Title: Temporins, Small Antimicrobial Peptides with Leishmanicidal Activity
Abstract: Leishmaniasis encompasses a wide range of infections caused by the human parasitic protozoan species belonging to the Leishmania genus. It appears frequently as an opportunistic disease, especially in virus-infected immunodepressed people. Similarly to other pathogens, parasites became resistant to most of the first-line drugs. Therefore, there is an urgent need to develop antiparasitic agents with new modes of action. Gene-encoded antimicrobial peptides are promising candidates, but so far only a few of them have shown anti-protozoa activities. Here we found that temporins A and B, 13-amino acid antimicrobial peptides secreted from the skin of the European red frog Rana temporaria, display anti-Leishmania activity at micromolar concentrations, with no cytolytic activity against human erythrocytes. To the best of our knowledge, temporins represent the shortest natural peptides having the highest leishmanicidal activity and the lowest number of positively charged amino acids (a single lysine/arginine) and maintain biological function in serum. Their lethal mechanism involves plasma membrane permeation based on the following data. (i) They induce a rapid collapse of the plasma membrane potential. (ii) They induce the influx of the vital dye SYTOX™ Green. (iii) They reduce intracellular ATP levels. (iv) They severely damage the membrane of the parasite, as shown by transmission electron microscopy. Besides giving us basic important information, the unique properties of temporins, as well as their membranolytic effect, which should make it difficult for the pathogen to develop resistance, suggest them as potential candidates for the future design of antiparasitic drugs with a new mode of action. Leishmaniasis encompasses a wide range of infections caused by the human parasitic protozoan species belonging to the Leishmania genus. It appears frequently as an opportunistic disease, especially in virus-infected immunodepressed people. Similarly to other pathogens, parasites became resistant to most of the first-line drugs. Therefore, there is an urgent need to develop antiparasitic agents with new modes of action. Gene-encoded antimicrobial peptides are promising candidates, but so far only a few of them have shown anti-protozoa activities. Here we found that temporins A and B, 13-amino acid antimicrobial peptides secreted from the skin of the European red frog Rana temporaria, display anti-Leishmania activity at micromolar concentrations, with no cytolytic activity against human erythrocytes. To the best of our knowledge, temporins represent the shortest natural peptides having the highest leishmanicidal activity and the lowest number of positively charged amino acids (a single lysine/arginine) and maintain biological function in serum. Their lethal mechanism involves plasma membrane permeation based on the following data. (i) They induce a rapid collapse of the plasma membrane potential. (ii) They induce the influx of the vital dye SYTOX™ Green. (iii) They reduce intracellular ATP levels. (iv) They severely damage the membrane of the parasite, as shown by transmission electron microscopy. Besides giving us basic important information, the unique properties of temporins, as well as their membranolytic effect, which should make it difficult for the pathogen to develop resistance, suggest them as potential candidates for the future design of antiparasitic drugs with a new mode of action. Leishmaniasis includes a wide variety of clinical symptoms caused by infection with different species of the genus Leishmania, a human protozoan parasite with a worldwide incidence of 12–14 million people affected. The disease is widespread mostly in tropical and subtropical countries (1Herwaldt B.L. Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1356) Google Scholar). Leishmania is a digenetic parasite with two major stages, differing widely in their antigenic pattern, metabolism, and plasma membrane composition (2Handman E. Adv. Parasitol. 1999; 44: 1-39Crossref PubMed Google Scholar): (i) the flagellated promastigote, and (ii) the intracellular nonflagellated amastigote. The promastigote dwells in the gut of the insect vector, the sandfly, and is transmitted by bite into the mammalian host. Then it invades the cells of the mononuclear phagocytic system and transforms into the amastigote, the pathological form of the parasite for vertebrates. At present, the only available treatment is based on chemotherapy with organic pentavalent antimonials as the first-line drugs (3Croft S.L. Yardley V. Curr. Pharm. Des. 2002; 8: 319-342Crossref PubMed Scopus (314) Google Scholar, 4Croft S.L. Coombs G.H. Trends Parasitol. 2003; 19: 502-558Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). However, their efficacy is rapidly eroding because of the increasing appearance of resistant clinical isolates (5Mbongo N. Loiseau P.M. Billion M.A. Robert-Gero M. Antimicrob. Agents Chemother. 1998; 42: 352-357Crossref PubMed Google Scholar, 6Seifert K. Matu S. Javier Perez-Victoria F. Castanys S. Gamarro F. Croft S.L. Int. J. Antimicrob. Agents. 2003; 22: 380-387Crossref PubMed Scopus (147) Google Scholar) and the severe side effects associated with the treatment (7Guerin P.J. Olliaro P. Sundar S. Boelaert M. Croft S.L. Desjeux P. Wasunna M.K. Bryceson A.D. Lancet Infect. Dis. 2002; 2: 494-501Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). Despite the availability of alternative drugs, such as amphotericin B or miltefosine, there is a real need for the development of new agents with new modes of action (4Croft S.L. Coombs G.H. Trends Parasitol. 2003; 19: 502-558Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). Among them, eukaryotic antimicrobial peptides (AMPs) 1The abbreviations used are: AMP, antimicrobial peptide; bisoxonol, bis-(1,3-diethylthiobarbituric) trimethine oxonol; CA(1–8)M(1–18), cecropin A-melittin hybrid (KWKLFKKIGIGAVLKVLTTGLPALISNH2); DMNPE-luciferin, d-luciferin-(1-(4,5-dimethoxy-2-nitrophenyl)-ethyl ester); HIFCS, heat-inactivated fetal calf serum; LPS, lipopolysaccharide; LPG, lipophosphoglycan; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.1The abbreviations used are: AMP, antimicrobial peptide; bisoxonol, bis-(1,3-diethylthiobarbituric) trimethine oxonol; CA(1–8)M(1–18), cecropin A-melittin hybrid (KWKLFKKIGIGAVLKVLTTGLPALISNH2); DMNPE-luciferin, d-luciferin-(1-(4,5-dimethoxy-2-nitrophenyl)-ethyl ester); HIFCS, heat-inactivated fetal calf serum; LPS, lipopolysaccharide; LPG, lipophosphoglycan; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. constitute a promising alternative. They are ubiquitous in pluricellular organisms, where they kill a wide variety of pathogens. Accordingly, they are located at the anatomical sites primarily involved in contact with microorganisms, such as mucosal, epithelial, or phagocytic cells (reviewed in Ref. 8Boman H.G. J. Intern. Med. 2003; 254: 197-215Crossref PubMed Scopus (848) Google Scholar). Skin secretions from Amphibia represent one of the richest sources for these molecules, because every species synthesizes its own particular set of defense peptides (9Simmaco M. Mignogna G. Barra D. Biopolymers. 1998; 47: 435-450Crossref PubMed Scopus (401) Google Scholar, 10Mor A. Drug Dev. Res. 2000; 50: 440-447Crossref Google Scholar, 11Nicolas P. Mor A. Annu. Rev. Microbiol. 