The NMR-derived Solution Structure of a New Cationic Antimicrobial Peptide from the Skin Secretion of the Anuran Hyla punctata

Abstract

Amphibian skin secretions constitute an important source of molecules for antimicrobial drug research in order to combat the increasing resistance of pathogens to conventional antibiotics. Among the various types of substances secreted by the dermal granular amphibian glands, there is a wide range of peptides and proteins, often displaying potent antimicrobial activities and providing an effective defense system against parasite infection. In the present work, we report the NMR solution structure and the biological activity of a cationic 14-residue amphiphilic α-helical polypeptide named Hylaseptin P1 (HSP1), isolated from the skin secretion of the hylid frog Hyla punctata. The peptide antimicrobial activity was verified against Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, whereas no significant lytic effect was detected toward red or white blood cells. Amphibian skin secretions constitute an important source of molecules for antimicrobial drug research in order to combat the increasing resistance of pathogens to conventional antibiotics. Among the various types of substances secreted by the dermal granular amphibian glands, there is a wide range of peptides and proteins, often displaying potent antimicrobial activities and providing an effective defense system against parasite infection. In the present work, we report the NMR solution structure and the biological activity of a cationic 14-residue amphiphilic α-helical polypeptide named Hylaseptin P1 (HSP1), isolated from the skin secretion of the hylid frog Hyla punctata. The peptide antimicrobial activity was verified against Candida albicans, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, whereas no significant lytic effect was detected toward red or white blood cells. Because of its role in respiration and osmoregulation, the skin of amphibians is made up of a thin layer of cells, densely populated by moisturizing glands, that facilitate the gas exchange necessary for survival of the animal. Despite being a poor mechanical barrier against biotic and physical stress, the epidermis, in particular the “stratum corneum,” has evolved a complex mechanism of defense. A wide range of chemically diverse components is rapidly secreted by the granular glands covering the skin. In addition to the psychotropic, toxic, and repellent effects that provide an effective strategy to keep predators away (1Duellman W.E. Trueb L. Biology of Amphibians. Johns Hopkins University Press, Baltimore1994Google Scholar), a large number of these secretions also contain potent antimicrobial agents that protect the amphibians against infections by molds, bacteria, and protozoa (2Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5449-5453Crossref PubMed Scopus (2030) Google Scholar, 3Bevins C.L. Zasloff M Annu. Rev. Biochem. 1990; 59: 395-414Crossref PubMed Scopus (521) Google Scholar, 4Stone D.M.J. Waugh R.J. Bowie J.H. Wallace J.C. Tyler M.J. J. Chem. Soc. Perkin Trans. 1992; 1: 3173-3178Crossref Scopus (51) Google Scholar). As for the antimicrobial agents, it is becoming clear that the cationic peptides represent an important component of this host-defense system, because they are rapidly expressed and secreted soon after the first contact with the pathogens, a process induced by bacterial products (5Hancock R.E.W. Scott M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8856-8861Crossref PubMed Scopus (825) Google Scholar). The binding of cationic peptides to the anionic lipids of biomembranes is the initial step in the permeabilization of the microbes cell wall, either via the formation of pores, involving the “barrel-stave” mechanism, or by the induction of phospholipids “flip-flop,” involving the “carpet-like” mechanism (6Chen H.M. Clayton W.W. Sawyer W.H. Eur. J. Biochem. 2001; 268: 1659-1669Crossref PubMed Scopus (31) Google Scholar, 7Epand R.M. Vogel H.J. Biochim. Biophys. Acta. 1999; 1462: 11-28Crossref PubMed Scopus (1151) Google Scholar). Indeed, these peptides share the propensity to acquire an amphiphilic α-helical structure when in hydrophobic environments, a structural feature essential for the initiation of the interaction with the phospholipid bilayer of the target membrane (8Simmaco M. Mignogna G. Barra D. Biopolymers. 1998; 47: 435-450Crossref PubMed Scopus (406) Google Scholar, 9Dathe M. Wieprecht T. Biochim. Biophys. Acta. 1999; 1462: 71-87Crossref PubMed Scopus (632) Google Scholar), that leads to membrane lysis and, as a final result, to inhibition of growth and cell death. The indiscriminate use of antibiotics has led to the emergence of highly resistant strains of microorganisms. Because of this, the screening for new naturally occurring antimicrobial agents is constantly growing and being expanded to include the chemicals involved in the defense of animals such as the arthropods and amphibians. The antimicrobial peptides found in amphibian skin secretions have been successfully tested in “in vitro” assays. So far, the Magainins (2Zasloff M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5449-5453Crossref PubMed Scopus (2030) Google Scholar), brevinins (10Morikawa N. Hagiwara K. Nakajima T. Biochem. Biophys. Res. Commun. 