Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex

Peptides 24 (2003) 955–961

Characterization of novel antimicrobial peptides from the skins of frogs of the Rana esculenta complex Mohamed F. Ali a , Floyd C. Knoop b , Hubert Vaudry c , J. Michael Conlon d,∗ a Department of Biomedical Sciences, Creighton University Medical School, Omaha, NE 68178, USA Department of Medical Microbiology and Immunology, Creighton University Medical School, Omaha, NE 68178, USA European Institute for Peptide Research, Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France d Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666, Al-Ain, United Arab Emirates b

c

Received 22 April 2003; accepted 5 June 2003

Abstract Rana esculenta is a hybridogenetic hybrid between Rana ridibunda and Rana lessonae and so is best considered as a complex of interbreeding species rather than a discrete single species. In this study, antimicrobial peptides were isolated from a pooled extract of the skins of specimens of the R. esculenta complex collected in the wild. In addition to several peptides belonging to the brevinin and esculentin families that have been previously isolated from skin secretions of a single specimen of R. esculenta, three newly described members of the brevinin-2 family (brevinin-2Ei, brevinin-2Ej, and brevinin-2Ek) and one member of the temporin family (temporin-1Ec) were purified and characterized. In addition, three structurally related peptides with no sequence similarity with antimicrobial peptides isolated from other species of ranid frogs, that potently and selectively inhibit the growth of the Gram-positive bacterium Escherichia coli (minimal inhibitory concentration (MIC < 5 ␮M)), were identified. 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 and so have been termed caerulein precursor-related fragments (CPRF-Ea, CPRF-Eb, and CPRF-Ec). The data suggest that there may be considerable polymorphism among specimens from different populations of the R. esculenta complex. It is proposed that the distribution and amino acid sequences of skin antimicrobial peptides may be useful markers for taxonomic classification of particular sub-populations and for an understanding of phylogenetic interrelationships. © 2003 Elsevier Inc. All rights reserved. Keywords: Antimicrobial peptide; Rana esculenta; Rana ridibunda; Rana lessonae; HPLC purification

1. Introduction The common edible frog Rana esculenta Linnaeus 1758 is widely distributed throughout Europe from France to the Volga river basin [8]. Its taxonomic status is not completely certain but it is generally regarded as having a hybrid origin arising from the marsh frog, Rana ridibunda Pallas 1771 and the pool frog, Rana lessonae Camerano 1882 [21]. R. ridibunda is a relatively large frog (size range for adult specimens 74–94 mm) that is distinguishable from the smaller R. lessonae (42–71 mm), not only on the basis of body size, but by numerous morphological and biochemical features. Studies in the field indicate that the original hybridizations that produce R. esculenta are between female R. ridibunda and male R. lessonae. Thereafter, R. esculenta lineages are ∗

Corresponding author. Tel.: +971-3-7039484; fax: +971-3-7672033. E-mail address: [email protected] (J.M. Conlon).

0196-9781/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0196-9781(03)00193-1

maintained largely by matings of R. esculenta females with R. lessonae males [6]. The ranges of the three species often overlap so that collection of specimens of the intermediate sized (54–89 mm) R. esculenta in the wild will almost inevitably contain individuals belonging R. ridibunda and R. lessonae species as well as the various hybrids. Hence, the term “R. esculenta complex” is used to describe the diverse interbreeding populations of the three species. 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 [10]. A previous study by Simmaco et al. [18] has described the isolation of multiple antimicrobial peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families from the electrically-stimulated skin secretions of a single specimen of R. esculenta. Similarly, analysis of an extract of gastric tissue from pooled specimens of R. esculenta collected in the wild led to the isolation of four

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antimicrobial peptides of the brevinin-2 family, two of which had been previously described by Simmaco et al. [18] and two with novel amino acid sequences [24]. In the present study, a pooled extract of the skin of approximately 100 specimens belonging to the R. esculenta complex collected in the field was analyzed for the presence of antimicrobial peptides. In addition to several components that have already been characterized, seven previously undescribed peptides were isolated and their primary structures and antimicrobial properties determined.

