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• W2036860085 abstract "We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing d- and l-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K4X7W, where X designates Gly, Ala, Val, or Leu (designated d-X peptides). The data revealed that PA-d-G and PA-d-A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PA-d-L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both d-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PA-d-L and PA-d-V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections. We report on the synthesis, biological function, and a plausible mode of action of a new group of lipopeptides with potent antifungal and antibacterial activities. These lipopeptides are derived from positively charged peptides containing d- and l-amino acids (diastereomers) that are palmitoylated (PA) at their N terminus. The peptides investigated have the sequence K4X7W, where X designates Gly, Ala, Val, or Leu (designated d-X peptides). The data revealed that PA-d-G and PA-d-A gained potent antibacterial and antifungal activity despite the fact that both parental peptides were completely devoid of any activity toward microorganisms and model phospholipid membranes. In contrast, PA-d-L lost the potent antibacterial activity of the parental peptide but gained and preserved partial antifungal activity. Interestingly, both d-V and its palmitoylated analog were inactive toward bacteria, and only the palmitoylated peptide was highly potent toward yeast. Both PA-d-L and PA-d-V lipopeptides were also endowed with hemolytic activity. Mode of action studies were performed by using tryptophan fluorescence and attenuated total reflectance Fourier transform infrared and circular dichroism spectroscopy as well as transmembrane depolarization assays with bacteria and fungi. The data suggest that the lipopeptides act by increasing the permeability of the cell membrane and that differences in their potency and target specificity are the result of differences in their oligomeric state and ability to dissociate and insert into the cytoplasmic membrane. These results provide insight regarding a new approach of modulating hydrophobicity and the self-assembly of non-membrane interacting peptides in order to endow them with both antibacterial and antifungal activities urgently needed to combat bacterial and fungal infections. Together with the growing number of individuals with impaired host defenses, invasive mycoses have emerged as major causes of morbidity and mortality in the last decade (1Groll A.H. Shah P.M. Mentzel C. Schneider M. Just-Nuebling G. Huebner K. J. Infect. 1996; 33: 23-32Abstract Full Text PDF PubMed Scopus (686) Google Scholar, 2Minamoto G.Y. Rosenberg A.S. Med. Clin. N. Am. 1997; 81: 381-409Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 3Walsh T.J. Hiemenz J.W. Anaissie E. Infect. Dis. Clin. North Am. 1996; 10: 365-400Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 4Ellis M. Richardson M. de Pauw B. Hosp. Med. 2000; 61: 605-609Crossref PubMed Scopus (30) Google Scholar, 5Odds F.C. Brown A.J. Gow N.A. Trends Microbiol. 2003; 11: 272-279Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar). Although the spectrum of fungal pathogens has changed, the vast majority of invasive fungal infections are still due to Aspergillus and Candida species (4Ellis M. Richardson M. de Pauw B. Hosp. Med. 2000; 61: 605-609Crossref PubMed Scopus (30) Google Scholar, 5Odds F.C. Brown A.J. Gow N.A. Trends Microbiol. 2003; 11: 272-279Abstract Full Text Full Text PDF PubMed Scopus (896) Google Scholar, 6Denning D.W. J. Antimicrob. Chemother. 1991; 28: 1-16Crossref PubMed Google Scholar). Because of their eukaryotic nature fungal cells have only a restricted set of unique targets. This makes it difficult to selectively target fungal cells. Two major families have been used for more than two decades to combat fungi; these include azoles, which inhibit sterol formation, and polyenes, which bind to mature membrane sterols. However, the development of fluconazole resistance among different pathogenic strains and the high toxicity of amphotericin B (7Alexander B.D. Perfect J.R. Drugs. 