1995; 49: 277-304Crossref PubMed Scopus (528) Google Scholar, 12Rinaldi A.C. Mangoni M.L. Rufo A. Luzi C. Barra D. Zhao H. Kinnunen P.K. Bozzi A. Giulio DiA. Simmaco M. Biochem. J. 2002; 368: 91-100Crossref PubMed Scopus (141) Google Scholar). Although AMPs differ significantly in their sequences, most of them share some features, such as a high positive charge and a potential to adopt an amphipathic α-helix and/or β-sheet structures upon their interaction with membranes. To date, there is compelling evidence that a common step in the microbial killing mechanism is their electrostatic interaction with the negatively charged cell membrane followed by its permeation/disruption (13Shai Y. Biochim. Biophys. Acta. 1999; 1462: 55-70Crossref PubMed Scopus (1549) Google Scholar, 14Shai Y. Biopolymers. 2002; 66: 236-248Crossref PubMed Scopus (1250) Google Scholar). During the last few years, a considerable number of studies have been carried out with AMPs on bacteria (15Friedrich C.L. Moyles D. Beveridge T.J. Hancock R.E.W. Antimicrob. Agents Chemother. 2000; 44: 2086-2092Crossref PubMed Scopus (399) Google Scholar, 16Wu M. Maier E. Benz R. Hancock R.E.W. Biochemistry. 1999; 38: 7235-7242Crossref PubMed Scopus (611) Google Scholar), fungi (17De Lucca A.J. Bland J.M. Jacks T.J. Grimm C. Walsh T.J. Med. Mycol. 1998; 36: 291-298Crossref PubMed Google Scholar, 18De Lucca A.J. Exp. Opin. Investig. Drugs. 2000; 9: 273-299Crossref PubMed Scopus (37) Google Scholar), viruses (19Wachinger M. Kleinschmidt A. von Winder D. Pechmann N. Ludvigsen A. Neumann M. Holle R. Salmons B. Erfle V. Brack-Werner R. J. Gen. Virol. 1998; 79: 731-740Crossref PubMed Scopus (171) Google Scholar, 20Robinson Jr., W.E. McDougall B. Tran D. Selsted M.E. J. Leukocyte Biol. 1998; 63: 94-100Crossref PubMed Scopus (172) Google Scholar), and tumor cells (21Baker M.A. Maloy W.L. Zasloff M. Jacob L.S. Cancer Res. 1993; 53: 3052-3057PubMed Google Scholar, 22Papo N. Shai Y. Biochemistry. 2003; 42: 9346-9354Crossref PubMed Scopus (160) Google Scholar) in an attempt to understand the parameters responsible for their activity. Nevertheless, reports on the activity and the mode of action of AMPs toward protozoan and metazoan parasites are very scarce (23Vizioli J. Salzet M. Trends Parasitol. 2002; 18: 475-476Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). For Leishmania, these include dermaseptins (24Feder R. Dagan A. Mor A. J. Biol. Chem. 2000; 275: 4230-4238Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and skin polypeptide tyrosine-tyrosine (25Vouldoukis I. Shai Y. Nicolas P. Mor A. FEBS Lett. 1996; 380: 237-240Crossref PubMed Scopus (70) Google Scholar), both isolated from the skin of frogs, gomesin, from the hemocytes of the tarantula spider, Acanthoscurria gomesiana (26Silva Jr., P.I. Daffre S. Bulet P. J. Biol. Chem. 2000; 275: 33464-33470Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar), and indolicidin (27Bera A. Singh S. Nagaraj R. Vaidya T. Mol. Biochem. Parasitol. 2003; 127: 23-35Crossref PubMed Scopus (123) Google Scholar), from granules of bovine neutrophils. In 1998, Diaz-Achirica et al. (28Diaz-Achirica P. Ubach J. Guinea A. Andreu D. Rivas L. Biochem. J. 1998; 330: 453-460Crossref PubMed Scopus (85) Google Scholar) and, more recently, Chicharro et al. (29Chicharro C. Granata C. Lozano R. Andreu D. Rivas L. Antimicrob. Agents Chemother. 2001; 45: 2441-2449Crossref PubMed Scopus (100) Google Scholar) and Luque-Ortega et al. (30Luque-Ortega J.R. Saugar J.M. Chiva C. Andreu D. Rivas L. Biochem. J. 2003; 375: 221-230Crossref PubMed Scopus (45) Google Scholar) investigated the leishmanicidal activity of highly positively charged cecropin A-melittin hybrid peptides. Their data suggested that the killing of the parasite was strongly correlated with plasma membrane permeabilization. Conversely, autophagic cell death has been described for indolicidin-treated parasites (27Bera A. Singh S. Nagaraj R. Vaidya T. Mol. Biochem. Parasitol. 