2. Materials and methods 2.1. Animals Adult specimens of frogs belonging to the R. esculenta complex were collected in different regions of Albania by a commercial enterprise and shipped to the University of Rouen. Approximately 100 animals of both sexes were anaesthetized by immersion in crushed ice and sacrificed by decapitation and pithing. Skin was immediately removed, frozen in liquid nitrogen, and freeze-dried. 2.2. Tissue extraction The dried tissue (27 g) was extracted by homogenization in ethanol/0.7 M HCl (3:1 v/v; 500 ml) at 0 ◦ C using a Waring blender. The homogenate was stirred for 2 h at 0 ◦ C and centrifuged (4000 × g for 30 min at 4 ◦ C). Ethanol

was removed from the supernatant under reduced pressure and, after further centrifugation (4000 × g for 30 min at 4 ◦ C), the extract was pumped onto eight Sep-Pak C-18 cartridges (Waters Associates) connected in series at a flow rate of 2 ml min−1 . Bound material was eluted with acetonitrile/water/trifluoroacetic acid (70.0:29.9:0.1 v/v/v) and freeze-dried. 2.3. Antimicrobial assays Aliquots (50 ␮l) of the fractions of chromatographic effluent (Fig. 1) were tested for their ability to inhibit the growth of Escherichia coli (ATCC 25922) and Staphylococcus aureus (NCTC 8325). Lyophilized aliquots of chromatographic effluent, reconstituted in Mueller–Hinton broth (50 ␮l) were incubated with an inoculum (50 ␮l of 5×105 colony forming units per milliliter) from an overnight culture of the bacteria in 96-well microtiter cell-culture plates for 18 h at 37 ◦ C in a humidified atmosphere of 5% CO2 in air. Incubations with Candida albicans (ATCC 90028) were carried out in RPMI 1640 medium for 48 h at 35 ◦ C. After incubation, the absorbance at 550 nm of each well was determined using a M.A. Bioproducts model MA308 microtiter plate reader. Minimal inhibitory concentrations (MICs), measured by a standard microdilution method [4], were taken as the lowest concentration of peptide where no visible growth was observed. The amounts of the peptides were determined by amino acid composition analyses. In order to monitor the validity of the assay, incubations with E. coli and S. aureus were carried out in parallel with

Fig. 1. Reverse-phase HPLC on a semipreparative Vydac C-18 column of an extract of the skin of frogs belonging to the Rana esculenta complex after partial purification on Sep-Pak cartridges. The bar denotes fractions with antimicrobial activity and the arrowheads show the retention times of (1) brevinin-2Ei, (2) brevinin-2Ej and brevinin-2Ek, (3) CPRP-Eb and CPRP-Ec, (4) CPRP-Ea, and (5) temporin-1Ec. The dashed line shows the concentration of acetonitrile in the eluting solvent.

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increasing concentrations of the broad-spectrum antibiotic, bacitracin, and incubations with C. albicans in parallel with amphotericin B. 2.4. Purification of the peptides The skin extract, after partial purification on Sep-Pak cartridges, was redissolved in 0.1% (v/v) trifluoroacetic acid/water (5 ml). The solution was chromatographed on a (1 cm × 25 cm) Vydac 218TP510 (C-18) reverse-phase HPLC column (separations group) equilibrated with 0.1% (v/v) trifluoroacetic acid/water at a flow rate of 2 ml min−1 . The concentration of acetonitrile in the eluting solvent was raised to 21% (v/v) over 10 min and to 63% (v/v) over 80 min using linear gradients. Absorbance was monitored at 214 and 280 nm and fractions (1 min) were collected. Aliquots (20 ␮l) of the fractions containing antimicrobial activity were analyzed by electrospray mass spectrometry using a Perkin-Elmer Sciex API 150EX single quadrupole instrument. The accuracy of mass determinations was ±0.02%. Those fractions containing components whose masses did not correspond to those of previously described antimicrobial peptides were successively chromatographed on a (0.46 cm × 25 cm) Vydac 214TP54 (C-4) column and a (0.46 cm × 25 cm) Vydac 219TP54 (phenyl) column. The concentration of acetonitrile in the eluting solvent was raised from 35 to 63% over 40 min and the flow rate was 1.5 ml min−1 . 2.5. Structural analysis The primary structures of the peptides were determined by automated Edman degradation using an Applied Biosystems