1997; 54: 657-678Crossref PubMed Scopus (143) Google Scholar, 8Mukherjee P.K. Chandra J. Kuhn D.M. Ghannoum M.A. Infect. Immun. 2003; 71: 4333-4340Crossref PubMed Scopus (418) Google Scholar, 9Kontoyiannis D.P. Mantadakis E. Samonis G. J. Hosp. Infect. 2003; 53: 243-258Abstract Full Text PDF PubMed Scopus (103) Google Scholar) have prompted the studies of new antifungal agents with new modes of actions. The investigation of antimicrobial peptides, from a wide range of biological sources, and their synthetic derivatives is a novel inroad to new antifungal agents. Antimicrobial peptides are gene-encoded and are part of the innate immunity to the microbial invasion of microorganisms of all types. The most studied group are short (<40 amino acids), positively charged, linear peptides whose possible mechanisms have been reviewed in detail (10Bechinger B. Biochim. Biophys. Acta. 1999; 1462: 157-183Crossref PubMed Scopus (439) Google Scholar, 11Blondelle S.E. Perez-Paya E. Houghten R.A. Antimicrob. Agents Chemother. 1996; 40: 1067-1071Crossref PubMed Google Scholar, 12Dathe M. Wieprecht T. Biochim. Biophys. Acta. 1999; 1462: 71-87Crossref PubMed Scopus (636) Google Scholar, 13Tossi A. Sandri L. Giangaspero A. Biopolymers. 2000; 55: 4-30Crossref PubMed Scopus (1056) Google Scholar, 14Oren Z. Shai Y. Biopolymers. 1998; 47: 451-463Crossref PubMed Scopus (766) Google Scholar, 15Hwang P.M. Vogel H.J. Biochem. Cell Biol. 1998; 76: 235-246Crossref PubMed Scopus (293) Google Scholar, 16Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6852) Google Scholar). It is believed that most of these peptides are targeted to biological membranes, increase their permeability, and kill the cells. However, other additional mechanisms were proposed for some of them (17Hancock R.E. Rozek A. FEMS Microbiol. Lett. 2002; 206: 143-149Crossref PubMed Google Scholar). Studies that investigated the mode of action of native antimicrobial peptides that are also endowed with antifungal activity are limited and included, for example, LL-37 and dermaseptines (18Oren Z. Lerman J.C. Gudmundsson G.H. Agerberth B. Shai Y. Biochem. J. 1999; 341: 501-513Crossref PubMed Scopus (493) Google Scholar, 19Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shai Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20Strahilevitz J. Mor A. Nicolas P. Shai Y. Biochemistry. 1994; 33: 10951-10960Crossref PubMed Scopus (122) Google Scholar). These studies showed that they self-associate in solution and reach the membrane as oligomers (18Oren Z. Lerman J.C. Gudmundsson G.H. Agerberth B. Shai Y. Biochem. J. 1999; 341: 501-513Crossref PubMed Scopus (493) Google Scholar, 19Ghosh J.K. Shaool D. Guillaud P. Ciceron L. Mazier D. Kustanovich I. Shai Y. Mor A. J. Biol. Chem. 1997; 272: 31609-31616Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20Strahilevitz J. Mor A. Nicolas P. Shai Y. Biochemistry. 1994; 33: 10951-10960Crossref PubMed Scopus (122) Google Scholar). Previously, we reported on a new approach for increasing the hydrophobicity and self-assembly in a solution of antibacterial peptides that are not active on fungi (21Avrahami D. Shai Y. Biochemistry. 2002; 41: 2254-2263Crossref PubMed Scopus (165) Google Scholar). We showed that peptide oligomerization in solution is crucial for the peptide to endow antifungal activity. In another study we examined the effect of multiple substitutions of different amino acids regarding the structure, binding, and biological function of positively charged diastereomers (include both d- and l-amino acids) of linear lytic peptides (22Avrahami D. Oren Z. Shai Y. Biochemistry. 