2003; 127: 23-35Crossref PubMed Scopus (123) Google Scholar). Here we report the biological function and the mode of action of temporin A (FLPLIGRVLSGIL-NH2) and temporin B (LLPIVGNLLKSLL-NH2), both isolated from the frog Rana temporaria, against Leishmania promastigotes and amastigotes. Temporins are short (13-residue) peptides, with an amidated C terminus and with only one positively charged amino acid. They were first identified in R. temporaria skin secretions (31Simmaco M. Mignogna G. Canofeni S. Miele R. Mangoni M.L. Barra D. Eur. J. Biochem. 1996; 242: 788-792Crossref PubMed Scopus (287) Google Scholar) and further detected in several North American Rana species, such as Rana clamitans (32Halverson T. Basir Y.J. Knoop F.C. Conlon J.M. Peptides (Elmsford). 2000; 21: 469-476Crossref PubMed Scopus (67) Google Scholar), Rana luteiventris (33Goraya J. Wang Y. Li Z. O'Flaherty M. Knoop F.C. Platz J.E. Conlon J.M. Eur. J. Biochem. 2000; 267: 894-900Crossref PubMed Scopus (150) Google Scholar), Rana pipiens (33Goraya J. Wang Y. Li Z. O'Flaherty M. Knoop F.C. Platz J.E. Conlon J.M. Eur. J. Biochem. 2000; 267: 894-900Crossref PubMed Scopus (150) Google Scholar), and Rana grylio (34Kim J.B. Halverson T. Basir Y.J. Dulka J. Knoop F.C. Abel P.W. Conlon J.M. Regul. Pept. 2000; 90: 53-60Crossref PubMed Scopus (51) Google Scholar). Temporins, together with indolicidin, are among the smallest AMPs isolated so far from animal sources. However, in contrast with indolicidin, temporins are nonhemolytic (35Selsted M.E. Novotny M.J. Morris W.L. Tang Y.Q. Smith W. Cullor J.S. J. Biol. Chem. 1992; 267: 4292-4295Abstract Full Text PDF PubMed Google Scholar, 36Subbalakshmi C. Krishnakumari V. Nagaraj R. Sitaram N. FEBS Lett. 1996; 395: 48-52Crossref PubMed Scopus (119) Google Scholar). Previous studies indicated that these molecules are active mainly against Gram-positive bacteria, Candida albicans, and some human tumor cell lines (12Rinaldi A.C. Mangoni M.L. Rufo A. Luzi C. Barra D. Zhao H. Kinnunen P.K. Bozzi A. Giulio DiA. Simmaco M. Biochem. J. 2002; 368: 91-100Crossref PubMed Scopus (141) Google Scholar). Our data show that temporins A and B have leishmanicidal activity at concentrations that are not toxic to human red blood cells. In addition, in contrast to most AMPs, temporins preserve biological function in serum. Furthermore, studies on their mode of action suggest that they act directly on the membrane of the parasite and destroy its integrity; therefore, it should make it difficult for the pathogen to develop resistance. Besides providing important basic information, the small size of temporins, their destructive mode of action, and their ability to maintain activity in serum suggest them as attractive templates for further development of new antiparasitic drugs. Reagents—Bis-(1,3-diethylthiobarbituric) trimethine oxonol (bisoxonol), SYTOX™ Green, d-luciferin, 1-(4,5-dimethoxy-2-nitrophenyll) ethyl ester (DMNPE-luciferin) were obtained from Molecular Probes (Leiden, The Netherlands). Other reagents of the highest quality were purchased from Sigma or Merck. Parasites—Promastigotes of L. donovani (MHOM/S.D./00/1S-2D) were grown in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (HIFCS), antibiotics, and 2 mm glutamine in a Bellco roller device (Ace Glass, Vineland, NJ) at 26 °C. The mutant R2D2 strain, defective in the biosynthesis of the repetitive phosphorylated disaccharide unit of lipophosphoglycan (LPG), was kindly provided by S. J. Turco (University of Kentucky School of Medicine). For this strain, 5 μg/ml of lectin ricin agglutinin (Ricinus communis) was added to the growth medium (37King D.L. Turco S.J. Mol. Biochem. Parasitol. 