Lipopolysaccharide, a Key Molecule Involved in the Synergism between Temporins in Inhibiting Bacterial Growth and in Endotoxin Neutralization
2008; Elsevier BV; Volume: 283; Issue: 34 Linguagem: Inglês
10.1074/jbc.m800495200
ISSN1083-351X
AutoresMaria Luisa Mangoni, Richard M. Epand, Yosef Rosenfeld, Adi Peleg, Donatella Barra, Richard M. Epand, Yechiel Shai,
Tópico(s)Aquaculture disease management and microbiota
ResumoLipopolysaccharide (LPS) is the major structural component of the outer membrane of Gram-negative bacteria and shields them from a variety of host defense factors, including antimicrobial peptides (AMPs). LPS is also recognized by immune cells as a pathogen-associated molecular pattern and stimulates them to secrete pro-inflammatory cytokines that, in extreme cases, lead to a harmful host response known as septic shock. Previous studies have revealed that a few isoforms of the AMP temporin, produced within the same frog specimen, can synergize to overcome bacterial resistance imposed by the physical barrier of LPS. Here we found that temporins can synergize in neutralizing the LPS-induced macrophage activation. Furthermore, the synergism between temporins, to overcome the protective function of LPS as well as its endotoxic effect, depends on the length of the polysaccharide chain of LPS. Importantly, mode of action studies, using spectroscopic and thermodynamic methods, have pointed out different mechanisms underlying the synergism of temporins in antimicrobial and anti-endotoxin activities. To the best of our knowledge, such a dual synergism between isoforms of AMPs from the same species has not been observed before, and it might explain the ability of such amphibians to resist a large repertoire of microorganisms. Lipopolysaccharide (LPS) is the major structural component of the outer membrane of Gram-negative bacteria and shields them from a variety of host defense factors, including antimicrobial peptides (AMPs). LPS is also recognized by immune cells as a pathogen-associated molecular pattern and stimulates them to secrete pro-inflammatory cytokines that, in extreme cases, lead to a harmful host response known as septic shock. Previous studies have revealed that a few isoforms of the AMP temporin, produced within the same frog specimen, can synergize to overcome bacterial resistance imposed by the physical barrier of LPS. Here we found that temporins can synergize in neutralizing the LPS-induced macrophage activation. Furthermore, the synergism between temporins, to overcome the protective function of LPS as well as its endotoxic effect, depends on the length of the polysaccharide chain of LPS. Importantly, mode of action studies, using spectroscopic and thermodynamic methods, have pointed out different mechanisms underlying the synergism of temporins in antimicrobial and anti-endotoxin activities. To the best of our knowledge, such a dual synergism between isoforms of AMPs from the same species has not been observed before, and it might explain the ability of such amphibians to resist a large repertoire of microorganisms. Naturally occurring host-defense antimicrobial peptides (AMPs) 2The abbreviations used are:AMPantimicrobial peptideOMouter membraneLPSlipopolysaccharideKDOketodeoxyoctonic acidMBHAamide 4-methyl benzhydrylamineF-moc9-fluorenylmethoxycarbonylDIEAN,N-diisopropylethylamineHOBTN-hydroxybenzotriazole hydrateHBTU2-(1H-benzotriazole-1-yl)-1,1,3,3 tetramethyluronium hexafluorophosphateDMFdimethylformamideCFUcolony-forming unitsMHMueller-Hinton mediumMICminimal inhibitory concentrationFICfractional inhibitory concentrationPBSphosphate-buffered salineTNF-αtumor necrosis factor αITCisothermal titration calorimetryQUELSquasi-elastic light scatteringPIPES1,4-piperazinediethanesulfonic acidILinterleukin. are produced across virtually all forms of life as a primitive component of their innate immune system and are constitutively or inducibly expressed in response to invasion by pathogens (1Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1526) Google Scholar, 2Ganz T. Nat. Rev. 