1992; 189: 184-190Crossref PubMed Scopus (244) Google Scholar), and dermaseptins (11Mor A. Nicolas P. Eur. J. Biochem. 1994; 219: 145-154Crossref PubMed Scopus (158) Google Scholar) are the best known broad-spectrum antimicrobial peptides obtained from frogs. In this work, we report the structural and functional characterization of a 14-residue cationic peptide isolated from the skin extract of Hyla punctata, a hylid frog commonly found mainly in the wet land areas of the Brazilian north and northeast regions. HPLC 1The abbreviations used are: HPLC, high performance liquid chromatography; HSP1, hylaseptin P1; RP-HPLC, reversed phase HPLC; Fmoc, N-(9-fluorenyl) methoxycarbonyl; NCCLS, National Committee for Clinical Laboratory Standards; MIC, minimal inhibitory concentration; MFC, minimal fungicidal concentration; TFE, trifluorethanol; NOE, nuclear effect Overhauser; DQF-COSY, double quantum filtered correlation spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; ROESY, rotating frame Overhauser spectroscopy; r.m.s.d., root mean square deviation; AFM, atomic force microscopy; LPS, lipopolysaccharides. —H. punctata specimens captured in Palmas, State of Tocantins, Brazil (IBAMA license 0637/91 A.C), were mildly stressed by electrical stimulation (around 6 V), and the skin secretion was collected in Milli-Q H2O. The crude extract was filtered, lyophilized, and fractionated by RP-HPLC (Shimadzu Co.) using a Vydac 218TP510 column. The fractions were eluted in a 60-min linear gradient of H2O (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid. The experiment was monitored at 216 and 280 nm, and samples were lyophilized and stored at -80 °C. Mass Spectrometry Analysis and de Novo Sequencing—Aliquots of the native and the synthetic peptides in 50 pm aqueous solutions were mass analyzed by matrix-assisted laser desorption ionization-time of flight/mass spectrometry, on an ABI 4700 Proteomics Analyzer with TOF-TOF optics (Applied Biosystems). The samples were mixed with a saturated matrix solution of α-cyano-4-hydroxycinnamic acid (1:3) and spotted on a MALDI-TOF/TOF sample plate. The MS and the MS/MS spectra were carried out in the reflector mode with external calibration, using the calibration mixture Sequazyme Standard kit (Applied Biosystems). Peptide de novo sequencing was performed by precursor ion fragmentation using N2 as collision-induced dissociation gas, and collision cell pressure was kept at 2.8 × 10-6 torr. Edman Degradation—The automatic N-terminal sequencing of HSP1 was performed on a Protein and Peptide Sequencer PPSQ-23 (Shimadzu Co.) following the manufacturer's instructions. Chemical Synthesis—The synthetic HSP1 was obtained by automatic solid phase Fmoc synthesis on a Pioneer Synthesis System (PerSeptive Biosystems), following the manufacturer's instructions. After chemical deprotection and lyophilization, the peptide was purified by preparative RP-HPLC, followed by analytical RP-HPLC, using Vydac columns (218TP1022 and 218TP54, respectively). The final HPLC fraction had its purity and amino acid sequence mass analyzed by MALDI-TOF/TOF. Antibacterial Assays—The three bacterial strains used to investigate the antimicrobial activity of the peptide HSP1 were Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25992, which were purchased from American Type Culture Collection. Microorganisms cultured at Laboratório SABIN de Análises Clínicas/Brasília-DF, Brazil, were grown in stationary culture at 37 °C and after that were transferred to Mueller-Hinton liquid medium, according to the National Committee for Clinical Laboratory Standards (NCCLS) to perform the assays (12National Committee for Clinical Laboratory Standards Performance Standards for Antimicrobial Susceptibility Testing, NCCLS-approved Standard M100-S9, National Committee for Clinical Laboratory Standards, Wayne, PA. 1999; Google Scholar). The bacterial liquid growth inhibition assay was performed as described by Bulet et al. (13Bulet P. Cociancich S. Dimarcq J.-L. Lambert J. Hoffmann D. Hoffmann J.A. J. Biol. Chem. 1991; 236: 24520-24525Abstract Full Text PDF Google Scholar). The peptide was dissolved up to 8-fold in Mueller-Hinton broth by serial dilution. The highest peptide concentration used for the assay was 256 μg/ml (195.2 μm) in an initial inoculum of 1.8 × 107 colony-forming units/ml. The final volume was 200 μl per well, 100 μl of the peptide and 100 μl of the inoculum. The experiment was carried out in stationary culture at 37 °C, and the spectrophotometric readings were performed 12 h after incubation. The minimal inhibitory concentration, as introduced by Park et al. (14Park C.B. Kim M.S. Kim S.C. Biochem. Biophys. Res. Commun. 