm) Vydac 214TP510 (C-4) column and a (1 cm 25 cm) Vydac 219TP510 (phenyl) column. The concentration of acetonitrile in the eluting solvent was raised from 21 to 56% over 50 min and the flow rate was 2.0 mL/min.
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peptides 27 (2006) 2111–2117
2.4.
Structural characterization
The primary structures of the peptides were determined by automated Edman degradation using an Applied Biosystems model 494 Procise sequenator. MALDI-TOF mass spectrometry was carried out using a Voyager DE-PRO instrument (Applied Biosystems) that was operated in reflector mode with delayed extraction and the accelerating voltage in the ion source was 20 kV. The instrument was calibrated with peptides of known molecular mass in the 2000–4000 Da range. The accuracy of mass determinations was 0.02%.
3.
Results
3.1.
Purification of the peptides
The skin extract from R. grahami, after concentration and partial purification on Sep-Pak C-18 cartridges, was chromatographed on a Vydac C-18 semipreparative reversephase HPLC column (Fig. 1). Aliquots of the fractions were tested for their ability to inhibit growth of Gram-negative bacteria, E. coli and Gram-positive bacteria, S. aureus. Antimicrobial activity was associated with the well-defined peaks designated 1–6. Subsequent structural analysis demonstrated that peak 1 contained esculentin-1GRa, peak 2 contained brevinin-2GRa, peak 3 contained brevinin-2GRb, peak 4 contained brevinin-2GRc and brevinin-1GRa, peak 5 contained nigrocin-2GRa and nigrocin-2GRb, and peak 6 contained nigrocin-2GRc. Under the conditions of assay, peaks 2 and 6 showed growth inhibitory activity against E. coli only. The antimicrobial peptides were purified to near homogeneity, as assessed by a symmetrical peak shape and mass spectrometry, by further chromatography on Vydac C-4 and Vydac phenyl columns. The methodology is illustrated by separation of nigrocin-2GRa and nigrocin-2GRb
Table 1 – Minimum inhibitory concentrations (mM) against microorganisms and concentrations producing 50% hemolysis of human erythrocytes (mM) of the endogenous peptides isolated from an extract of the skin of Rana grahami Peptide Esculentin-1GRa Brevinin-2GRa Brevinin-2GRb Brevinin-1GRa Brevinin-2GRc Nigrocin-2GRa Nigrocin-2GRb Nigrocin-2GRc
S. aureus
6 12.5 6 25 12.5 25 3 50
12.5 50 25 12.5 50 >100 12.5 >100
C. albicans
LC50
>50 >100 >100 ND >100 >100 50 >100
210 140 180 ND 100 295 40 >500
on a Vydac C-4 column (Fig. 2A) and final purification of each component on a Vydac phenyl column (Fig. 2B and C). The final yields of purified peptides (nmol) were esculentin-1GRa 590, brevinin-2GRa 570, brevinin-2GRb 510, brevinin-2GRc 480, brevinin-1GRa 55, nigrocin-2GRa 950, nigrocin-2GRb 110, and nigrocin-2GRc 520.
3.2.
Structural characterization
The primary structures of the antimicrobial peptides isolated from R. grahami were established by automated Edman degradation and their amino acid sequences are shown in Fig. 3. MALDI-TOF mass spectrometry was used to confirm the proposed sequences and to demonstrate the presence of a disulfide bridge in the esculentin-1, brevinin-1, brevinin-2, and nigrocin-2 peptides (Fig. 3).
3.3.
Antimicrobial and hemolytic activities
The abilities of the endogenous peptides isolated from R. grahami skin to inhibit the growth of S. aureus, E. coli, and C. albicans, and to lyse human erythrocytes are quantified in Table 1.
4.
Fig. 1 – Reverse-phase HPLC on a preparative Vydac C-18 column of an extract of skin Rana grahami. Peptides in the peaks designated 1–6 were associated with antimicrobial activity and were subjected to further purification. The dashed line shows the concentration of acetonitrile in the eluting solvent.