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There is compelling evidence that unlike current antibiotics, which interact strongly with specific target molecules such as proteins, many AMPs act by a nonspecific mechanism and kill microbes by disrupting their plasma membrane (3Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6852) Google Scholar, 10Shai Y. Biopolymers. 2002; 66: 236-248Crossref PubMed Scopus (1297) Google Scholar, 11Yeaman M.R. Yount N.Y. Pharmacol. Rev. 2003; 55: 27-55Crossref PubMed Scopus (2376) Google Scholar, 12Lohner K. Blondelle S.E. Comb. Chem. High Throughput Screen. 2005; 8: 241-256Crossref PubMed Scopus (191) Google Scholar). However, before reaching the plasma membrane, they must traverse the cell wall. In Gram-negative bacteria, this cell wall is surrounded by an asymmetric outer membrane (OM) containing primarily the amphiphilic lipopolysaccharide (LPS, endotoxin) in its outer leaflet. The LPS barrier is believed to be stabilized by LPS-associated cations (Mg2+) through salt bridges that neutralize the repulsive forces of adjacent LPS molecules. This leads to the formation of an oriented and tightly cross-linked leaflet that protects bacteria from a variety of host-defense hydrophobic molecules (13Nikaido H. Science. 1994; 264: 382-388Crossref PubMed Scopus (1271) Google Scholar, 14Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar), including some AMPs (15Rosenfeld Y. Barra D. Simmaco M. Shai Y. Mangoni M.L. J. Biol. Chem. 2006; 281: 28565-28574Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 16Papo N. Shai Y. J. Biol. Chem. 2005; 280: 10378-10387Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 17Sal-Man N. Oren Z. Shai Y. Biochemistry. 2002; 41: 11921-11930Crossref PubMed Scopus (69) Google Scholar). Note that for all enterobacterial and most nonenterobacterial strains, LPS is composed of three parts: (i) a hydrophobic moiety, also referred to as lipid A, consisting of fatty acid chains linked to two phosphorylated glucosamine residues; (ii) an oligosaccharide core, covalently bound to the lipid A via ketodeoxyoctonic acid (KDO); and (iii) a hydrophilic O-antigenic domain, composed of repeating saccharide units, which protrudes into the surrounding medium. The composition and the number of the O-antigen repeat units vary among different bacterial species (18Trent M.S. Stead C.M. Tran A.X. Hankins J.V. J. Endotoxin Res. 2006; 12: 205-223Crossref PubMed Scopus (269) Google Scholar, 19Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Zahringer U. Seydel U. Di Padova F. Schreier M. Brade H. Faseb J. 1994; 8: 217-225Crossref PubMed Scopus (1334) Google Scholar). antimicrobial peptide outer membrane lipopolysaccharide ketodeoxyoctonic acid amide 4-methyl benzhydrylamine 9-fluorenylmethoxycarbonyl N,N-diisopropylethylamine N-hydroxybenzotriazole hydrate 2-(1H-benzotriazole-1-yl)-1,1,3,3 tetramethyluronium hexafluorophosphate dimethylformamide colony-forming units Mueller-Hinton medium minimal inhibitory concentration fractional inhibitory concentration phosphate-buffered saline tumor necrosis factor α isothermal titration calorimetry quasi-elastic light scattering 1,4-piperazinediethanesulfonic acid interleukin. The immune system has evolved to recognize LPS as a pathogen-associated molecular pattern (PAMP). Upon its recognition, LPS stimulates the innate immune cells, inducing the secretion of pro-inflammatory cytokines (e.g. TNF-α, IL-1, IL-6), mainly by mononuclear cells and macrophages (20Kubo Y. Fukuishi N. Yoshioka M. Kawasoe Y. Iriguchi S. Imajo N. Yasui Y. Matsui N. 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This can rapidly lead to the development of septic shock, which, in extreme cases, may lead to death (25Cohen J. Nature. 2002; 420: 885-891Crossref PubMed Scopus (2179) Google Scholar, 26Angus D.C. Wax R.S. Crit. Care Med. 2001; 29: S109-S116Crossref PubMed Scopus (695) Google Scholar). In contrast with these conventional antibiotics, several AMPs possess dual functions: they kill bacteria and neutralize the endotoxic effect of LPS, although the exact mechanism is not yet well understood (27Hancock R.E. Diamond G. Trends Microbiol. 2000; 8: 402-410Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar, 28Scott M.G. Davidson D.J. Gold M.R. Bowdish D. Hancock R.E. J. Immunol. 