2001; 40: 12591-12603Crossref PubMed Scopus (72) Google Scholar). For this purpose, a group of diastereomeric peptides with the sequence K4X7W were synthesized, where X designates one of the aliphatic amino acids, Gly, Ala, Val, or Leu. The results revealed that the peptides containing Ala and Gly are not active against microorganisms, concomitant with their inability to bind membranes. This is in agreement with the notion that a threshold of hydrophobicity along with a defined structure is required for antimicrobial activity. Here, we conjugated palmitic acid to this series of diastereomers and investigated their biological function and mode of action. Very interestingly, we found that the most potent antibacterial and antifungal lipopeptides are those derived from the peptides containing Ala and Gly. Studies on the function and structure of the peptides together with their interaction with bacteria, fungi, and model membranes shed light on their mode of action. Furthermore, these studies demonstrated that a lipophilic tail can compensate for the hydrophobicity and amphipathic structure of the peptidic chain previously shown to be a prerequisite for antimicrobial activity. Materials—4-Methylbenzhydrylamine (MBHA) Rink amide resin was obtained from Calbiochem-Novabiochem AG (Switzerland). Other reagents used for peptide synthesis include trifluoroacetic acid (Sigma), piperidine (Merck), N,N-diisopropylethylamine (Sigma), N-hydroxybenzotriazole hydrate (Aldrich), and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and dimethylformamide (peptide synthesis grade, Bio-lab Ltd.). Egg phosphatidylcholine (PC), 1The abbreviations used are: PC, phosphatidylcholine; Fmoc, fluorenylmethoxycarbonyl; ATR-FTIR, attenuated total reflectance Fouriertransform infrared; CD, circular dichroism; hRBC, human red blood cells; PBS, phosphate buffered saline; PI, phosphatidylinositol; PE, phosphatidylethanolamine; HPLC, high performance liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; MIC, minimal inhibitory concentration; diS-C3-5, 3-3′-dipropylthiadicarbocyanine iodide; PA, palmitoylated. phosphatidylinositol (PI), and phosphatidylethanolamine (PE) ergosterol were purchased from Lipid Products (South Nutfield, UK). 3-3′-Dipropylthiadicarbocyanine iodide (diS-C3-5) and calcein were purchased from Molecular Probes (Junction City, OR). All other reagents were of analytical grade. Buffers were prepared in double-distilled water. Amphotericin B and trypsin (bovine pancreas) were purchased from Sigma. RPMI 1640 was purchased from Biological Industries (Beit Haemek, Israel). Proteinase K (fungal) was purchased from Beckman Instruments. Peptide Synthesis, Acylation, and Purification—Peptides were synthesized by a Fmoc solid phase method on Rink amide 4-methylbenzhydrylamine resin using an ABI 433A automatic peptide synthesizer. The lipophilic acid was attached to the N terminus of a resin-bound peptide using standard Fmoc chemistry. Briefly, after removal of the Fmoc from the N terminus of the peptide with a solution of 20% piperidine in dimethylformamide, the fatty acid (7 eq, 1 m in dimethylformamide)) was coupled to the resin under similar conditions used for the coupling of an amino acid. The peptides were cleaved from the resin by 95% trifluoroacetic acid and purified by reverse phase high performance liquid chromatography (HPLC) on a C18 (for non-lipidic peptides) or C4 (for the lipopeptides) Bio-Rad semi-preparative column (250 × 10 mm, 300-Å pore size, 5-μm particle size). The purified peptides were shown to be homogeneous (>99%) by analytical reverse phase HPLC. The elution time of the lipopeptides increased by ∼20 min, indicating an increase in hydrophobicity owing to the attachment of the fatty acid. Electrospray mass spectroscopy was used to confirm their molecular weight, and amino acid analysis was used to confirm the composition of the peptidic moiety. Antifungal Activity—The antifungal activity of the peptides and their lipophilic acid conjugated analogs was preformed according to the conditions of National Committee for Clinical Laboratory Standards document M27-A. The peptides were examined in sterile 96-well plates (Nunc F96 microtiter plates) in a final volume of 200 μl as follows. 