E. coli
Discussion
Mainland China is home to a large number of species belonging to the genus Rana [19], many of which are severely threatened because of the rapid pace of urbanization. The present study has led to the isolation from an extract of the skin of the Chinese frog R. grahami of seven peptides with antimicrobial activity whose primary structures indicate that they belong to four previously described families—nigrocin-1, brevinin-1, brevinin-2, and esculentin-1. A comparison of their amino acid sequences with orthologs from other Asian ranids provides some insight into the phylogenetic placement of this species. Nigrocin-2 was first isolated from the skin of the pond frog R. nigromaculata [24]. The peptide shows little structural similarity to other antimicrobial peptides but, in common with most antimicrobial peptides from ranid frogs, nigrocin-2 lacks stable secondary structure in aqueous solution but adopts an amphipathic a-helical conformation in a membrane-mimetic solvent such as 50% trifluoroethanol. Prior to
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peptides 27 (2006) 2111–2117
Fig. 2 – Separation of nigrocin-2GRa (peak 1) and nigrocin-2GRb (peak 2) on a Vydac C-4 column (Panel A), and purification to near homogeneity on a Vydac phenyl column of nigrocin-2GRa (Panel B) and nigrocin-2GRb (Panel C). The arrowheads show where peak collection began and ended.
the present study, nigrocin-2 had not been detected in the skin of any other ranid frog investigated and so the isolation of orthologs with a high degree of amino acid sequence similarity (Fig. 4) from R. grahami provides good evidence for a close
phylogenetic relationship between this species and R. nigromaculata. Structure–activity relationships within the nigrocin-2 molecule have never been investigated. Nigrocin-2GRb shows
Fig. 3 – Amino acid sequences, observed molecular masses (Mr (obs)), and calculated molecular masses (Mr (calc)) of the antimicrobial peptides isolated from an extract of the skin of R. grahami.
peptides 27 (2006) 2111–2117
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Fig. 4 – A comparison of the primary structures of peptides belonging to the nigrocin-2, brevinin-2, and brevinin-1 families. Nigrocin-1 and -2 are from R. nigromaculata [24], brevinin-1 and -2 are from R. porosa brevipoda [20], peptides designated -GR are from R. grahami (this study), -PR are from R. pirica [8], -TS are from R. tsuchimensis [5], and -OK are from R. okinavana [7]. Percent amino acid sequence identities are shown. The shaded residues denote conserved domains in the peptides. In order to maximize structural similarity, residue deletions denoted by (*) have been introduced in some sequences.
greatest structural similarity to nigrocin-2 and, as shown in Table 1, this peptide, like nigrocin-2 [24], shows relatively high potency against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria and against the opportunistic yeast pathogen C. albicans. In contrast, nigrocin-2GRa and -2CRc show much lower potency against E. coli and are inactive (MIC > 100 mM) against S. aureus and C. albicans. The antimicrobial activities of a peptide against bacteria and fungi are determined by a complex interaction between cationicity, hydrophobicity, a-helicity, and amphipathicity [31]. The bacterial cytoplasmic cell membrane is rich in acidic (phospho) lipids whereas the plasma membrane of mammalian cells contains a much higher proportion of zwitterionic phosphatidyl-choline and sphingomyelin phospholipids. An
increase in peptide cationicity, therefore, should promote interaction with the more negatively charged bacterial cell membrane and increase antimicrobial potency. The positive charge on the peptide is also believed to facilitate interaction with, and passage across, the bacterial cell wall, both in the case of Gram-negative bacteria that contain negatively charged lipopolysaccharides and of Gram-positive bacteria that contain negatively charged teichoic and teichuronic acids [31]. In these peptides, the conserved Lys5 residue in nigrocin-2 and nigrocin-2GRb is replaced by Gly and the Lys12 residue is replaced by His (nigrocin-2GRa) and Asn (nigrocin-2GRc). It is proposed that this decrease in cationicity is primarily responsible for the observed decrease in antimicrobial potency.
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peptides 27 (2006) 2111–2117
Fig. 5 – A comparison of the primary structures of peptides belonging to the esculentin-1 family. Esculentin-1 is from R. esculenta [26]; peptides designated -PL are from R. palustris [3] and -AR are from R. aerolata [1].