1988; 28: 285-293Crossref PubMed Scopus (69) Google Scholar). Promastigotes of the 3-Luc L. donovani strain were obtained from the aforementioned parental strain by transfection with the pX63NEO-3Luc expression vector encoding the gene for a cytoplasmic form of Photinus pyralis luciferase (30Luque-Ortega J.R. Saugar J.M. Chiva C. Andreu D. Rivas L. Biochem. J. 2003; 375: 221-230Crossref PubMed Scopus (45) Google Scholar). These parasites were grown under the same conditions as the parental strain, except for the additional supplement of 50 μg/ml geneticin to the growth medium. Leishmania pifanoi axenic amastigotes were grown at 32 °C in M199 medium (31100, Invitrogen) supplemented with 20% HIFCS, 5% trypticase, and 50 μg/ml hemin, pH 7.2 (38Pan A.A. McMahon-Pratt D. Honigberg B.M. J. Parasitol. 1984; 70: 834-835Crossref PubMed Scopus (10) Google Scholar). Peptide Synthesis—Temporin A and temporin B were synthesized by the standard Nα-Fmoc (N-(9-fluorenyl)methoxycarbonyl) amino acid derivatives with a [5-(4-Fmoc-aminomethyl-3,5-dimethoxy-phenoxy)]valeric acid polyethyleneglycol resin on an automated peptide synthesizer (Pioneer, Applied Biosystems, Foster City, CA). The purity of the peptides was confirmed by high pressure liquid chromatography analysis, and their sequence was determined by both automated Edman degradation, using a protein sequencer (Applied Biosystems, model AB476A), and mass spectral analysis with a matrix-assisted laser desorption ionization time-of-flight Voyager DE (Applied Biosystems). The concentration of the peptides was determined by quantitative amino acid analysis after acid hydrolysis using a Beckman System Gold instrument, equipped with an ion-exchange column and ninhydrin derivatization. Antibacterial Activity—The antibacterial activity of the peptides was tested by using an inhibition zone assay on agarose plates seeded with viable bacteria, according to Hultmark et al. (39Hultmark D. Engstrom A. Andersson K. Steiner H. Bennich H. Boman H.G. EMBO J. 1983; 2: 571-576Crossref PubMed Scopus (338) Google Scholar). 2-Fold serial dilution of the peptides in 20% ethanol (v/v) and in 33% heat-inactivated human serum was used (40Oren Z. Hong J. Shai Y. J. Biol. Chem. 1997; 272: 14643-14649Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). Activity of the Peptides on Leishmania Promastigotes and Amastigotes—Procyclic promastigotes and amastigotes were harvested at a late exponential phase, whereas metacyclic promastigotes were from cultures at a stationary phase. Parasites were then washed twice with Hanks' medium (136 mm NaCl; 4.2 mm Na2HPO4; 4.4 mm KH2PO4; 5.4 mm KCl; 4.1 mm NaHCO3), pH 7.2, supplemented with 20 mm d-glucose (Hanks-Glc) (41J.H. Hanks W.J. Proc. Soc. Exp. Biol. Med. 1958; 98: 188-192Crossref PubMed Scopus (196) Google Scholar) and resuspended in the same buffer at a final concentration of 2 × 107 parasites/ml. Aliquots of this suspension (120 μl) were incubated with the peptides for 60 min at 26 and 32 °C for promastigotes and amastigotes, respectively, and subsequently divided into 2 further aliquots (100 and 10 μl), which were used in the following two assays (29Chicharro C. Granata C. Lozano R. Andreu D. Rivas L. Antimicrob. Agents Chemother. 