2002; 169: 3883-3891Crossref PubMed Scopus (576) Google Scholar, 29Bommineni Y.R. Dai H. Gong Y.X. Soulages J.L. Fernando S.C. Desilva U. Prakash O. Zhang G. Febs J. 2007; 274: 418-428Crossref PubMed Scopus (71) Google Scholar, 30Mookherjee N. Rehaume L.M. Hancock R.E. Expert Opin. Ther. Targets. 2007; 11: 993-1004Crossref PubMed Scopus (91) Google Scholar). Most living organisms produce a single AMP at the site of infection, but some animal species, particularly those from Amphibia, synthesize and secrete different isoforms of the same AMP. Among such isoforms, temporins represent the largest family, with more than 50 members (31Mangoni M.L. Marcellini H.G. Simmaco M. J. Pept. Sci. 2007; 13: 603-613Crossref PubMed Scopus (47) Google Scholar). They are among the smallest amphipathic α-helical AMPs found in nature to date (10–14 amino acids) and contain only a few positively charged amino acids (net charge at a neutral pH ranging from 0 to +3) (32Simmaco M. Mignogna G. Canofeni S. Miele R. Mangoni M.L. Barra D. Eur. J. Biochem. 1996; 242: 788-792Crossref PubMed Scopus (303) Google Scholar, 33Mangoni M.L. Cell Mol. Life Sci. 2006; 63: 1060-1069Crossref PubMed Scopus (144) Google Scholar). The majority of them act by increasing the permeability of the microbial membrane (34Mangoni M.L. Rinaldi A.C. Di Giulio A. Mignogna G. Bozzi A. Barra D. Simmaco M. Eur. J. Biochem. 2000; 267: 1447-1454Crossref PubMed Scopus (153) Google Scholar, 35Mangoni M.L. Papo N. Barra D. Simmaco M. Bozzi A. Di Giulio A. Rinaldi A.C. Biochem. J. 2004; 380: 859-865Crossref PubMed Scopus (135) Google Scholar, 36Mangoni M.L. Saugar J.M. Dellisanti M. Barra D. Simmaco M. Rivas L. J. Biol. Chem. 2005; 280: 984-990Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Recently, the physiological significance of the existence of multiple forms of temporins has been addressed and has revealed that temporins A and B strongly synergize in killing Gram-negative bacteria, when each is combined with temporin L (15Rosenfeld Y. Barra D. Simmaco M. Shai Y. Mangoni M.L. J. Biol. Chem. 2006; 281: 28565-28574Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Temporin L has also been studied for its potential to suppress the endotoxic effect of LPS (37Giacometti A. Cirioni O. Ghiselli R. Mocchegiani F. Orlando F. Silvestri C. Bozzi A. Di Giulio A. Luzi C. Mangoni M.L. Barra D. Saba V. Scalise G. Rinaldi A.C. Antimicrob. Agents Chemother. 2006; 50: 2478-2486Crossref PubMed Scopus (61) Google Scholar). In this study we investigated: (i) the role of the carbohydrate region of LPS in the mechanism underlying the synergistic effect of temporins against Gram-negative bacteria; and (ii) the potential of temporins to synergize in the detoxification of LPS, and whether the underlying molecular mechanism is similar to that of the synergism against Gram-negative bacteria. Importantly, we found a strong synergism between different isoforms of temporins in the inhibition of TNF-α release from macrophages stimulated with LPS. In addition, our data emphasize a different mode of action for the synergistic effect of these AMPs in antimicrobial and anti-endotoxin activities, which show dependence on the size of the polysaccharide chain of the bacterial LPS. Materials—Rink amide 4-methyl benzhydrylamine (MBHA) resin and 9-fluorenylmethoxycarbonyl (F-moc)-protected amino acids were obtained from Calbiochem-Novabiochem. Other reagents used for peptide synthesis included trifluoroacetic acid (Sigma), piperidine (Merck), N,N-diisopropylethylamine (DIEA, Sigma), N-hydroxybenzotriazole hydrate (HOBT, Sigma), 2-(1H-benzotriazole-1-yl)-1,1,3,3 tetramethyluronium hexafluorophosphate (HBTU, peptide synthesis grade, Bio-Lab), and dimethylformamide (DMF, peptide synthesis grade, Bio-Lab). Proteinase K and lipopolysaccharides from Escherichia coli O111:B4 (LPS O111:B4) and from E. coli O26:B6 (LPS O26:B6) were purchased from Sigma. All buffers were prepared in double glass-distilled water. Peptide Synthesis, Fluorescent Labeling, and Purification—Peptides were synthesized by an F-moc solid phase method on Rink amide resin, using an ABI 433A automatic peptide synthesizer. Cleavage of the peptides from the MBHA resin resulted in the amidation of the C terminus. To label the peptides, the F-moc protecting group was removed from the N terminus of the resin-bound peptides, by incubation