As well as decreased antimicrobial potency, nigrocin-2GRa and nigrocin-2GRc showed appreciably reduced hemolytic activity against human erythrocytes (Table 1). Studies with model compounds have demonstrated a direct correlation between degree of amphipathicity and hemolytic activity in peptides that form an a-helix [10,30]. It is probable, therefore, that the decrease in amphipathic produced by replacing polar residues on the hydrophilic face of the a-helix by neutral amino acids is responsible for the decrease in hemolytic activity. The conclusion that R. grahami is evolutionarily related to R. nigromaculata is supported by comparison of the amino acid sequences of the brevinin-2 peptides (Fig. 4). Although termed nigrocin-1 [24], this peptide is clearly a member of the brevinin-2 family (Fig. 4). Amino acid sequence similarity between nigrocin-1 and brevinin-2GRa and -GRb is relatively high and the three peptides share a four-residue deletion. By way of comparison, the sequences of orthologs with lower sequence similarity from the Japanese brown frog, R. pirica [8] are included in Fig. 4. Brevinin-2, first isolated from an extract of the skin of the Japanese pond frog R. porosa brevipoda [20], has a wide distribution in those species of Asian and European ranid frogs examined to-date but has yet to be identified in a North American species [6]. In general, 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 three amino acid residues (Cys27, Lys28, and Cys33) are invariant in the peptide. Brevinin-2GRa and GRb also share a high degree of sequence similarity with brevinin-2 suggesting a close phylogenetic relationship of R. grahami with R. porosa brevipoda. Brevinin-1, also first isolated from the skin of R. porosa brevipoda [20], is amongst the most widely distributed of all the antimicrobial peptides from ranid frogs and has been identified in the skins of numerous Eurasian and North American species [6]. The amino acid sequence of the peptide has been very poorly conserved across species with no residue invariant. Brevinin-1GRa like brevinin-1 contains 24 amino acid residues and sequence similarity between the two peptides is relatively high (71%) (Fig. 4). However, several molecular variants of brevinin-1 have been isolated, particular from the skins of the Japanese brown frogs (Fig. 5), that contain amino acid deletions and lack the C-terminal disulfide-bridged cyclic domain [5,7,8]. The 46 amino acid residue peptide esculentin-1 has been isolated from the skin of frogs belonging to the R. esculenta complex from Europe [26] and North Africa [18] and also from North American species belonging to the Nenirana group (R.
palustris (pickerel frog) [3] and R. areolata (crawfish frog) [1]). Identification of an ortholog in the skin of an Asian frog demonstrates that esculentin-1 has a widespread, although sporadic, distribution among ranid frogs. As shown in Fig. 5, evolutionary pressure has acted primarily to conserve the polar residues in the molecule (Lys, Arg, Glu, Asp) although hydrophobic amino acids are generally replaced by one of similar properties. Consistent with previous data [1,3,26], esculentin-1GRa shows growth-inhibitory activity against both Gram-negative and Gram-positive bacteria and is relatively non-hemolytic. After this study was completed, a report [33] appeared describing the isolation of two peptides identical in structure to nigrocin-2GRa and nigrocin-2GRc from R. grahami skin secretions. Regretably, these peptides were described as grahamin 1 and grahamin 2 despite the fact that the authors were aware that the peptides belong to the nigrocin-2 family. It is essential that workers in the field adopt a consistent terminology otherwise the study of complex array of Rana antimicrobial peptides will degenerate into chaos.
Acknowledgments This work was supported by an Individual Research Grant (0103-8-11/06) and a Faculty Support Grant (NP/06/02) from the United Arab Emirates University.
references
[1] Ali MF, Lips KR, Knoop FC, Fritzsch B, Miller C, Conlon JM. Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata. Biochim Biophys Acta 2002;1601:55–63. [2] Austin JD, Lougheed SC, Moler PE, Boag PT. Phylogenetics, zoogeography, and the role of dispersal and vicariance in the evolution of the Rana catesbeiana (Anura: Ranidae) species group. Biol J Linnean Soc 2003;80:601–24. [3] Basir YJ, Knoop FC, Dulka J, Conlon JM. Multiple antimicrobial peptides and peptides related to bradykinin and neuromedin N isolated from the skin secretions of the North American pickerel frog, Rana palustris. Biochim Biophys Acta 2000;1543:95–105. [4] Conlon JM. The therapeutic potential of antimicrobial peptides from frog skin. Rev Med Micro 2004;15:17–25. [5] Conlon JM, Al-Ghaferi N, Abraham B, Sonnevend A, Coquet L, Leprince J, et al. Antimicrobial peptides from the skin of the Tsushima brown frog Rana tsushimensis. Comp Biochem Physiol 2006;143:42–9.
