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• W1996289330 abstract "A 37-residue cationic antimicrobial peptide named mesentericin Y 10537 was purified to homogeneity from cell-free culture supernatant of the Gram-positive bacterium Leuconostoc mesenteroides. The complete amino acid sequence of the peptide, KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW, has been established by automated Edman degradation, mass spectrometry, and solid phase synthesis. Mesentericin Y 10537 contains a single intramolecular disulfide bond that forms a 6-membered ring within the molecule. Mesentericin Y 10537 was synthesized by the solid phase method. The synthetic replicate was shown to be indistinguishable from the natural peptide with respect to electrophoretic and chromatographic properties, mass spectrometry analysis, automated amino acid sequence determination, and antimicrobial properties. At nanomolar concentrations, synthetic mesentericin Y 10537 is active against Gram+ bacteria in the genera Lactobacillus and Carnobacterium. Most interestingly, the peptide is inhibitory to the growth of the food-borne pathogen Listeria. CD spectra of mesentericin Y 10537 in low polarity medium, which mimic the lipophilicity of the membrane of target organisms, indicated 30–40%α-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning over residues 17–31. To reveal the molecular basis of the specificity of mesentericin Y 10537 targetting and mode of action, NH2- or COOH-terminally truncated analogs together with point-substituted analogs were synthesized and evaluated for their ability to inhibit the growth of Listeria ivanovii. In sharp contrast with broad spectrum α-helical antimicrobial peptides from vertebrate animals, which can be shortened to 14–18 residues without deleterious effect on potency, molecular elements responsible for anti-Listeria activity of mesentericin Y 10537 are to be traced at once to the NH2-terminal tripeptide KYY, the disulfide bridge, the putative α-helical domain 17–31, and the COOH-terminal tryptophan residue of the molecule. It is proposed that the amphipathic helical domain of the peptide interacts with lipid bilayers, leading subsequently to alteration of the membrane functions, whereas residues 1–14 form part of a recognition structure for a membrane-bound receptor, which may be critical for peptide targetting. Because mesentericin Y 10537 is easy to synthesize at low cost, it may represent a useful and tractable tool as a starting point for the design of more potent analogs that may be of potential applicability in foods preservation. A 37-residue cationic antimicrobial peptide named mesentericin Y 10537 was purified to homogeneity from cell-free culture supernatant of the Gram-positive bacterium Leuconostoc mesenteroides. The complete amino acid sequence of the peptide, KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW, has been established by automated Edman degradation, mass spectrometry, and solid phase synthesis. Mesentericin Y 10537 contains a single intramolecular disulfide bond that forms a 6-membered ring within the molecule. Mesentericin Y 10537 was synthesized by the solid phase method. The synthetic replicate was shown to be indistinguishable from the natural peptide with respect to electrophoretic and chromatographic properties, mass spectrometry analysis, automated amino acid sequence determination, and antimicrobial properties. At nanomolar concentrations, synthetic mesentericin Y 10537 is active against Gram+ bacteria in the genera Lactobacillus and Carnobacterium. Most interestingly, the peptide is inhibitory to the growth of the food-borne pathogen Listeria. CD spectra of mesentericin Y 10537 in low polarity medium, which mimic the lipophilicity of the membrane of target organisms, indicated 30–40%α-helical