100 μl of a suspension containing fungi at a concentration of 2 × 103 colony-forming units/ml in culture medium (RPMI 1640, 0.165 m MOPS, pH 7.0, with l-glutamine, without NaHCO3 medium) was added to 100 μl of water containing the peptide in serial 2-fold dilutions. The fungi were incubated for 24 h for opportunistic fungi or 48–72 h for Candida albicans and Cryptococcus neoformans at 35 °C using a Binder KB115 incubator. Growth inhibition was determined by measuring the absorbance at 620 nm in a microplate autoreader El309 (Bio-tek Instruments). Antifungal activity is expressed as the minimal inhibitory concentration (MIC), the concentration at which no growth was observed. The fungi used were Aspergillus fumigatus ATCC 26430, Aspergillus flavus ATCC 9643, Aspergillus niger ATCC 9642, C. albicans ATCC 10231, and C. neoformans ATCC MYA-422. Antibacterial Activity—The antibacterial activity of the peptides was examined in sterile 96-well plates (Nunc F96 microtiter plates) in a final volume of 100 μl as follows. Aliquots (50 μl) of a suspension containing bacteria at a concentration of 106 colony-forming units/ml in culture medium (LB medium) were added to 50 μl of water containing the peptide in serial 2-fold dilutions in water (prepared from a stock solution of 1 mg/ml peptide in water). Inhibition of growth was determined by measuring the absorbance at 492 nm with a Microplate autoreader El309 (Bio-tek Instruments) after an incubation of 18–20 h at 37 °C. Antibacterial activities were expressed as the MIC, the concentration at which no growth was observed after 18–20 h of incubation. The bacteria used were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii ATCC 19606, Bacillus subtilis ATCC 6051, and Staphylococcus aureus ATCC 6538P. Hemolysis of Human Red Blood Cells (hRBC)—Fresh hRBC with EDTA were rinsed 3 times with PBS (35 mm phosphate buffer/0.15 m NaCl, pH 7.3) by centrifugation for 10 min at 800 × g and resuspended in PBS. Lipopeptides dissolved in PBS were then added to 50 μl of a solution of the stock hRBC in PBS to reach a final volume of 100 μl (final erythrocyte concentration, 4% v/v). The resulting suspension was incubated with agitation for 60 min at 37 °C. The samples were then centrifuged at 800 × g for 10 min. The release of hemoglobin was monitored by measuring the absorbance of the supernatant at 540 nm. Controls for 0 hemolysis (blank) and 100% hemolysis consisted of hRBC suspended in PBS and Triton 1%, respectively. Preparation of Liposomes—Small unilamellar vesicles were prepared by sonication as described earlier (23Shai Y. Bach D. Yanovsky A. J. Biol. Chem. 1990; 265: 20202-20209Abstract Full Text PDF PubMed Google Scholar). Briefly, dry lipids were dissolved in CHCl3:MeOH (2:1 v/v). The solvents were then evaporated under a stream of nitrogen and then lyophilized overnight. The lipids were resuspended in the appropriate buffer (7 mg/ml) with vortexing, and the resulting lipid dispersions were sonicated (10–30 min) in a bath-type sonicator (G1125SP1 sonicator, Laboratory Supplies Company Inc.), until the turbidity had cleared. The vesicles were visualized using a JEOL JEM 100B electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan). Lipid films were prepared from a mixture of zwitterionic phospholipids and ergosterol (PC/PE/PI/ergosterol, 5:2.5: 2.5:1 w/w), a lipid composition used to mimic the major components of the outer leaflet of C. albicans (24Schneiter R. Brugger B. Sandhoff R. Zellnig G. Leber A. Lampl M. Athenstaedt K. Hrastnik C. Eder S. Daum G. Paltauf F. Wieland F.T. Kohlwein S.D. J. Cell Biol. 1999; 146: 741-754Crossref PubMed Scopus (382) Google Scholar). Circular Dichroism (CD) Spectroscopy—The CD spectra of the lipopeptides were measured with a Aviv 202 spectropolarimeter. The spectra were scanned with a thermostatic quartz optical cell with a path length of 1 mm. Each spectrum was recorded in an average time of 5 s at steps of 0.2 nm at a wavelength range of 260 to 190 nm. The lipopeptides were scanned at a concentration of 10–100 μm in PBS (35 mm phosphate buffer, 0.15 m NaCl, pH 7.3) and 100 μm in the presence of 1% lysophosphatidylcholine micelles. Fractional helicities (25Wu C.S. Ikeda K. Yang J.T. Biochemistry. 1981; 20: 566-570Crossref PubMed Scopus (251) Google Scholar, 26Greenfield N. Fasman G.D. Biochemistry. 