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[6] Conlon JM, Kolodziejek J, Nowotny N. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim Biophys Acta 2004;1696:1–14. [7] Conlon JM, Sonnevend A, Jouenne T, Coquet L, Cosquer D, Vaudry H, et al. A family of acyclic brevinin-1 peptides from the skin of the Ryukyu brown frog Rana okinavana. Peptides 2005;26:185–90. [8] Conlon JM, Sonnevend A, Patel M, Al-Dhaheri K, Nielsen PF, Kolodziejek J, et al. A family of brevinin-2 peptides with potent activity against Pseudomonas aeruginosa from the skin of the Hokkaido frog, Rana pirica. Regul Pept 2004;118:135–41. [9] Conlon JM, Sonnevend A, Patel M, Vijayasarathy C, Nowotny N, Zilahi E, et al. A melittin-related peptide from the skin of the Japanese frog, Rana tagoi with antimicrobial and cytolytic properties. Biochem Biophys Res Commun 2003;306:496–500. [10] Dathe M, Wieprecht T, Nikolenko H, Handel L, Maloy WL, MacDonald DL, et al. Hydrophobicity, hydrophobic moment and angle subtended by charged residues modulate antibacterial and haemolytic activity of amphipathic helical peptides. FEBS Lett 1997;403:208–12. [11] Dubois A. Notes sur la classification des Ranidae (Amphibiens Anoures). Bull Soc Linne´enne Lyon 1992;61:305–52. [12] Duellman WE, Trueb L. Biology of amphibians Baltimore and London: The Johns Hopkins University Press; 1994. [13] Hillis DM, Ammerman LK, Dixon MT, de Sa RO. Ribosomal DNA and the phylogeny of frogs. Herpetol Monogr 1993;7:118–31. [14] Hillis DM, Wilcox TP. Phylogeny of the New World true frogs (Rana). Mol Phylogenet Evol 2005;34:299–314. [15] Isaacson T, Soto A, Iwamuro S, Knoop FC, Conlon JM. Antimicrobial peptides with atypical structural features from the skin of the Japanese brown frog Rana japonica. Peptides 2002;23:419–25. [16] Kim JB, Iwamuro S, Knoop FC, Conlon JM. Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris. J Peptide Res 2001;58:349–56. [17] Macey JR, Strasburg JL, Brisson JA, Vredenburg VT, Jennings M, Larson A. Molecular phylogenies of western North American frogs of the Rana boylii species group. Mol Phylogenet Evol 2001;19:131–43. [18] Marenah L, Flatt PR, Orr DF, Shaw C, Abdel-Wahab YH. Skin secretions of Rana saharica frogs reveal antimicrobial peptides esculentins-1 and -1B and brevinins-1E and -2EC with novel insulin releasing activity. J Endocrinol 2006;188:1–9. [19] Matsui M, Ota H. On Chinese herpetology. Herpetologica 1995;51:234–50.
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/peptides
Antimicrobial peptides from diverse families isolated from the skin of the Asian frog, Rana grahami J. Michael Conlon a,*, Nadia Al-Ghaferi a, Bency Abraham a, Hu Jiansheng b, Pascal Cosette c,d, Je´roˆme Leprince c,e, Thierry Jouenne c,d, Hubert Vaudry c,e a
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 17666 Al-Ain, United Arab Emirates b School of Life Science, Yunnan University, Kunming, Yunnan Province, China c European Institute for Peptide Research, CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France d CNRS UMR-6522, CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France e INSERM U-413, CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France
article info
abstract
Article history:
Seven peptides with antimicrobial activity were isolated in pure form from an extract of the
Received 15 February 2006
skin of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. The peptides were
Received in revised form
identified as belonging to the nigrocin-2 (three peptides), brevinin-1 (one peptide), brevi-
4 March 2006
nin-2
Accepted 7 March 2006
(GLFGKILGVGKKVLCGLSGMC) containing three lysine residues, represented the peptide
Published on line 18 April 2006
with highest potency against microorganisms (MIC = 3 mM against Escherichia coli, 12.5 mM
Keywords:
activity against human erythrocytes (LD50 = 40 mM). In contrast, nigrocin-2GRa (GLLSGIL-
Antimicrobial peptides
GAGKHIVCGLSGLC) and nigrocin-2GRc (GLLSGILGAGKNIVCGLSGLC), with only a single
Frog skin
lysine residue, showed weak antimicrobial and hemolytic activity. Phylogenetic relation-
Brevinin
ships among Eurasian ranid frogs are less well understood than those of North American
Esculentin
ranids but the primary structures of the R. grahami antimicrobial peptides suggest a close
(three
peptides),
and
esculentin-1
(one
peptide)
families.
Nigrocin-2GRb
against Staphylococcus aureus and 50 mM against Candida albicans) and the greatest hemolytic
relationship of this species with the Japanese pond frogs R. nigromaculata and R. porosa
Nigrocin
brevipoda. # 2006 Elsevier Inc. All rights reserved.
1.