conformation, and predictions of secondary structure suggested that the peptide can be configured as an amphipathic helix spanning over residues 17–31. To reveal the molecular basis of the specificity of mesentericin Y 10537 targetting and mode of action, NH2- or COOH-terminally truncated analogs together with point-substituted analogs were synthesized and evaluated for their ability to inhibit the growth of Listeria ivanovii. In sharp contrast with broad spectrum α-helical antimicrobial peptides from vertebrate animals, which can be shortened to 14–18 residues without deleterious effect on potency, molecular elements responsible for anti-Listeria activity of mesentericin Y 10537 are to be traced at once to the NH2-terminal tripeptide KYY, the disulfide bridge, the putative α-helical domain 17–31, and the COOH-terminal tryptophan residue of the molecule. It is proposed that the amphipathic helical domain of the peptide interacts with lipid bilayers, leading subsequently to alteration of the membrane functions, whereas residues 1–14 form part of a recognition structure for a membrane-bound receptor, which may be critical for peptide targetting. Because mesentericin Y 10537 is easy to synthesize at low cost, it may represent a useful and tractable tool as a starting point for the design of more potent analogs that may be of potential applicability in foods preservation. The production of gene-encoded antimicrobial peptides as an immune strategy is widely used in nature and has been conserved in evolution. As a first line of defense against infections, vertebrate and invertebrate animals have developed chemical defense systems based on cationic antimicrobial peptides 22–46 residues long that are synthesized and secreted by nonmyeloid cells (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Google Scholar, 2Nicolas P. Mor A. Annu. Rev. Microbiol. 1995; 49: 277-304Google Scholar, 3Boman H.G. Hultmark D. Annu. Rev. Microbiol. 1987; 41: 103-126Google Scholar, 4Hoffmann J.A. Hetru C. Immunol. Today. 1992; 13: 411-415Google Scholar). The microbicidal effects of these broad spectrum peptide antibiotics very likely result from their capacity to interact with membranes and to permeate the target cells. Gene-encoded antibiotics are considered ancestral effectors of immunity because microbicidal peptides, named bacteriocins, have also been used by a number of Gram-positive and Gram-negative bacteria for millions of years for containing the proliferation of organisms that are closely related or confined within the same ecological niche (5Jung G. Angew Chem. Int. Ed. Engl. 1991; 30: 1051-1068Google Scholar, 6James R. Ladzunski C. Pattus F. Bacteriocins, Microcins and Lantibiotics. Springer -Verlag, Berlin1992Google Scholar, 7Kolter R. Moreno F. Annu. Rev. Microbiol. 1992; 46: 141-163Google Scholar, 8Bierbaum G. Sahl H.G. Int. J. Med. Microbiol. Virol. Parasitol. Infect. Dis. 1993; 278: 1-22Google Scholar, 9Klaenhammer T.R. FEMS Microbiol. Rev. 1993; 12: 39-86Google Scholar, 10Nettles C.G. Barefoot S.F. J. Food Prot. 1993; 56: 338-356Google Scholar, 11Jack R.W. Tagg J.R. Ray B. Microbiol. Rev. 1995; 59: 171-200Google Scholar, 12Venema K. Venema G. Kok J. Trends Microbiol. 1995; 3: 299-304Google Scholar), helping the producing microbe to compete for limited resources. Gene-encoded peptides of the chemical defense so far isolated from eukaryotic and prokaryotic organisms differ in several respects from the “classical” antibiotics or secondary metabolites and may provide a wholly new approach to fighting infectious diseases and nocosomial infections (13Chin G.J. Marx J. Science. 