1969; 8: 4108-4116Crossref PubMed Scopus (3332) Google Scholar) were calculated as, [θ]222-[θ]2220[θ]222100-[θ]2220(Eq. 1) where [θ]222 is the experimentally observed mean residue ellipticity at 222 nm, and values for [θ]2220 and [θ]222100, corresponding to 0 and 100% helix content at 222 nm, are estimated to be –2,000 and –32,000 deg·cm2/dmol, respectively (25Wu C.S. Ikeda K. Yang J.T. Biochemistry. 1981; 20: 566-570Crossref PubMed Scopus (251) Google Scholar). In Vivo Transmembrane Potential Depolarization Assay in Bacteria and Yeast—Membrane destabilization, which results in the collapse of transmembrane potential, was detected fluorimetrically by using a fluorescence dye (27Sims P.J. Waggoner A.S. Wang C.H. Hoffmann J.R. Biochemistry. 1974; 13: 3315-3330Crossref PubMed Scopus (767) Google Scholar) (see details below for bacteria and yeasts). The dye binds the plasma membrane owing to the cell transmembrane potential, resulting in a quenching of the dye fluorescence. Peptide-induced membrane permeation caused a dissipation of the transmembrane potential that was monitored by an increase in fluorescence due to release of the dye. Experiments were performed in sterile 96-well plates (Nunc F96 microtiter plates) in a final volume of 100 μl as follows. 50 μl of the cell suspensions and the dye were added to 50 μl of water containing the peptide in serial 2-fold dilutions. Membrane depolarization was monitored by an increase in the fluorescence of diS-C3-5 (excitation wavelength λex = 622 nm, emission wavelength λem = 670 nm). Transmembrane Potential Depolarization on the yeast C. neoformans—The assay was performed with intact yeast. More specifically, cells were centrifuged at 4000 × g for 5 min at 4 °C in a SS-34 rotor after incubation at 35 °C with agitation for 24 h in RPMI buffer (RPMI 1640, 0.165 m MOPS, pH 7.0, with l-glutamine and without NaHCO3). The cells were resuspended in PBS (without Ca2+ and Mg2+) to an inoculum of 4 × 103 colony-forming units/ml. Cells were incubated with 1 μm diS-C3-5 followed by fluorescence dequenching until a stable base line was achieved (∼50 min), indicating the incorporation of the dye onto the yeast membrane. Transmembrane Potential Depolarization in E. coli—Spheroplasts of the Gram-negative bacteria E. coli D21 were prepared by the osmotic shock procedure (28Frate M.C. Lietz E.J. Santos J. Rossi J.P. Fink A.L. Ermacora M.R. Eur. J. Biochem. 2000; 267: 3836-3847Crossref PubMed Scopus (22) Google Scholar). Bacteria were grown at 37 °C with agitation to the mid-log phase. Cells from cultures grown (A600 = 0.8) were harvested by centrifugation and washed twice with 10 mm Tris/H2SO4, 25% sucrose, pH 7.5. Cells were resuspended in the washing buffer containing 1 mm EDTA. After 10 min of incubation at 20 °C with rotary mixing, the cells were collected by centrifugation and resuspended immediately in cold (0 °C) water. After 10 min of incubation at 4 °C with rotary mixing, the spheroplasts were collected by centrifugation. The spheroplasts were then resuspended to A600 of 0.05 in a buffer containing 20 mm glucose, 5 mm HEPES, 1 m KCl, pH 7.3. The cells were incubated with 1 μm diS-C3-5 followed by fluorescence dequenching until a stable base line was achieved (∼2 h), indicating the incorporation of the dye into the bacteria membrane. ATR-FTIR Spectroscopy—Spectra were obtained with a Bruker equinox 55 FTIR spectrometer equipped with a deuterated triglyceride sulfate detector and coupled with an ATR device after removing the trifluoroacetate (CF3COO–) counterions, as has been described in detail in other studies (21Avrahami D. Shai Y. Biochemistry. 