Introduction
Molecular techniques of phylogenetic analysis, particularly the comparison of nucleotide sequences of orthologous genes, are becoming increasingly important for an understanding of the evolutionary history of amphibia [13]. The elucidation of phylogenetic relationships among frogs belonging to the genus Rana (Neobatrachia, Ranidae), often referred to as ‘‘true frogs’’, poses a particularly challenging problem for the taxonomist. Ranid frogs represent a diverse group comprising
more than 250 species that are distributed worldwide, except for the Polar Regions, southern South America and most of Australia [12]. Morphological differences between species are often slight and the fossil record is poor so that ‘‘classical’’ techniques based upon morphological analyses alone are generally unable to provide unambiguous resolution of issues relating to phylogenetic placement. Peptides with broad-spectrum antibacterial and antifungal activities are synthesized in the skins of most, but not all, species of ranid frogs and represent a component of the
* Corresponding author. Tel.: +791 3 7137484; fax: +791 3 7672033. E-mail address: [email protected] (J.M. Conlon). 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.03.002
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animal’s system of innate immunity that defends the animal against invasion by pathogenic microorganisms [6,23,25]. On the basis of common structural features, these antimicrobial peptides may be grouped together in families that share a common evolutionary origin but the variation in amino acid sequences of homologous peptides is considerable [4,6]. Virtually no single peptide from one species is found with an identical amino acid sequence in another and there is an extremely wide variation in antimicrobial potency and specificity for different microorganisms. Consequently, determination of the primary structures of these peptides can be used to complement morphological and other types of molecular analysis to provide valuable insight into phylogenetic relationships. The New World is home to about one-quarter of known species of ranid frogs and they are regarded as belonging to a monophyletic group [14]. As a result of a series of comprehensive analyses based upon comparisons of nucleotide sequences from the mitochondrial genome, their evolutionary history is becoming much better understood. In particular, the molecular systematics of the Rana catesbiana species group (Aquarana) [2], Rana boylii group (Amerana) [17], Rana pipiens complex (Pantherana) [14], and the Mexican leopard frogs of the Rana berlandieri group [32] have been studied in detail. The molecular systematics of the Eurasian ranids has not been as extensively investigated. Phylogenetic relationships based upon mitochondrial gene sequences have been assessed for the Japanese brown frogs (R. dybowskii, R. japonica, R. okinavana, R. ornativentris, R. pirica, R. sakuraii, R. tagoi, and R. tsushimensis) [29], Japanese pond frogs (R. nigromaculata, R. porosa porosa, and R. p. brevipoda) [28], and for selected pond frogs distributed in Eurasia [27]. Previous work from the author’s laboratory has focussed upon a systemic study of the primary structures and biological properties of antimicrobial peptides from Japanese brown frogs [5,7–9,15,16]. We now extend this work to investigate the structures and properties of the antimicrobial peptides present in an extract of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. This species, also known as the diskless-fingered odorous frog, is found in evergreen forests in mountainous regions of southern Shanxi, Sichuan, Guizhou, and Yunnan, China and in Vietnam. According to the classification system of Dubois [11], R. grahami is placed in the Hylarana group.
2.
Materials and methods
2.1.
Tissue collection and extraction
Adult male specimens of the Yunnanfu Kunming frog Rana grahami (n = 3; length 6.6–7.3 cm, body weight 28–31 g) were collected under permit in summer, 2005 in Guandu county, Kunming, Yunnan province, China. The animals were anesthetized by immersion in ice–water and sacrificed by pithing. Skin was immediately removed and freeze-dried for shipment to UAE. University. The dried tissue (1.41 g) was extracted by homogenization in ethanol/0.7 M HCl (3:1 v/v; 100 mL) at 0 8C using a Waring blender. The homogenate was
stirred for 2 h at 0 8C and centrifuged (4000 g for 30 min at 4 8C). Ethanol was removed from the supernatant under reduced pressure and, after further centrifugation (4000 g for 30 min at 4 8C), the extract was pumped onto 4 Sep-Pak C18 cartridges (Waters Associates) connected in series at a flow rate of 2 mL/min. Bound material was eluted with acetonitrile/ water/trifluoroacetic acid (70.0:29.9:0.1, v/v/v) and freezedried.
2.2.