1994; 264: 359Google Scholar). Whereas many antibiotics disable or kill pathogens over a period of days by inhibiting essential enzymes, most gene-encoded antimicrobial peptides kill microorganisms rapidly by destroying or permeating the microbial membrane and impairing the ability to carry out anabolic processes (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Google Scholar, 2Nicolas P. Mor A. Annu. Rev. Microbiol. 1995; 49: 277-304Google Scholar). In addition, antimicrobial peptides are of relatively small size and made as pre-proproteins that are processed to the mature peptide by dedicated pathways. These peptides are thus unlikely to face the same antimicrobial resistance mechanisms that limit current antibiotic use. In this regard, the bacteriocins produced by lactic acid bacteria have gained much attention as potentially useful food additives against food-borne pathogens (9Klaenhammer T.R. FEMS Microbiol. Rev. 1993; 12: 39-86Google Scholar, 10Nettles C.G. Barefoot S.F. J. Food Prot. 1993; 56: 338-356Google Scholar, 12Venema K. Venema G. Kok J. Trends Microbiol. 1995; 3: 299-304Google Scholar). Class I bacteriocins (lantibiotics) undergo extensive post-translational modifications and contain very unusual amino acids (5Jung G. Angew Chem. Int. Ed. Engl. 1991; 30: 1051-1068Google Scholar). Nisin, for instance, is a 34-residue peptide produced by Lactococcus lactis that is very active against most Gram-positive bacteria, including genera Lactococcus, Lactobacillus, Bacillus, Micrococcus, and Listeria and Staphylococcus aureus and Clostridium botulinum. In contrast, class II nonlanthionine-containing bacteriocins, such as the lactococcins, the pediocins, the lactacins, and leucocin A, are 36–44-amino acid peptides that are minimally modified (14Holo H. Nilssen O. Nes I.F. J. Bacteriol. 1991; 173: 3879-3887Google Scholar, 15Henderson J.T. Chopko A.L. van Wassenaar P.D. Arch. Biochem. Biophys. 1992; 295: 5-12Google Scholar, 16Hastings J.W. Sailer M. Johnson K. Roy K.L. Vederas J.C. Stiles M.E. J. Bacteriol. 1991; 173: 7491-7500Google Scholar, 17Barefoot S.F. Klaenhammer T.R. Antimicrob. Agents Chemother. 1984; 26: 328-334Google Scholar, 18Muriana P.M. Klaenhammer T.R. Appl. Environ. Microbiol. 1991; 57: 114-121Google Scholar). Most of these class II bacteriocins are potent against Listeria monocytogenes, Gram-positive pathogenic bacteria that are responsible for severe infections of the central nervous system following the absorption of contaminated dairy products (9Klaenhammer T.R. FEMS Microbiol. Rev. 1993; 12: 39-86Google Scholar). The approval and use of nisin as an additive in processed cheese spreads raised the interesting possibility that direct addition of bacteriocins, especially those belonging to class II that are easier and cheaper to produce either by chemical synthesis or genetic engineering, may provide a novel means of preserving foods from pathogenic bacteria. In recent years, a wealth of information has been gained about the effectiveness of class II bacteriocins against undesirable bacteria in vitro. However, most of these data have been obtained through the use of cell-free culture supernatants of bacteriocin-producing bacterial strains or semi-purified bacteriocins, and no class II bacteriocin has been chemically synthesized and assayed for antimicrobial activity. In addition, little if anything is known with regard to structural and conformational determinants that confer stability and activity to these peptides (11Jack R.W. Tagg J.R. Ray B. Microbiol. Rev. 1995; 59: 171-200Google Scholar). These studies would provide molecular models for the conception of more potent structural analogues and a starting point for the design of new preventive or therapeutic agents. 