1996; 218: 408-413Crossref PubMed Scopus (244) Google Scholar), was on three using the antibiotics had inhibitory against the three bacterial by effects and the used in the were for 12 h in the of HSP1 and on solid culture containing The were and after and h assays were performed in S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Assays—The peptide HSP1 was tested against a Candida and of from the The in assays were performed using the broth according to the from the J.H. M.S. A. M. M.G. PubMed Scopus Google Scholar). Candida grown on of were in the cell concentration was to × per and to per The solution containing the peptide was to μl per and The final of peptide in the from to μg/ml to After μl per well, × per the was at °C for and the was at nm, using an automatic Chem. 1999; PubMed Scopus Google Scholar). of peptides and was as the first concentration of peptide in which in the was that in the Committee for Clinical Laboratory Standards for Susceptibility for Standard National Committee for Clinical Laboratory Standards, PA. Google Scholar). the minimal fungicidal samples were from the containing the and from containing the The samples were in The were with a and at 37 °C for The was as the concentration of the peptide in which no growth was detected A. M.S. G. 1999; PubMed Scopus Google Scholar, S. G. D. J. PubMed Scopus Google Scholar). activity of HSP1 was using blood from a collected on concentration and at × for at The was by the Committee of the Laboratório SABIN de Análises and with of The of was three in and in the to 107 × J. K. J. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). of HSP1 were from to to in 50 μl of and μl of the was followed by at 37 °C for The samples were on and at × and at °C L. J. J. A. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the was at nm, and the total was obtained by the addition of on the J. K. J. Biochim. Biophys. Acta. 1999; PubMed Scopus Google Scholar). peptide HSP1 was dissolved in 100 μl of and with blood a peptide of 50 μl were collected after and of and analyzed by a System in order to the blood cell total and the mean volume after to The was obtained using the K. S. M. L. PubMed Scopus Google Scholar). were performed using a to a system for were by the peptide to the concentration of in and In the carried out in the of the concentration was by addition of the volume of a solution at were carried out in the range of per sample were performed in a cell at °C. After of the of the and for in the of the was to the mean NMR sample for NMR was by HSP1 in μl of a mixture to a peptide concentration of NMR were carried out in a at for at a of °C. The chemical were to the peptide were used U. G. J. Chem. Soc. Scopus Google Scholar), total correlation L. J. Scopus Google Scholar), nuclear Overhauser enhancement A. K. Biochem. Biophys. Res. Commun. PubMed Scopus (2030) Google Scholar, S. Scopus Google Scholar), and rotating frame Overhauser A. J. Scholar) The spectra were using a sequence at a of and of spectra with a and were to the were with 100 were in the mode using the of et al. J. Scholar). The spectra was and with of complex for induction were for The experiment was collected with with of complex was by the in was carried out using the M. J. J. 1992; PubMed Scopus Google Scholar). were on a using the S. G. J. A. J. PubMed Scopus Google Scholar). to the were in both to a × a was after and The experiment was to × matrix to a for were from the spectra and to a calibration was made using the of 1.8 for the and the were as medium, and to the of and were to the of the for the in structure of HSP1 was using the in the P. K. J. Biol. PubMed Scopus Google Scholar). The was on the by et al. P. J. Chem. PubMed Scopus Google Scholar). of with from which the with the target were used to and to in the process was were In the final of a total of 100 was and the with the target were for After these with the target with no and no were with force P. M. PubMed Scopus Google Scholar, Scholar) and In the of were used the was In the final we used the was were carried out on a using the with the as The of the final was analyzed using the J.A. J. 1996; Google Scholar) and P. J. N. R.J. T. Biol. 1998; PubMed Scopus Google Scholar) coli ATCC in phase were as described coli in broth were with HSP1 μm) at 37 °C for After were collected and in following at × for were in the of The were by in solution and After for the samples were at × for and the was in μl of The was and The was on the with a and was on the of the for The used in this experiment was a in the contact mode and at A with a of square and a with was The for the contact force of the on the was to The was to and initial of the crude extract from punctata skin secretion a the of a of collected fractions were lyophilized and to mass The fraction containing the peptide of was named Hylaseptin P1 and structure was by de novo sequencing and by automatic Edman in a for both and The sequence was and its mass was with an of and and and in the HSP1 in with the of Edman A of the with the N-terminal of the peptides from the from that the mass in a new Chemical phase Fmoc synthesis of which was purified by preparative and analytical RP-HPLC, using a gradient