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model 491 Procise sequenator (Foster City, CA). Amino acid compositions were determined by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using a Waters AccQ Tag system with fluorescence detection and separation of the amino acid derivatives by reverse-phase HPLC. Hydrolysis in 5.7 M HCl (24 h at 110 ◦ C) of approximately 1 nmol of peptide was carried out.

3. Results 3.1. Purification of the peptides The skin extract, after partial purification on Sep-Pak C-18 cartridges, was chromatographed on a Vydac C-18 semipreparative reverse-phase HPLC column and the elution profile is shown in Fig. 1. Aliquots of the fractions were tested for their ability to inhibit growth of the Gram-negative bacterium, E. coli and the Gram-positive bacterium, S. aureus. Antimicrobial activity was associated with fractions with retention times between 54 and 78 min. Analysis of these fractions by electrospray mass spectrometry led to the identification of peptides with molecular masses (amu) corresponding to the previously characterized esculentin-1a (4800), esculentin-2a (3712), brevinin-1E (2676), brevinin-2Ec (3519), brevinin-2Ef (3365), brevinin-2Eg (3371), and brevinin-2Eh (3442). These components were not purified further. Six peptides with growth inhibitory activity against E. coli and one peptide with activity against S. aureus had molecular masses that did not correspond to those of previously described antimicrobial peptides from R. esculenta. These peptides were purified to near homogeneity, as assessed by

Fig. 2. Separation of brevinin-1E (peak 1) and peptide CPRF-Ea (peak 2) on an analytical Vydac C-4 column. The arrows show where peak collection began and ended.

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Peptide

Primary Structure

Mass (a.m.u.)

Brevinin-2Ei

GILSTIKDFAIKAGKGAAKGLLEMASCKLSGQC

3309

Brevinin-2Ej

GIFLDKLKNF

AKGVAQSLLNKASCKLSGQC

3181

Brevinin-2Ek

GLWNTKLEA

GLLFAMGKLDLKRCLKAGGC

3122

CPRF-Ea

GLGSILGKILNVAGKVGKTIGKVADAVGNKE

3008

CPRF-Eb

GLGSFLKNAIKIAGKVGSTIGKVADAIGNKE

3058

CPRF-Ec

GLGSFFKNAIKIAGKVGSTIGKVADAIGNKE

3091

Temporin-1Ec

FLPVIAGLLSKLF.NH2

1417

Fig. 3. Amino acid sequences of the previously undescribed peptides with antimicrobial activity isolated from an extract of the skins of frogs belonging to the Rana esculenta complex. The sequences are aligned so as to maximize structural similarity.

a symmetrical peak shape and a single component on mass spectrometry, by further chromatography on analytical Vydac C-4 and phenyl columns. The same methodology was used for all purifications and is illustrated by a representative separation of brevinin-1E and CPRF-Ea on a Vydac C-4 column (Fig. 2). The final yields of the purified peptides (nmol), determined by amino acid composition analysis, were brevinin-2Ei (87), brevinin-2Ej (11), brevinin-2Ek (4), temporin-1Ec (8), CPRF-Ea (10), CPRF-Eb (14), and CPRF-Ec (12). 3.2. Peptide characterization The primary structures of the peptides with antimicrobial activity that had not been previously identified in R. esculenta tissues were determined by automated Edman degradation and are shown in Fig. 3. The molecular masses and the amino acid compositions of the peptides were consistent Table 1 Minimal inhibitory concentrations (MICs) of peptides isolated from the skin of frogs of the Rana esculenta complex MICs (␮M)

Brevinin-2Ei Brevinin-2Ej Temporin-1Ec CPRF-Ea CPRF-Eb CPRF-Ec NA: not active at 100 ␮M.