2001; 45: 2441-2449Crossref PubMed Scopus (100) Google Scholar). (i) Inhibition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction to insoluble formazan by mitochondrial reductases was used as a short term viability parameter of the parasites and assayed immediately after peptide incubation (28Diaz-Achirica P. Ubach J. Guinea A. Andreu D. Rivas L. Biochem. J. 1998; 330: 453-460Crossref PubMed Scopus (85) Google Scholar). To the 100-μl aliquot of parasite suspension, 1 ml of Hanks-Glc was added to slow down peptide activity. The parasites were then collected by centrifugation, resuspended in 100 μl of a 0.5 mg/ml MTT solution in Hanks-Glc, transferred into a 96-well culture microplate, and incubated for 2 h at 26 or 32 °C for promastigotes or amastigotes, respectively. The reduced formazan was solubilized by the addition of an equal volume of 10% (w/v) SDS, incubated overnight at 26 °C, and measured in a 450 Bio-Rad Microplate Reader equipped with a 595-nm filter. (ii) To measure inhibition of promastigotes proliferation, the 10-μl aliquot was added to 100 μl of RPMI medium, devoid of phenol red, and supplemented with 10% HIFCS. The surviving parasites were allowed to proliferate for 72 h; then 100 μl of MTT solution (1 mg/ml in Hanks-Glc) was added, and its reduction was measured as described above. For amastigotes, the inhibition of proliferation was performed in M199 growth medium. After 5 days, the cells were centrifuged, washed with Hanks', and resuspended in 0.5 mg/ml MTT solution. All assays were performed in triplicate, and the experiments were repeated twice. The results were normalized to those of the corresponding controls in the absence of the peptide. Modification of Bioenergetic Parameters—We carried out two different assays. (i) The collapse of membrane potential was estimated by the potentially sensitive anionic dye bisoxonol, as its fluorescence increases after its insertion into the membrane, once the cell is depolarized. Assays were performed under standard conditions, in the presence of 0.1 μm bisoxonol. Peptides were added at different concentrations, and changes in fluorescence were recorded continuously for about 30 min using a Fluorostat Galaxy microplate reader (BMG Labotechnologies, Offenburg, Germany), equipped with excitation and emission filters at 540 and 580 nm, respectively. Maximal depolarization was considered as that obtained with 2.5 μm CA(1–8)M(1–18) cecropin A-melittin hybrid (28Diaz-Achirica P. Ubach J. Guinea A. Andreu D. Rivas L. Biochem. J. 1998; 330: 453-460Crossref PubMed Scopus (85) Google Scholar). All assays were done in triplicate. (ii) For in vivo monitoring of intracellular ATP variation, the variation of intracellular ATP level was monitored as described previously (30Luque-Ortega J.R. Saugar J.M. Chiva C. Andreu D. Rivas L. Biochem. J. 2003; 375: 221-230Crossref PubMed Scopus (45) Google Scholar). Briefly, promastigotes transfected with the pX63NEO-3Luc expression vector were incubated for 15 min at 26 °C with 25 μm DMNPE-luciferin, a free membrane-permeable caged luciferase substrate. Changes in luminescence were measured in the Fluorostat Galaxy microplate reader fitted with luminescence optics, with readings averaging every 10 s. Peptides were added once the luminescence reached a plateau. This point was considered as time 0, and its luminescence value was taken as 100%. The decrease in luminescence was monitored continuously for 60 min. All assays were performed in triplicate, and the luminescence values were normalized to those of the corresponding controls in the absence of the peptide. Permeation of the Plasma Membrane of the Promastigotes and Amastigotes—We adapted the procedure described by Thevissen et al. (42Thevissen K. Terras F.R. Broekaert W.F. Appl. Environ. Microbiol. 