2002; 41: 2254-2263Crossref PubMed Scopus (165) Google Scholar). The sample was hydrated by exposure of the samples to an excess of deuterium oxide (D2O). The H/D exchange was considered complete due to the complete shift of the amide II band. Any contribution of D2O vapor to the absorbance spectra near the amide I peak region was eliminated by subtraction of the spectra of pure lipids equilibrated with D2O under the same conditions. ATR-FTIR Data Analysis—To resolve overlapping bands, we processed the spectra using PEAKFIT™ (Jandel Scientific, San Rafael, CA) software. Second-derivative spectra were calculated to identify the positions of the component bands in the spectra. These wave numbers were used as initial parameters for curve fitting with gaussian component peaks. Positions, bandwidths, and amplitudes of the peaks were varied until a good agreement between the calculated sum of all components and the experimental spectra was achieved (r2 > 0.999). The relative amounts of different secondary structure elements were estimated by dividing the areas of individual peaks assigned to a particular secondary structure by the whole area of the resulting amide I band. Analysis of the Polarized ATR-FTIR Spectra—The ATR electric fields of incident light were calculated (29Ishiguro R. Kimura N. Takahashi S. Biochemistry. 1993; 32: 9792-9797Crossref PubMed Scopus (83) Google Scholar, 30Harrick N.J. Internal Reflection Spectroscopy. Inter-science, New York1967Google Scholar) as follows. Ex=2cosθsin2θ-n212(1-n212)[(1+n212)sin2θ-n212](Eq. 2) Ey=2cosθ1-n212(Eq. 3) Ez=2sinθcosθ(1-n212)[(1+n212)sin2θ-n212](Eq. 4) where θ is the angle of a light beam to the prism normal at the point of reflection (45°), and n21 = n2/n1 (n1 and n2 are the refractive indices of ZnSe, taken as 2.4, and the membrane sample, taken as 1.5, respectively). Under these conditions, Ex, Ey, and Ez are 1.09, 1.81, and 2.32, respectively. The electric field components together with the dichroic ratio (defined as the ratio between absorption of parallel (to a membrane plane), Ap, and perpendicularly polarized incident light, As) are used to calculate the orientation order parameter, f, by the formula f=2(Ex2-RATREy2+Ez2)(3cos2α-1)(Ex2RATREy2-2Ez2)(Eq. 5) where α is the angle between the transition moment of the amide I vibration of an α-helix and the helix axis. Several values ranging from 27° to 40° were reported in the literature for α (31Tamm L.K. Tatulian S.A. Q. Rev. Biophys. 1997; 30: 365-429Crossref PubMed Scopus (631) Google Scholar). We used the values of 27° (29Ishiguro R. Kimura N. Takahashi S. Biochemistry. 1993; 32: 9792-9797Crossref PubMed Scopus (83) Google Scholar, 32Rothschild K.J. Clark N.A. Science. 1979; 204: 311-312Crossref PubMed Scopus (86) Google Scholar) and 39° (33Bradbury E.M. Brown L. Downie A.R. Elliott A. Fraser R.D.B. Hanby W.E. J. Mol. Biol. 1962; 5: 230-247Crossref PubMed Scopus (122) Google Scholar) for α. Lipid order parameters were obtained from the symmetric (∼2853 cm–1) and antisymmetric (∼2922 cm–1) lipid stretching modes using the same equations, setting α = 90° (29Ishiguro R. Kimura N. Takahashi S. Biochemistry. 1993; 32: 9792-9797Crossref PubMed Scopus (83) Google Scholar). Tryptophan Fluorescence Measurements—To determine the environment of the peptides we measured changes in the intrinsic tryptophan fluorescence in PBS and upon binding to vesicles. A peptide (1 μm) was added to PBS or PBS containing 1 mm PC/PE/PI/ergosterol (5:2.5:2.5:1) small unilamellar vesicles. Emission spectra were measured on a SLM-Aminco Series 2 Spectrofluorimeter with the excitation set at 280 nm using a 4-nm slit and recorded in the range of 300–400 nm (4-nm slit). In these studies small unilamellar vesicles were used to minimize differential light-scattering effects, (34Mao D. Wallace B.A. Biochemistry. 1984; 23: 2667-2673Crossref PubMed Scopus (180) Google Scholar) and the lipid/peptide molar ratio was kept high (1000:1) so spectral contributions of free peptide would be negligible. Four diastereomeric peptides were synthesized corresponding to the sequence KX3KWX2KX2K (X = Gly, Ala, Val, or Leu). They were designed to create a perfect amphipathic α-helical structure in their l form, as revealed by the Schiffer and Edmundson wheel projection (35Vogel H. FEBS Lett. 1981; 134: 37-42Crossref PubMed Scopus (166) Google Scholar). Each diastereomer contained four d-amino acids at positions 3, 4, 8,10, when X = Ala, Val, or Leu, or alternatively at positions 1, 5, 9, 12, when X = Gly. A Trp was