9-Fluorenylmethoxycarbonyl (Fmoc)-protected 1The abbreviations used are: Fmoc9-fluorenylmetoxycarbonylHPLChigh performance liquid chromatographyMICminimal inhibitory concentrationAcmacetamidomethylTricineN-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycineTFEtrifluoroethanol.L-amino acids and polyethylene glycol polystyrene-graft copolymer support (substituted at 0.18 molar eq/g) were from Milligen (Bedford, MA). Chemicals for peptide synthesis (dimethylformamide, dichloromethane, diisopropylcarbodiimide, hydroxybenzotriazol, piperidin, trifluroacetic acid, and acetonitrile) were obtained from commercial sources and were of the highest purity available. 9-fluorenylmetoxycarbonyl high performance liquid chromatography minimal inhibitory concentration acetamidomethyl N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine trifluoroethanol. Leuconostoc mesenteroides Y 105 was grown aerobically to the late exponential phase at 30°C for 18–20 h in lactobacilli MRS broth (DIFCO Laboratories, Inc., Detroit, MI). The indicator strain Listeria ivanovii 496 was grown at 30°C for 18 h in Tryptic Soy broth (DIFCO Laboratories). Lactobacilli MRS broth (1.5 liters) was inoculated with 2 ml of an overnight culture of L. mesenteroides Y 105 and incubated at 30°C. After incubation for 22 h (A600 = 2.2), the cells were removed by centrifugation at 6,000 × g for 20 min at 4°C, and the cell-free culture supernatant was fractionated with ammonium sulfate at 60% for 18 h at 4°C. After centrifugation at 11,000 × g for 30 min at 4°C, the resulting precipitate was dissolved in 45 ml 10% acetic acid and loaded on a calibrated gel filtration column (Sephadex G-50; 2.5 × 100 cm) equilibrated in 10% acetic acid. Fractions (11 ml) were collected at a flow rate of 15 ml/h and assayed for anti-Listeria activity as described below. Active fractions were pooled and evaporated under vacuum. The dried extract was dissolved in 15 ml of 10% acetonitrile containing 20 mM ammonium acetate, pH 6.7, and further fractionated on Sep-pak C-18 cartridges (Waters). After washing with 10% acetonitrile containing 20 mM ammonium acetate, the extract was eluted with 5 ml each of 50% acetonitrile in 20 mM ammonium acetate and 80% acetonitrile in 20 mM ammonium acetate. Fractions which displayed anti- Listeria activity were lyophilized, solubilized with 5 ml in 0.07% trifluoroacetic acid/water and loaded an a Lichrospher C-18 reverse-phase HPLC column (5 µm; 4.6 × 250 mm). After an initial 3-min wash in 25% acetonitrile in 0.1% trifluoroacetic acid/water, elution was achieved in 50 min at a flow rate of 0.8 ml/min with a 25–50% linear gradient of acetonitrile in 0.07% trifluoroacetic acid/water. Fractions were monitored for absorbance at 280 and 220 nm and for activity against the indicator strain L. ivanovii 496. The active fractions were further purified to homogeneity on HPLC using the same column and solvent system and lyophilized. Quantification of free thiols was achieved with the Ellman's reagent as described previously (19Nicolas P. Delfour A. Bousseta H. Morel A. Rholam M. Cohen P. Biochem. Biophys. Res. Commun. 1986; 140: 565-573Google Scholar). Sequence analyses were carried out on an Applied Biosystem 470 gas phase sequencer. Phenylthiohydantoin amino acids were detected with an on-line Applied Biosystem 120 A analyzer. Data collection and analysis were performed with an Applied Biosystem 900 A module calibrated with 25 pmol of phenylthiohydantoin amino acid standards. Alternatively, analysis were carried out on a Milligen 6600 solid phase sequencer after covalent binding of the samples (250 pmol) to Sequelon arylamide membranes. Phenylthiohydantoin amino acids were detected with an on-line HPLC column (Waters MS HPLC; SequaTag C-18 phenylthiohydantoin analysis column; 350 mm × 3.9 mm) developed with ammonium acetate (pH 4.8) and acetonitrile and calibrated with 15 pmol of phenylthiohydantoin amino acid standards. Data collection and analysis were performed with a Maxima-phenylthiohydantoin chromatography analysis software package (Dynamic Solution Corp., Division of Waters Chromatography, Milford MA). Mass spectral analyses were performed using a quadrupole-coupled electrospray mass spectrometer (VG Platform). The mass scale was calibrated using myoglobin. The accuracy was ± 0.1 atomic mass unit. Samples (25 pmol) were dissolved in a water/acetonitrile (1:1, v/v) mixture containing 0.2% formic acid and introduced via a capillary using a microliter syringe. An electrospray voltage of 5 kV was applied to the internal wall of the source at the origin of the liquid dispersion for an electrospray formation and ion extraction. Ions were detected and analyzed in the positive mode as a function of their m/z ratio. Fast atom bombardment spectrometry was carried out on a Kratos high field spectrometer operating at an accelerating voltage of 8 KV. Ions were analyzed in the positive mode as a function of their m/z ratio. Mesentericin Y 10537, [Lys10] mesentericin Y 10537, [Acm, Cys9,14] mesentericin Y 10537, leucocin A, [Ser9,14] mesentericin Y 10537, mesentericin Y 10537-[4-37], mesentericin Y 10537-[15-37], mesentericin Y 10537-[1-36], mesentericin Y 10537-[1-14]-[28-37], and mesentericin Y 10537-[1-8]-[28-37] were prepared by stepwise solid phase synthesis using 9-Fmoc polyamide active ester chemistry on a Milligen 9050 pepsynthesizer. All Nα-Fmoc-amino acids were from Milligen. Polyethylene glycol polystyrene resins were used for all peptides but mesentericin-[1-36], leucocin A, and [Lys10] mesentericin Y 10537, for which 4-hydroxymethylphenoxyacetic acid-linked polyamide/kieselguhr resin (pepsin Ka) were used. Side chain protections were tert-butyloxycarbonyl for lysine and histidine; 2,2,5,7,8-pentamethyl-6-chromansulfonyl for arginine; O-tert-butyl ester for glutamic acid; O-tert-butyl ether for serine, threonine, and tyrosine; and trityl for asparagine, cysteine, and histidine. Synthesis was carried out using a double-coupling protocol: Nα-Fmoc-amino acids (4.4 molar excess) were coupled for 30–60 min with 0.23 M diisopropylcarbodiimide in a mixture of dimethylformamide and dichloromethane (60:40, v/v). Acylation was checked after each coupling step by the Kaiser test. Cleavage of the peptidyl resins and side chain deprotection were carried out at a concentration of 40 mg of peptidyl resin in 1 ml of a mixture composed of trifluoroacetic acid, phenol, thioanisole, water, and ethyl methyl sulfide (82.5:5:5:5:2.5, v/v/v/v/v) for 2 h at room temperature. After filtering to remove the resin and ether precipitation at 20°C, the crude peptides were recovered by centrifugation at 5,000 × g for 10 min, washed three times with cold ether, dried under nitrogen, dissolved in 20% acetic acid, and lyophilized. To perform air oxidation of the thiols of cysteine residues, crude peptides were dissolved in distilled water, adjusted to pH 8, and allowed to stand under gentle stirring at 20°C. After 24 h, less than 5% free thiols remained as assessed by Ellman's method (20Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Google Scholar). After lyophilization, the crude oxidized peptides were purified by preparative reverse-phase HPLC on a Waters RCM compact preparative cartridge Deltapak C-18 (300 Å; 25 × 100 mm) eluted at a flow rate of 8 ml/min by a multistep linear gradient of acetonitrile in 0.1% trifluoroacetic acid in water. Homogeneity of the synthetic peptides was assessed by solid phase sequence analysis, mass spectrum analysis, and analytical HPLC on a Lichrospher ODS 2 column (5 µm, 4.6 × 250 mm) eluted at a flow rate of 0.8 ml/min by a linear gradient of acetonitrile in 0.1% trifluoroacetic acid/water. A summary of the production and characterization of the synthetic peptides is shown in Table I.Table I.Purity, yield, molecular mass, and cysteine oxydation state of synthetic mesentericin Y 10537 and related analogsSynthetic peptideYieldaYield of crude peptide recovered after ether precipitation and air oxydation, based on molar eq of starting resin.PuritybAs assessed by HPLC analysis on a Lichrospher ODS2 column (5 µm, 4.6 × 250 mm).Molecular masscMass spectral analysis was performed using an electrospray ionization spectrometer. Note that the molecular mass corresponds to those expected theoretically for oxidized peptides.Cysteine oxydation statedAs assessed by Ellman's method (20).