Antimicrobial and hemolytic assays
Purification of the peptides was monitored by incubating lyophilized aliquots of chromatographic effluent in Mueller– Hinton broth (50 mL) with an inoculum (50 mL of 106 colony forming units per milliliter) from a log-phase culture of reference strains Staphylococcus aureus (NCTC 8325) and Escherichia coli (ATCC 25922) in 96-well microtiter cell-culture plates for 18 h at 37 8C in a humidified atmosphere of air. Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 h at 35 8C using an inoculum (50 mL) of 5 104 colony forming units per milliliter. After incubation, the absorbance at 630 nm of each well was determined using a microtiter plate reader. In order to monitor the validity and reproducibility of the assays, incubations with bacteria were carried out in parallel with increasing concentrations of bacitracin and incubations with C. albicans in parallel with amphotericin B. Minimum inhibitory concentration (MIC) was measured by standard microdilution methods [21,22] and was taken as the lowest concentration of peptide where no visible growth was observed. Peptides in the concentration range 15–500 mM were incubated with washed human erythrocytes (2 107 cells) from a healthy donor in Dulbecco’s phosphate-buffered saline, pH 7.4 (100 mL) for 1 h at 37 8C. After centrifugation (12,000 g for 15 s), the absorbance at 450 nm of the supernatant was measured. A parallel incubation in the presence of 1% (v/v) Tween-20 was carried out to determine the absorbance associated with 100% hemolysis. The LC50 value was taken as the mean concentration of peptide producing 50% hemolysis in three independent experiments.
2.3.
Peptide purification
The skin extract, after partial purification on Sep-Pak cartridges, was redissolved in 0.1% (v/v) trifluoroacetic acid/ water (2 mL) and injected onto a (2.2 cm 25 cm) Vydac 218TP1022 (C-18) reverse-phase HPLC column (Separations Group) equilibrated with 0.1% (v/v) trifluoroacetic acid/water at a flow rate of 6.0 mL/min. The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min and to 63% (v/v) over 60 min using linear gradients. Absorbance was monitored at 214 and 280 nm, and fractions (1 min) were collected. The abilities of freeze-dried aliquots (50 mL) of the fractions to inhibit the growth of S. aureus and E. coli were determined as described in the previous section. Fractions with antimicrobial activity were successively chromatographed on a (1 cm 25 cm) Vydac 214TP510 (C-4) column and a (1 cm 25 cm) Vydac 219TP510 (phenyl) column. The concentration of acetonitrile in the eluting solvent was raised from 21 to 56% over 50 min and the flow rate was 2.0 mL/min.
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2.4.
Structural characterization
The primary structures of the peptides were determined by automated Edman degradation using an Applied Biosystems model 494 Procise sequenator. MALDI-TOF mass spectrometry was carried out using a Voyager DE-PRO instrument (Applied Biosystems) that was operated in reflector mode with delayed extraction and the accelerating voltage in the ion source was 20 kV. The instrument was calibrated with peptides of known molecular mass in the 2000–4000 Da range. The accuracy of mass determinations was 0.02%.
3.
Results
3.1.
Purification of the peptides
The skin extract from R. grahami, after concentration and partial purification on Sep-Pak C-18 cartridges, was chromatographed on a Vydac C-18 semipreparative reversephase HPLC column (Fig. 1). Aliquots of the fractions were tested for their ability to inhibit growth of Gram-negative bacteria, E. coli and Gram-positive bacteria, S. aureus. Antimicrobial activity was associated with the well-defined peaks designated 1–6. Subsequent structural analysis demonstrated that peak 1 contained esculentin-1GRa, peak 2 contained brevinin-2GRa, peak 3 contained brevinin-2GRb, peak 4 contained brevinin-2GRc and brevinin-1GRa, peak 5 contained nigrocin-2GRa and nigrocin-2GRb, and peak 6 contained nigrocin-2GRc. Under the conditions of assay, peaks 2 and 6 showed growth inhibitory activity against E. coli only. The antimicrobial peptides were purified to near homogeneity, as assessed by a symmetrical peak shape and mass spectrometry, by further chromatography on Vydac C-4 and Vydac phenyl columns. The methodology is illustrated by separation of nigrocin-2GRa and nigrocin-2GRb
Table 1 – Minimum inhibitory concentrations (mM) against microorganisms and concentrations producing 50% hemolysis of human erythrocytes (mM) of the endogenous peptides isolated from an extract of the skin of Rana grahami Peptide Esculentin-1GRa Brevinin-2GRa Brevinin-2GRb Brevinin-1GRa Brevinin-2GRc Nigrocin-2GRa Nigrocin-2GRb Nigrocin-2GRc
S. aureus
6 12.5 6 25 12.5 25 3 50
12.5 50 25 12.5 50 >100 12.5 >100
C. albicans
LC50
>50 >100 >100 ND >100 >100 50 >100
210 140 180 ND 100 295 40 >500
on a Vydac C-4 column (Fig. 2A) and final purification of each component on a Vydac phenyl column (Fig. 2B and C). The final yields of purified peptides (nmol) were esculentin-1GRa 590, brevinin-2GRa 570, brevinin-2GRb 510, brevinin-2GRc 480, brevinin-1GRa 55, nigrocin-2GRa 950, nigrocin-2GRb 110, and nigrocin-2GRc 520.