E. coli

S. aureus

3 2 NA 4 5 4

NA NA 8 NA NA NA

with their proposed structures. Brevinin-2Ek was isolated as its [Met15 ]sulfoxide derivative. 3.3. Antimicrobial activities The abilities of brevinin-2Ei, brevinin-2Ej, temporin-1Ec, and the CPRF-E peptides to inhibit the growth of the Gram-positive bacterium S. aureus and the Gram-negative bacterium E. coli are compared in Table 1. No peptide, at concentrations up to 100 ␮M, inhibited the growth of the fungal pathogen, C. albicans. There was insufficient pure material to determine the activities of brevinin-2Ek. 4. Discussion The present study extends the work of Simmaco et al. [17,18,20] and has led to the isolation of seven previously undescribed antimicrobial peptides from the skins of frogs of the R. esculenta complex. The amino acid sequence of three peptides indicates that they are members of the brevinin-2 family, first identified in the skin of the Asian frog Rana brevipoda porsa [13]. Their primary structures are compared in Fig. 4 with those of brevinin-2 peptides previously isolated from the skin secretions of a single specimen of R. esculenta (brevinin-2E, brevinin-2Ea, brevinin-2Eb, brevinin-2Ec, and brevinin-2Ed) [18] and from an extract of R. esculenta gastric tissue (brevinin-2Ec, brevinin-2Ef, brevinin-2Eg, and brevinin-2Eh) [24]. The amino acid sequence of brevinin-2Ee has been deduced from the nucleotide sequence of a cloned cDNA prepared from a R. esculenta skin library [18].

M.F. Ali et al. / Peptides 24 (2003) 955–961

959

Brevinin-2 Peptides Brevinin-2E

GI MDTLKNLAKTAGKGALQSLLNKASCKLSGQC

Brevinin-2Ea

-- L-------IS-A---A-G-V-----------

Brevinin-2Eb

-- L----------------G-VKM---------

Brevinin-2Ec

--L--K---F-------V-----TA---------

Brevinin-2Ed

-- L-S------N--

-I-------------

Brevinin-2Ee

-- F-K---F--GVA

---------------

Brevinin-2Ef

-- --------------------KM---------

Brevinin-2Eg

-- ---------------------H---------

Brevinin-2Eh

-- ---------------------H------K--

Brevinin-2Ei

-- LS-I-DF-IK-----AKG--EM---------

Brevinin-2Ej

-- FLDKLKNF

A--VA---------------

Brevinin-2Ek

-L WN- -LE-

-LLFAMGK-DLKR-LKA-GC

Temporin Peptides Temporin-1Ec

FLPVIAGLLSKLF

Peptide A1

---A---I--Q--

Peptide B9

---L-----G---

Fig. 4. A comparison of the primary structures of the brevinin-2 and temporin peptides isolated from frogs of the R. esculenta complex. (– – –) Denotes residue identity.

The amino acid sequence of a fourth previously undescribed peptide indicates that it is a member of the temporin family of antimicrobial peptides, first identified in the skin of the European frog R. temporaria [19] but subsequently isolated from several species of Asian and North American ranid frogs [23]. Earlier work by Simmaco et al. [20] resulted in the isolation of two peptides, termed peptide A1 and peptide B9, with potent hemolytic activity from R. esculenta skin that are clearly members of the temporin family (Fig. 4). Consequently, the peptide isolated in this study is designated temporin-1Ec to indicate that it is the third member of the family to be identified in frogs of the R. esculenta complex. Consistent with previous data from