%%atomic mass units%Mesentericin Y 1053747>953868.24 ± 0.1>99Leucocin A16>953930.80 ± 0.5>98[Lys10] mesentericin Y 105377>953895.30 ± 0.3>95[Acm Cys9,14] mesentericin Y 1053749>954012.40 ± 0.1not applicable[Ser9,14] mesentericin Y 1053742>953838.10 ± 0.3not applicableMesentericin Y 10537-[4-37]48>953414.00 ± 0.3>95Mesentericin Y 10537-[15-37]49>952371.10 ± 0.1not applicableMesentericin Y 10537-[1-36]15>953680.24 ± 0.1>95Mesentericin Y 10537-[1-14]-[28-37]24>95NDeND, not determined.>95Mesentericin Y 10537-[1-8]-[28-37]38>952010.20 ± 03not applicablea Yield of crude peptide recovered after ether precipitation and air oxydation, based on molar eq of starting resin.b As assessed by HPLC analysis on a Lichrospher ODS2 column (5 µm, 4.6 × 250 mm).c Mass spectral analysis was performed using an electrospray ionization spectrometer. Note that the molecular mass corresponds to those expected theoretically for oxidized peptides.d As assessed by Ellman's method (20Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Google Scholar).e ND, not determined. Open table in a new tab Assays were performed by an antagonism well diffusion method in Tryptic Soy-buffered agar plates (pH 7.4) to avoid organic acid inhibition (21Tagg J.R. Dajani A.S. Wannamaker L.W. Bacteriol. Rev. 1976; 40: 722-756Google Scholar) inoculated with a 1% (v/v) stationnary phase culture of the indicator strain L. ivanovii 496. Wells (diameter, 5 mm) were punched in the agar plates, and serial 2-fold dilutions of the peptides to be assayed were added to each well to give a final volume of 50 µl. The plate cultures were incubated at 30°C for 18 h. Inhibition of the growth of the indicator bacteria appeared as clear circular zones surrounding the wells. The reciprocal of the highest peptide dilution showing a 1-mm zone of inhibition around the well was arbitrarily defined as the number of units of bacteriocin activity. Each unit of bacteriocin activity was determined from two independent experiments performed in duplicate. Synthetic peptides were weighted in a microbalance and solubilized in water at the desired primary dilution. Concentrations were determined by measuring the optical density of primary dilutions at 280 nm. Reversibility of growth inhibition was assessed in liquid medium as follows. The indicator bacteria culture medium was incubated at 30°C to early exponential growth phase (108 cells/ml). Synthetic peptides at a final concentration of 3.5 µM were then added to the culture. After various incubation times, 1-ml aliquots of the suspension were drawn and centrifuged at 12,000 × g for 2 min. To verify the reversibility of the inhibition, the pellets were resuspended in 1 ml of sterile water, and serial dilutions were pour-plated with suitable agar medium. Colony-forming units were counted after incubation at 30°C for 48 h. To prepare mesentericin Y 10537-[21-37], mesentericin Y 10537-[29-37], mesentericin Y 10537-[1-28], and mesentericin Y 10537-[1-20], synthetic mesentericin Y 10537 (0.5 mg/ml in 100 mM ammonium bicarbonate, pH 7.8) was incubated either with endoproteinases Glu-C or Arg-C at an enzyme to substrate ratio of 1 to 20 (w/w). After incubation for 3 h at 37°C, the mixtures were heated at 100°C for 10 min. The resulting peptide fragments were separated by reverse-phase HPLC using a