3.2.
Structural characterization
The primary structures of the antimicrobial peptides isolated from R. grahami were established by automated Edman degradation and their amino acid sequences are shown in Fig. 3. MALDI-TOF mass spectrometry was used to confirm the proposed sequences and to demonstrate the presence of a disulfide bridge in the esculentin-1, brevinin-1, brevinin-2, and nigrocin-2 peptides (Fig. 3).
3.3.
Antimicrobial and hemolytic activities
The abilities of the endogenous peptides isolated from R. grahami skin to inhibit the growth of S. aureus, E. coli, and C. albicans, and to lyse human erythrocytes are quantified in Table 1.
4.
Fig. 1 – Reverse-phase HPLC on a preparative Vydac C-18 column of an extract of skin Rana grahami. Peptides in the peaks designated 1–6 were associated with antimicrobial activity and were subjected to further purification. The dashed line shows the concentration of acetonitrile in the eluting solvent.
E. coli
Discussion
Mainland China is home to a large number of species belonging to the genus Rana [19], many of which are severely threatened because of the rapid pace of urbanization. The present study has led to the isolation from an extract of the skin of the Chinese frog R. grahami of seven peptides with antimicrobial activity whose primary structures indicate that they belong to four previously described families—nigrocin-1, brevinin-1, brevinin-2, and esculentin-1. A comparison of their amino acid sequences with orthologs from other Asian ranids provides some insight into the phylogenetic placement of this species. Nigrocin-2 was first isolated from the skin of the pond frog R. nigromaculata [24]. The peptide shows little structural similarity to other antimicrobial peptides but, in common with most antimicrobial peptides from ranid frogs, nigrocin-2 lacks stable secondary structure in aqueous solution but adopts an amphipathic a-helical conformation in a membrane-mimetic solvent such as 50% trifluoroethanol. Prior to
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Fig. 2 – Separation of nigrocin-2GRa (peak 1) and nigrocin-2GRb (peak 2) on a Vydac C-4 column (Panel A), and purification to near homogeneity on a Vydac phenyl column of nigrocin-2GRa (Panel B) and nigrocin-2GRb (Panel C). The arrowheads show where peak collection began and ended.
the present study, nigrocin-2 had not been detected in the skin of any other ranid frog investigated and so the isolation of orthologs with a high degree of amino acid sequence similarity (Fig. 4) from R. grahami provides good evidence for a close
phylogenetic relationship between this species and R. nigromaculata. Structure–activity relationships within the nigrocin-2 molecule have never been investigated. Nigrocin-2GRb shows
Fig. 3 – Amino acid sequences, observed molecular masses (Mr (obs)), and calculated molecular masses (Mr (calc)) of the antimicrobial peptides isolated from an extract of the skin of R. grahami.
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Fig. 4 – A comparison of the primary structures of peptides belonging to the nigrocin-2, brevinin-2, and brevinin-1 families. Nigrocin-1 and -2 are from R. nigromaculata [24], brevinin-1 and -2 are from R. porosa brevipoda [20], peptides designated -GR are from R. grahami (this study), -PR are from R. pirica [8], -TS are from R. tsuchimensis [5], and -OK are from R. okinavana [7]. Percent amino acid sequence identities are shown. The shaded residues denote conserved domains in the peptides. In order to maximize structural similarity, residue deletions denoted by (*) have been introduced in some sequences.
greatest structural similarity to nigrocin-2 and, as shown in Table 1, this peptide, like nigrocin-2 [24], shows relatively high potency against both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria and against the opportunistic yeast pathogen C. albicans. In contrast, nigrocin-2GRa and -2CRc show much lower potency against E. coli and are inactive (MIC > 100 mM) against S. aureus and C. albicans. The antimicrobial activities of a peptide against bacteria and fungi are determined by a complex interaction between cationicity, hydrophobicity, a-helicity, and amphipathicity [31]. The bacterial cytoplasmic cell membrane is rich in acidic (phospho) lipids whereas the plasma membrane of mammalian cells contains a much higher proportion of zwitterionic phosphatidyl-choline and sphingomyelin phospholipids. An
increase in peptide cationicity, therefore, should promote interaction with the more negatively charged bacterial cell membrane and increase antimicrobial potency. The positive charge on the peptide is also believed to facilitate interaction with, and passage across, the bacterial cell wall, both in the case of Gram-negative bacteria that contain negatively charged lipopolysaccharides and of Gram-positive bacteria that contain negatively charged teichoic and teichuronic acids [31]. In these peptides, the conserved Lys5 residue in nigrocin-2 and nigrocin-2GRb is replaced by Gly and the Lys12 residue is replaced by His (nigrocin-2GRa) and Asn (nigrocin-2GRc). It is proposed that this decrease in cationicity is primarily responsible for the observed decrease in antimicrobial potency.