our laboratory [1,5,9], temporin-1Ec inhibited the growth of the Gram-positive bacterium S. aureus but was not active against the Gram-negative bacterium E. coli. The most novel aspect of the present study is the isolation of three structurally related peptides (CPRF-Ea, CPRF-Eb, and CPRF-Ec) that show no significant amino acid sequence similarity to any antimicrobial peptide previously isolated from the tissues or skin secretions of frogs of the genus Rana. A BLAST search (National Center for Biotechnology Information, Bethesda, MD) [3] indicated that the CPRF-E peptides showed limited sequence similarity with homologous exon gene products that encode the N-terminal flanking peptides of preprocaerulein, preproxenopsin, and preprolevitide

CPRF-Ea

GLGSILGKIL NVAGKVGKTIGKVADAVGNKE

CPRF-Eb

GLGSFLKNAI KIAGKVGSTIGKVADAIGNKE

CPRF-Ec

GLGSFFKNAI KIAGKVGSTIGKVADAIGNKE

CPF

GXXSXLGKAL KAXLKIGXXXLGGXPQQ

XPF

GWASKIGQTLGKIA KVGLKQLIQPK

XPF

GWASKIGQTLGKIA KVGLQGLMQPK

LPF

GWASKIGQTLGKIA KVGLQGLMQPK

XT-7

GLLGPLL

KIAAKVGSNLL

Fig. 5. A comparison of the primary structures of the CPRF-E peptides from R. esculenta 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. The shaded residues indicate sequence identity with one or more CPRF-E peptides.

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[12] (Fig. 5). These genes are expressed in the skin of the African clawed frog, Xenopus laevis [14,15,22] and it has been suggested that they may have evolved from a common ancestral gene by a series of duplication events [12]. The tetraploidization that occurred in the X. laevis genome [11] together with probable multiple gene and exon duplications has resulted in a complex array of peptides derived from preprocaerulein. Analysis of skin cDNA libraries has revealed the existence of four types of preprocaerulein, termed 1, 1 , III, and IV according to the number of copies of caerulein in the sequence, with the result that X. laevis skin secretions contain at least 11 structurally-related but not identical spacer peptides (termed caerulein precursor fragments CPF) [15]. The consensus sequence of these peptides illustrating the conserved residues is shown in Fig. 5. Three peptides (XT-1, XT-6, and XT-7) showing limited structural similarity to the X. laevis caerulein spacer fragments were identified in skin secretions of the diploid frog Xenopus tropicalis [2] and, as shown in Fig. 5, the CPRF-E peptides show significant amino acid sequence similarity with peptide XT-7. There is no evidence for expression of preprocaerulein-related genes in ranid frogs and so the biosynthetic relationship between the CPRF-E peptides and the Xenopus CPF, xenopsin precursor fragments (XPF), and levitide precursor fragments (LPF) is unknown. The single specimen of the R. esculenta complex used by Simmaco et al. [18] was collected in Italy in the region of Naples (D. Barra, personal communication) whereas the specimens used in this study were collected in various regions of Albania and probably comprise a range of interbreeding individuals from R. ridibunda, R. lessonae, and their R. esculenta hybrids. This species diversity is reflected in the different spectrum of antimicrobial peptides identified in the two studies. Skin secretions may be collected from ranid frogs in the wild under non-invasive conditions (mild electrical stimulation [7] or injection of norepinephrine [16]). It is proposed that analysis of the antimicrobial peptide components present in the secretions by mass spectrometry coupled with HPLC separation will provide a valuable method for taxonomic classification of different sub-populations of the R. esculenta complex and will facilitate an understanding of their phylogenetic relationships. Acknowledgments This work was supported by a grant from the National Science Foundation (EPS-9720643). The authors thank Donald Babin and Eva Lovas, Creighton University Medical School for amino acid analyses and mass spectrometry measurements. References [1] Ali MF, Lips KR, Knoop FC, Fritzsch B, Miller C, Conlon JM. Antimicrobial peptides and protease inhibitors in the skin

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