C-18 column eluted at a flow rate of 0.8 ml/min for 60 min with a 8–56% linear gradient of acetonitrile containing 0.07% trifluoroacetic acid. Fractions (0.8 ml) were monitored for absorbance at 220 and 280 nm and for activity against the indicator strain. Identity of each peptide peak was assessed by amino acid sequence analysis and mass spectrometry. Purified mesentericin Y 10537 preparations were examined using 16% polyacrylamide gel and 0.1 M Tris-Tricine, pH 8.8, to allow suitable resolution of small peptides. Solution samples (1–5 µg) were dissolved (v/v) in sample buffer (2×) containing 5% SDS, 12% glycerol, 2%β-mercaptoethanol, 10% Coomassie Brilliant Blue G, and 5% 1 M Tris-Cl, pH 6.8, and heated for 10 min at 100°C. Electrophoresis was done at constant voltage of 100 V for 2 h. Gels were fixed in 50% (v/v) methanol and 10% (v/v) acetic acid for 20 min and stained with Coomassie Brilliant Blue R-250 (Bio-Rad). To test for activity, stained or unstained polyacrylamide gels were washed with water for 12–16 h, placed into steril Petri dishes, and overlaid with 20 ml of Tryptic soy broth agar (15 g/liter) inoculated with 200 µl of a stationary phase culture of L. ivanovii. Dishes were incubated for 18 h at 30°C and examined for the size of the growth inhibition zones (22Bhunia A.K. Johnson M.C. Ray B. J. Ind. Microbiol. 1987; 2: 319-322Google Scholar). Peptide samples were dissolved in water (0.05 mg/ml) or in 25–75% trifluoroethanol/water (v/v). Spectra were obtained at room temperature using a quartz cuvette of 1-mm path length in a Jobin-Yvon Mark IV instrument linked to a Minc digital II microprocessor. Spectra represented average values from six separate recordings. The content of α-helix, β-sheet, and unordered structure were estimated as described (23Yang J.T. Wu C.S.C. Martinez H.M. Methods Enzymol. 1986; 130: 208-269Google Scholar). A bacteriocin was purified to homogeneity from a cell-free culture supernatant of L. mesenteroides by a four-step protocol involving ammonium sulfate precipitation, size fractionation, Sep-pak filtration, and reverse-phase HPLC. Activity against the indicator strain L. ivanovii was used as a functional assay. The absorbance profile at a wavelength of 280 nm of a gel filtration fractionation on a Sephadex G-50 column of a 60% ammonium sulfate precipitate of a cell-free culture supernatant of L. mesenteroides is shown in Fig. 1, along with the anti-Listeria activity profile. Fractions 15–20 containing the peak of the wide zone of anti-Listeria activity were pooled and fractionated on Sep-pak C-18 cartridges. The active material eluting at 50% acetonitrile was further purified by reverse-phase HPLC. As depicted in Fig. 2, the initial anti-Listeria activity from G-50 was recovered after a series of HPLC runs as a symetrical sharp peak eluting at 24.49 min and accounting for >95% of the eluted material. Inspection of the near UV spectra of the peak indicated the presence of the classical tryptophan signature (Fig. 2). Analysis of the purified peptide by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Brilliant Blue and overlay anti-Listeria assay revealed only a single active band in the 3.5-kDa size zone (Fig. 3), indicating that the bacteriocin has been purified to homogeneity. The concentration of peptide producing a 1-mm zone of growth inhibition against L. ivanovii in the well diffusion assay was estimated to be in the nanomolar range (see Table III). The purified bacteriocin (final yield, 17 µg/liter of culture supernatant) was directly subjected to amino acid sequence analysis and mass spectral analysis.Fig. 2Final purification step of mesentericin Y 10537performed on an analytical Lichrospher C-18 reverse-phase HPLC column (5 µm; 250 × 4.6 mm). A, elution was achieved in 50 min at a flow rate of 0.8 ml/min