with piperidine for 12 min, whereas all the other reactive amine groups of the attached peptides were kept protected. The resin-bound peptides were washed twice with DMF, and then treated with rhodamine-N-hydroxysuccinimide (2 equiv), in anhydrous DMF containing 2% DIEA, leading to the formation of a resin-bound N-rhodamine peptide. After 24 h, the resin was washed thoroughly with DMF and then with methylene chloride. The three rhodamine-labeled temporins A (rho-temporin A), B (rho-temporin B), and L (rho-temporin L) were then cleaved from the resin. All the peptides were purified by reversed-phase high performance liquid chromatography (RP-HPLC) on a C18 reversed-phase Bio-Rad semi-preparative column (250 × 10 mm, 300 Å pore size, 5-μm particle size). The column was eluted with a 40-min linear gradient of 20–60% acetonitrile in water, containing 0.05% trifluoroacetic acid (v/v), at a flow rate of 1.8 ml/min. The purified peptides were further subjected to amino acid analysis and electrospray mass spectrometry to confirm their composition and molecular weights. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)—LPS from E. coli O111:B4 and E. coli O26:B6 were separated by SDS-PAGE with a gel containing 12% acrylamide and 0.5% SDS (38Peterson A.A. McGroarty E.J. J. Bacteriol. 1985; 162: 738-745Crossref PubMed Google Scholar). Samples of 100 μg of LPS were applied to the gel, and LPS bands visualized by immunoblot analysis using anti-LPS core antibodies (mouse IgG, Hycult, Biotechnology, Netherlands). Antibacterial Activity of the Peptides—Susceptibility testing was performed by the microbroth dilution method according to the procedures outlined by the National Committee for Clinical Laboratory Standards (2001) using sterile 96-well plates. Aliquots (50 μl) of bacteria in mid-log phase at a concentration of 2 × 106 colony-forming units (CFU)/ml in culture medium (Mueller-Hinton, MH) were added to 50 μl of MH broth containing the peptide in serial 2-fold dilutions in 20% ethanol. Inhibition of growth was determined by measuring the absorbance at 600 nm with a 450-Bio-Rad Microplate Reader after an incubation of 18–20 h at 30 °C. Antibacterial activities were expressed as the minimal inhibitory concentration (MIC), the concentration of peptide at which 100% inhibition of growth was observed after 18–20 h of incubation. The ranges of peptide dilutions used were 0.15–40 μm for temporin L and 0.2–100 μm for temporins A and B. Synergism between temporins was evaluated by the checkerboard titration method, by adding combinations of two temporins, in a serial 2-fold dilution, to wells of a sterile flat-bottomed 96-well plate, each containing 1 × 105 CFU in a final volume of 100 μl. Briefly, wells in the first row were loaded with temporin L at twice the highest final concentration used in the experiment, and 2-fold dilutions were prepared across rows starting from the first one. Therefore, all wells in the same row kept temporin L at a constant concentration, whereas the concentration of temporin A or B varied in a serial 2-fold dilution. Note that the first column and the last row wells were loaded only with the single peptides (temporin L, A, or B, respectively), in a 2-fold dilution. The fractional inhibitory concentration (FIC) index for combinations of two peptides was calculated according to Equation 1,Σ(FICA+FICB)/n=Σ(A/MICA+B/MICB)/n(Eq. 1) where A and B are the MICs of drug A and drug B in the combination, MICA and MICB are the MICs of drug A and drug B alone, FICA and FICB are the FICs of drug A and drug B, and n is the number of wells per plate used to calculate the FIC. The FIC indices were interpreted as follows (39Lewis R.E. Diekema D.J. Messer S.A. Pfaller M.A. Klepser M.E. J. Antimicrob. Chemother. 2002; 49: 345-351Crossref PubMed Scopus (176) Google Scholar): FIC ≤ 0.5, synergy; 0.5 < FIC <1, additivity; 1 ≤ FIC < 4, indifference; FIC ≥ 4, antagonism. The following Gram-negative bacterial strains were used: E. coli O111:B4, E. coli O26:B6, and the cell wall-defective mutant strains of E. coli D21, i.e. D21 e7, D21 f1, and D21 f2, with shorter LPS carbohydrate chain length (40Boman H.G. Monner D.A. J. Bacteriol. 