1999; 65: 5451-5458Crossref PubMed Google Scholar) to assess the permeability of the Leishmania membrane. Briefly, the parasites were incubated with 1 μm SYTOX™ Green in Hanks-Glc for 15 min in the dark. After peptide addition, the increase in fluorescence, due to the binding of the dye to intracellular DNA, was measured in the same microplate reader described above, using 485- and 520-nm filters for excitation and emission wavelengths, respectively. Maximal membrane permeation was defined as that obtained after the addition of 0.1% Triton X-100 (29Chicharro C. Granata C. Lozano R. Andreu D. Rivas L. Antimicrob. Agents Chemother. 2001; 45: 2441-2449Crossref PubMed Scopus (100) Google Scholar). Electron Microscopy—Parasites were incubated with the peptides for 1 h and washed twice with phosphate-buffered saline. Afterward, parasites were fixed for 1 h with 5% (w/v) glutaraldehyde in phosphate-buffered saline, included with 2.5% (w/v) osmium tetraoxide, and gradually dehydrated in ethanol (30, 50, 70, 90, and 100% (v/v) for 30 min each) and propylene oxide (1 h). Finally, they were embedded in Epon 812 resin and observed with a Philips 2200 electron microscope as described previously (28Diaz-Achirica P. Ubach J. Guinea A. Andreu D. Rivas L. Biochem. J. 1998; 330: 453-460Crossref PubMed Scopus (85) Google Scholar). Other Methods—Hydrophobicity was calculated according to the normalized scale of Eisenberg (43Eisenberg D. Annu. Rev. Biochem. 1984; 53: 595-623Crossref PubMed Scopus (731) Google Scholar). LC50 (the concentration of peptide required to inhibit half of the maximum MTT reduction) and its 95% confidence limits were determined by the Litchfield and Wilcoxon procedure using the PHARM/PCS version 4 software package for PCs. The number of experiments analyzed was indicated in the legend of each figure. Temporins were tested for their activity and plausible mode of action on both promastigote and amastigote stages of Leishmania. The ability of a drug to kill the mammalian intracellular stage of the parasite is crucial for its potential to be developed as a therapeutic agent. Note that this form has been shown to be more resistant to other cationic AMPs than the corresponding form present in the insect host (29Chicharro C. Granata C. Lozano R. Andreu D. Rivas L. Antimicrob. Agents Chemother. 2001; 45: 2441-2449Crossref PubMed Scopus (100) Google Scholar). Inhibition of MTT Reduction in Promastigotes by Temporins A and B—Temporins A and B were tested for their ability to inhibit MTT reduction in Leishmania promastigotes at a concentration range of 5–25 μm. The assay was done either after 1-h incubation of the parasites with the peptides (short term effect) or after proliferation of the remaining viable protozoa at a 72-h incubation, as described under “Experimental Procedures.” The results are given in Fig. 1. The data shown in Fig. 1A reveal that, at a given peptide concentration, the percentage of inhibition for the short time effect was practically similar to the inhibition of proliferation, without taking into account the S.D. Moreover, both peptides exhibited a similar antiparasitic effect as indicated also by their corresponding LC50 values (Table I). The lethal concentrations against three bacterial strains (the Gram-negative Escherichia coli D21, the Gram-positive Bacillus megaterium Bm11, and Staphylococcus aureus Cowan I) as well as human erythrocytes are included in Table I for comparison. These results indicate that although the antibacterial potency of temporin A was ∼2-fold higher than that of temporin B, both peptides were similarly active toward the promastigotes, and at least 5-fold more efficient than cecropin A, a potent 36-residue AMP (44Steiner H. Hultmark D. Engstrom A. Bennich H. Boman H.G. Nature. 