with a 25–50% linear gradient of acetonitrile in 0.07% trifluoroacetic acid/water. Synthetic mesentericin Y 10537 eluted at 24.49 min under the same experimental conditions (arrow). The solid line indicates absorbance at 280 nm, and the dashed line represents the percentage of acetonitrile. B, UV absorbance spectrum of the peak eluting at 24.49 min.View Large Image Figure ViewerDownload (PPT)Fig. 3SDS-polyacrylamide gel electrophoresis analysis of purified mesentericin Y 10537. Electrophoresis was performed in a 16% polyacrylamide gel in 0.1 M Tris-Tricine buffer, pH 8.8. The gel was stained with Coomassie Brilliant Blue R-250, washed with water for 12–16 h, placed into steril Petri dishes, overlaid with Tryptic soy broth agar containing L. ivanovii 496, and incubated for 18 h at 30°C. A, molecular size standards (ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; trypsin inhibitor, 21.5 kDa; lysozyme, 14.3 kDa; aprotinin, 6.5 kDa; insulin β-chain, 3.4 kDa; insulin α-chain, 2.35 kDa). B, purified mesentericin Y 10537.View Large Image Figure ViewerDownload (PPT)Table III.In vitro ability of synthetic mesentericin Y 10537 and related analogs to inhibit the growth of L. ivanoviiPeptideAmino acid sequenceaAmino acid residues are indicated by the single-letter code.Relative potencybThe relative potency of a peptide analog to inhibit the growth of L. ivanovii is expressed as the ratio (MIC for mesentericin Y 10537/MIC for the tested peptide) × 100.%Mesentericin Y 10537KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW100Mesentericin Y 10537cNatural mesentericin Y 10537 isolated from L. mesenteroides.KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW96Leucocin AKYYGNGVHCTKSGCSVNWGEAFSAGVHRLANGGNGFW50[Lys10] mesentericin Y 10537KYYGNGVHCKKSGCSVNWGEAASAGIHRLANGGNGFW65[Acm Cys9,14] mesentericin Y 10537KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW0.042[Ser9,14] mesentericin Y 10537KYYGNGVHSTKSGSSVNWGEAASAGIHRLANGGNGFW0.0049Mesentericin Y 10537-[4-37]GNGVHCTKSGCSVNWGEAASAGIHRLANGGNGFW0.0022Mesentericin Y 10537-[15-37]SVNWGEAASAGIHRLANGGNGFW<3.10−4Mesentericin Y 10537-[21-37]dProduced by treatment of synthetic mesentericin Y 10537 by Glu-C endoproteinase.AASAGIHRLANGGNGFW<3.10−4Mesentericin Y 10537-[29-37]eProduced by treatment of synthetic mesentericin Y 10537 by Arg-C endoproteinase.LANGGNGFW<3.10−4Mesentericin Y 10537-[1-36]KYYGNGVHCTKSGCSVNWGEAASAGIHRLANGGNGF0.01Mesentericin Y 10537-[1-28]eProduced by treatment of synthetic mesentericin Y 10537 by Arg-C endoproteinase.KYYGNGVHCTKSGCSVNWGEAASAGIHR<3.10−4Mesentericin Y 10537-[1-20]dProduced by treatment of synthetic mesentericin Y 10537 by Glu-C endoproteinase.KYYGNGVHCTKSGCSVNWGE<3.10−4Mesentericin Y 10537-[1-14]-[28-37]KYYGNGVHCTKSGCRLANGGNGFW<3.10−4Mesentericin Y 10537-[1-8]-[28-37]KYYGNGVHRLANGGNGFW<3.10−4a Amino acid residues are indicated by the single-letter code.b The relative potency of a peptide analog to inhibit the growth of L. ivanovii is expressed as the ratio (MIC for mesentericin Y 10537/MIC for the tested peptide) × 100.c Natural mesentericin Y 10537 isolated from L. mes" @default.
• W1996289330 created "2016-06-24" @default.
• W1996289330 creator A5014668083 @default.
• W1996289330 creator A5026092329 @default.
• W1996289330 creator A5026647128 @default.
• W1996289330 creator A5039882384 @default.
• W1996289330 creator A5042674789 @default.
• W1996289330 creator A5064537568 @default.
• W1996289330 creator A5086683190 @default.
• W1996289330 date "1996-06-01" @default.
• W1996289330 modified "2023-10-14" @default.
• W1996289330 title "Covalent Structure, Synthesis, and Structure-Function Studies of Mesentericin Y 10537, a Defensive Peptide from Gram-positive Bacteria" @default.
• W1996289330 cites W1485776927 @default.
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