of and the native peptide was as a The purified synthetic HSP1 was also by MS/MS in order to its amino acid sequence and peptide HSP1 was tested against and for The was S. aureus ATCC 25923, and the strains were P. aeruginosa ATCC and coli ATCC 25992, obtained from the SABIN Laboratory culture The HSP1 were with for the The that HSP1 with against both and S. aureus P. aeruginosa and coli μm) activity of minimal peptide for total inhibition of cell In liquid were performed according to the of were performed in aureus ATCC no no no aeruginosa ATCC coli ATCC MIC, minimal peptide for total inhibition of cell In liquid were performed according to the of were performed in no in a new In the of HSP1 were for the inhibition of growth of Candida In this the were also Candida ATCC and albicans, isolated from and resistant to were tested with HSP1 and fungicidal at the The HSP1 against both strains were for of which the because this drug is known for its activity and against ATCC and isolated from HSP1 are by and minimal inhibitory minimal fungicidal concentration, by SABIN were performed according to the of performed in after h of resistant to was this is a drug ATCC was this is a drug MIC, minimal inhibitory MFC, minimal fungicidal concentration, by SABIN were performed according to the of performed in after h of resistant to was this is a drug in a new As for and that the and are for in this to a it is necessary to that they are The effect of is followed by of and skin a of effects 1999; PubMed Scopus Google Scholar, M. 2001; PubMed Scopus Google Scholar, S. J.H. 2001; PubMed Scopus Google Scholar). In the of its is by such as A. K. G. J. Sci. Scholar). for these that for of high and this HSP1 an The activity of HSP1 was by the peptide with in The concentration of 256 μg/ml (195.2 μm) used to HSP1 against the growth of the various a minimal effect to this concentration is the for HSP1 against S. The of the blood also that HSP1 in white a and In after h of with HSP1, at the used to and the number of blood types significant there was no in the mean the spectra of HSP1 in and in the of In the peptide is in a as by the at 200 The addition of a to an α-helical structure that a A is when the peptide is in the of In this was to the of HSP1 to a NMR the spectra that the peptide an α-helical in both and its solution structure was in a were performed on the use of and K. NMR of and Scholar). chemical and for the peptide were in the number 6 the of the chemical The from to the of an in that The of the for the peptide is in The with a and and a the peptide is in an α-helical with the of the chemical and with the of the and range for HSP1 in mixture at °C, The of the are by the of and are as medium, and to of and of 100 that were on the target were using the After on the were used to represent the solution structure of The of the the peptide is for the and for the the structural of the solution of for the of the solution of from in a new As by the from for and the a and The the high of the of HSP1, which of and in the the of the final of HSP1 and, the high of the that the peptide as an amphiphilic force was to the effect of HSP1 on the coli ATCC of the cells. The of a on the bacterial when using the peptide at concentration which was in the sample the initial of of the cell of the that the inhibition of bacterial growth is with the of the cell The dermal glands of antimicrobial peptides that protect the against and in The amino acid of these peptides are both and that the antimicrobial The of antimicrobial peptides with such in sequence and a wide of the of in providing with against microbes and, at the the of resistance to In this is the of drug D. Nicolas P. M. Eur. J. Biochem. PubMed Scopus Google Scholar). A sequence in the for the peptide HSP1 with the N-terminal of the antimicrobial lytic peptides known as from the skin secretion of the (10Morikawa N. Hagiwara K. Nakajima T. Biochem. Biophys. Res. 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A. 1987; 84: 5449-5453Crossref PubMed Scopus (2030) Google Scholar). In the membrane on the of with HSP1, with the of the cells, the that the of HSP1 to cell pathogens is on its interaction with the cell to a process of permeabilization and in cell these are in with the that antimicrobial activity of peptides is with to the membrane of pathogens (6Chen H.M. Clayton W.W. Sawyer W.H. Eur. J. Biochem. 2001; 268: 1659-1669Crossref PubMed Scopus (31) Google Scholar, 7Epand R.M. Vogel H.J. Biochim. Biophys. Acta. 1999; 1462: 11-28Crossref PubMed Scopus (1151) Google Scholar, 1999; PubMed Google Scholar). In the the of cationic antimicrobial peptides has and there is a growing of that role in defense against microbes is as important to the as cells, and (5Hancock R.E.W. Scott M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8856-8861Crossref PubMed Scopus (825) Google Scholar). The present of the cationic antimicrobial peptide HSP1, obtained from an frog in the Brazilian is an of important is the of and is the of are in to the of the peptide on the cell and to its