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Fig. 5 – A comparison of the primary structures of peptides belonging to the esculentin-1 family. Esculentin-1 is from R. esculenta [26]; peptides designated -PL are from R. palustris [3] and -AR are from R. aerolata [1].
As well as decreased antimicrobial potency, nigrocin-2GRa and nigrocin-2GRc showed appreciably reduced hemolytic activity against human erythrocytes (Table 1). Studies with model compounds have demonstrated a direct correlation between degree of amphipathicity and hemolytic activity in peptides that form an a-helix [10,30]. It is probable, therefore, that the decrease in amphipathic produced by replacing polar residues on the hydrophilic face of the a-helix by neutral amino acids is responsible for the decrease in hemolytic activity. The conclusion that R. grahami is evolutionarily related to R. nigromaculata is supported by comparison of the amino acid sequences of the brevinin-2 peptides (Fig. 4). Although termed nigrocin-1 [24], this peptide is clearly a member of the brevinin-2 family (Fig. 4). Amino acid sequence similarity between nigrocin-1 and brevinin-2GRa and -GRb is relatively high and the three peptides share a four-residue deletion. By way of comparison, the sequences of orthologs with lower sequence similarity from the Japanese brown frog, R. pirica [8] are included in Fig. 4. Brevinin-2, first isolated from an extract of the skin of the Japanese pond frog R. porosa brevipoda [20], has a wide distribution in those species of Asian and European ranid frogs examined to-date but has yet to be identified in a North American species [6]. In general, 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 three amino acid residues (Cys27, Lys28, and Cys33) are invariant in the peptide. Brevinin-2GRa and GRb also share a high degree of sequence similarity with brevinin-2 suggesting a close phylogenetic relationship of R. grahami with R. porosa brevipoda. Brevinin-1, also first isolated from the skin of R. porosa brevipoda [20], is amongst the most widely distributed of all the antimicrobial peptides from ranid frogs and has been identified in the skins of numerous Eurasian and North American species [6]. The amino acid sequence of the peptide has been very poorly conserved across species with no residue invariant. Brevinin-1GRa like brevinin-1 contains 24 amino acid residues and sequence similarity between the two peptides is relatively high (71%) (Fig. 4). However, several molecular variants of brevinin-1 have been isolated, particular from the skins of the Japanese brown frogs (Fig. 5), that contain amino acid deletions and lack the C-terminal disulfide-bridged cyclic domain [5,7,8]. The 46 amino acid residue peptide esculentin-1 has been isolated from the skin of frogs belonging to the R. esculenta complex from Europe [26] and North Africa [18] and also from North American species belonging to the Nenirana group (R.
palustris (pickerel frog) [3] and R. areolata (crawfish frog) [1]). Identification of an ortholog in the skin of an Asian frog demonstrates that esculentin-1 has a widespread, although sporadic, distribution among ranid frogs. As shown in Fig. 5, evolutionary pressure has acted primarily to conserve the polar residues in the molecule (Lys, Arg, Glu, Asp) although hydrophobic amino acids are generally replaced by one of similar properties. Consistent with previous data [1,3,26], esculentin-1GRa shows growth-inhibitory activity against both Gram-negative and Gram-positive bacteria and is relatively non-hemolytic. After this study was completed, a report [33] appeared describing the isolation of two peptides identical in structure to nigrocin-2GRa and nigrocin-2GRc from R. grahami skin secretions. Regretably, these peptides were described as grahamin 1 and grahamin 2 despite the fact that the authors were aware that the peptides belong to the nigrocin-2 family. It is essential that workers in the field adopt a consistent terminology otherwise the study of complex array of Rana antimicrobial peptides will degenerate into chaos.
Acknowledgments This work was supported by an Individual Research Grant (0103-8-11/06) and a Faculty Support Grant (NP/06/02) from the United Arab Emirates University.
references
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