1981; 292: 246-248Crossref PubMed Scopus (1080) Google Scholar). Most interestingly, we found that, in contrast to most natural AMPs (40Oren Z. Hong J. Shai Y. J. Biol. Chem. 1997; 272: 14643-14649Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), temporins maintained high activity in serum (Table I). Furthermore, temporins inhibited MTT reduction of the metacyclic promastigotes (Fig. 1B), the circulating form of the parasite in the blood of an infected mammal for about 24 h before becoming amastigote, to a slightly lower extent than that found for the procyclic promastigotes (Fig. 1A).Table IAntimicrobial and hemolytic activity of temporin A, temporin B, and cecropin APeptideL. donovaniaL. donovani promastigotes were incubated with the corresponding peptides at different concentrations, and viability was evaluated by MTT reduction as described under “Experimental Procedures.” Values are expressed as LC50. 95% confidence limits determined by the Litchfield and Wilcoxon procedure are included in parentheses.E. coli D21bValues are taken from Ref. 31.B. megaterium Bm11cNumbers in parentheses were obtained in 33% human serum. S. aureus Cowan I is resistant to 33% human serum, whereas B. megaterium Bm11 is only slightly sensitive. Controls were done in 33% human serum without peptide.S. aureus Cowan IcNumbers in parentheses were obtained in 33% human serum. S. aureus Cowan I is resistant to 33% human serum, whereas B. megaterium Bm11 is only slightly sensitive. Controls were done in 33% human serum without peptide.Human erythrocytesbValues are taken from Ref. 31.LC50 (μm)Temporin A8.4 (7.5-9.4)11.91.1 (1.8)2.7 (3.4)>120Temporin B8.6 (7.2-8.9)21.01.3 (2.5)6.2 (6.7)>120Cecropin A>50dValues are taken from Ref. 28.0.30.5 (not active)>300>400a L. donovani promastigotes were incubated with the corresponding peptides at different concentrations, and viability was evaluated by MTT reduction as described under “Experimental Procedures.” Values are expressed as LC50. 95% confidence limits determined by the Litchfield and Wilcoxon procedure are included in parentheses.b Values are taken from Ref. 31Simmaco M. Mignogna G. Canofeni S. Miele R. Mangoni M.L. Barra D. Eur. J. Biochem. 1996; 242: 788-792Crossref PubMed Scopus (287) Google Scholar.c Numbers in parentheses were obtained in 33% human serum. S. aureus Cowan I is resistant to 33% human serum, whereas B. megaterium Bm11 is only slightly sensitive. Controls were done in 33% human serum without peptide.d Values are taken from Ref. 28Diaz-Achirica P. Ubach J. Guinea A. Andreu D. Rivas L. Biochem. J. 1998; 330: 453-460Crossref PubMed Scopus (85) Google Scholar. Open table in a new tab The Influence of External Parameters on the Activity of Temporins on Leishmania Promastigotes—To better understand the interaction of these peptides with the surface of the parasite, we carried out a systematic variation of the conditions used in the short term standard assay for determining the ability of temporins to inhibit MTT reduction. (i) To study the role of electrostatic interactions, we tested temporins in the absence and presence of 50 μg/ml heparin, a strongly anionic polysaccharide. Temporin A, at 15 μm, caused 90.8 ± 2.3% inhibition of MTT reduction in the absence of heparin (Fig. 1A), while after a 15-min incubation with heparin, prior to the addition of the peptide to the promastigotes, the inhibition was 78.4 ± 3.2%. This effect was clearly weaker than that obtained for other peptides having a higher cationic character such as CA(1–8)M(1–18) (28Diaz-Achirica P. Ubach J. Guinea A. Andreu D. Rivas L. Biochem. J. 1998; 330: 453-460Crossref PubMed Scopus (85) Google Scholar). In another assay, temporin A was assayed at 10 μm for its ability to inhibit the MTT reduction using two isosmotic incubation media as follows: the standard Hanks' medium (140