introduced in each peptide to serve as an intrinsic fluorescent probe. All four peptides were amidated at their C termini and conjugated at their N termini to palmitic acid (PA), a linear saturated chain of 16 carbons (CH3(CH2)14COOH). Table I shows the sequence of the peptides and their designations and molecular weights.Table IPeptide sequences and designationsPeptide designationPeptide formulaSequenceMolecular weightPA-d-GPA-d-K1,5,9,12-K4G7W(CH3(CH2)14CO)-KGGGKWGGKGGK-NH21353PA-d-APA-d-A3,4,8,10-K4A7W(CH3(CH2)14CO)-KAAAKWAAKAAK-NH21451PA-d-VPA-d-V3,4,8,10-K4V7W(CH3(CH2)14CO)-KVVVKWVVKVVK-NH21647PA-d-LPA-d-L3,4,8,10-K4L7W(CH3(CH2)14CO)-KLLLKWLLKLLK-NH21746 Open table in a new tab Antibacterial activity was assayed against three strains of Gram-negative (P. aeruginosa, A. baumannii, and E. coli) and two strains of Gram-positive (S. aureus, B. subtilis) bacteria. Table II presents the MIC of the peptides against the tested bacteria. The data revealed that both d-V and its palmitoylated analog were devoid of antibacterial activity. However, in contrast to d-V, d-L was highly active toward all bacteria tested, but its palmitoylated analog was also devoid of antibacterial activity. Most interestingly, whereas both d-G and d-A were devoid of antibacterial activity, their palmitoylated analogs PA-d-G and PA-d-A were highly active toward all the bacteria tested.Table IIMIC of the peptides against bacteriaMIC (μm)Peptide designationGram-positive bacteriaGram-negative bacteriaS. aureus (ATCC 6538P)B. subtilis (ATCC 6051)P. aeruginosa (ATCC 27853)A. baumannii (ATCC 19606)E. coli (ATCC 25922)d-G>100>100>100>100>100PA-d-G12.53.126.252525d-A>100>100>100>100>100PA-d-A12.53.126.256.256.25d-V>10060>100>100>100PA-d-V>100>100>100>100>100d-L301.257.51515PA-d-L>100ND>100>100>100 Open table in a new tab We further tested the antifungal activity of the peptides against representative pathogenic yeasts and fungi. The list includes C. albicans, C. neoformans, A. fumigatus, A. flavus, and A. niger. The results are summarized in Table III. The data revealed that in all cases palmitoylation significantly increased the antifungal activity. Similar to the antibacterial activity, the lipopeptides PA-d-G and PA-d-A were the most active toward all the tested fungi and yeast, whereas the hydrophobic lipopeptides, PA-d-V and PA-d-L, are only partially active.Table IIIMIC of the peptides against yeasts and fungiMIC (μm)Peptide designationYeastFungiC. albicans (ATCC 10231)C. neoformans (ATCC MYA-422)A. fumigatus (ATCC 26430)A. flavus (ATCC 9643)A. niger (ATCC 9642)d-G>100>100>100>100>100PA-d-G3.1256.2512.51006.25d-A>100>100>100>100>100PA-d-A12.51.561008010d-V>10050>100>100>100PA-d-V3.1253.125>100>100>100d-L253.125>100>100>100PA-d-L>1003.1256.25>10025 Open table in a new tab The hemolytic activity of the lipopeptides against a highly diluted solution of human erythrocytes (4%) is shown in Fig. 1. The data revealed that PA-d-G and PA-d-A, which were highly active against bacteria, yeasts, and fungi showed low hemolytic activity. In contrast, PA-d-V and PA-d-L, which were practically devoid of antibacterial activity and only partially killed yeasts and fungi, were highly hemolytic. We monitored the fluorescence emission spectrum of the intrinsic tryptophan in aqueous solution. We found that the signal of the Trp emission in PA-d-V was fully quenched (Table IV), possibly because of close proximity between the tryptophans, which causes self-quenching. Because tryptophan is located along the peptide chain, we can assume that the peptidic moiety exists as an oligomer in solution, as suggested previously for the non-lipidic form (22Avr" @default.
• W2036860085 created "2016-06-24" @default.
• W2036860085 creator A5070485974 @default.
• W2036860085 creator A5077610815 @default.
• W2036860085 date "2004-03-01" @default.
• W2036860085 modified "2023-10-11" @default.
• W2036860085 title "A New Group of Antifungal and Antibacterial Lipopeptides Derived from Non-membrane Active Peptides Conjugated to Palmitic Acid" @default.
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