1975; 121: 455-464Crossref PubMed Google Scholar, 41Farnaud S. Spiller C. Moriarty L.C. Patel A. Gant V. Odell E.W. Evans R.W. FEMS Microbiol. Lett. 2004; 233: 193-199Crossref PubMed Google Scholar, 42Marvin H.J. ter Beest M.B. Witholt B. J. Bacteriol. 1989; 171: 5262-5267Crossref PubMed Google Scholar). The Effect of LPS on the Oligomeric State of the Peptides as Determined by Rhodamine Fluorescence Dequenching Measurements—Rhodamine-labeled peptides (final concentration, 3 μm for temporin A and 1.5 μm for both temporins B and L) were added to 100 μl of phosphate buffered saline (PBS), and changes in the intensity of the fluorescence emission were followed upon the addition of different concentrations of LPS O26:B6, using a microplate counter (Wallac 1420 Victor 3™, Perkin Elmer) with excitation and emission wavelengths set at 485 and 590 nm, respectively. Proteinase K (80 μg/ml in PBS) was then added, and the resulting fluorescence was monitored. An increase in fluorescence indicates that the peptide exists as an oligomer (43Papo N. Oren Z. Pag U. Sahl H.G. Shai Y. J. Biol. Chem. 2002; 277: 33913-33921Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). All fluorescence measurements were performed at 30 °C. Evaluation of TNF-α Release from RAW264.7 Macrophages—RAW264.7 macrophages were cultured overnight in 96-well plates (2 × 105 cells/well). The medium was then removed followed by the addition of fresh medium to each well. The cells were stimulated with LPS O111:B4 or LPS O26:B6 (10 ng/ml final concentration), in the presence of 2.5, 5, or 10 μm temporin A, B, or L. In another set of experiments, the effects of all combinations of temporins (A+B, A+L, or B+L, 2.5 μm of each) were tested. Cells that were stimulated with LPS alone and untreated cells served as controls. The cells were incubated for 4 h at 37 °C. Afterwards, samples of the medium from each treatment were collected. Concentrations of pro-inflammatory cytokines (TNF-α, IL-6) in the samples were evaluated using mouse enzyme-linked immunosorbent assay kits according to the manufacturer's protocol (BIOSOURCE (TNF-α); Biolegend (IL-6)). All experiments were repeated three times. Isothermal Titration Calorimetry (ITC)—LPS O26:B6, or LPS O111:B4, was diluted in 20 mm PIPES buffer, pH 7.4, containing 0.14 m NaCl and 1 mm EDTA, incorporated into the calorimeter cell (1.8 ml) and equilibrated to 38 °C in a MicroCal VP-ITC instrument (MicroCal Inc, Southampton, MA). Solutions of temporins A, B, and L were made at a concentration of 350 μm in matching buffer and placed in the syringe. A volume of 10 μl of peptide (1.9 μm in the cell) was successively titrated into LPS with constant stirring at 300 rpm. Mixtures of temporins A+L, B+L, or A+B, containing 175 μm of each peptide were also injected into LPS in separate experiments. At the end of the titrations, the content of the cell was removed and kept for further processing. LPS is known to be a heterogeneous mixture in nature (38Peterson A.A. McGroarty E.J. J. Bacteriol. 1985; 162: 738-745Crossref PubMed Google Scholar) (see also Fig. 1), making it difficult to establish its molecular weight. In these studies, LPS O26:B6 and LPS O111:B4 were quantified with the Purpald reagent, which is a specific reagent for the detection of aldehydes, using glycerol as a standard (44Lee C.H. Tsai C.M. Anal. Biochem. 1999; 267: 161-168Crossref PubMed Scopus (145) Google Scholar). The molecular weight for the LPS monomers was determined assuming LPS is a single molecular species, which is very useful as an arbitrary mode of comparison among data found with different amounts of LPS. The obtained values (13,000 and 20,000 kDa for LPS O26:B6 and LPS O111: B4, respectively) were in agreement with those previously reported in the literature (45Ribi E. Anacker R.L. Brown R. Haskins W.T. Malmgren B. Milner K.C. Rudbach J.A. J. Bacteriol. 1966; 92: 1493-1509Crossref PubMed Google Scholar, 46McIntire F.C. Barlow G.H. Sievert H.W. Finley R.A. Yoo A.L. Biochemistry. 1969; 8: 4063-4067Crossref PubMed Scopus (36) Google Scholar, 47Srimal S. Surolia N. Balasubramanian S. Surolia A. Biochem. J. 1996; 315: 679-686Crossref PubMed Scopus (141) Google Scholar). Data were analyzed and plotted using the program Origin 5.0 and fitted with programs provided by the manufacturer. Quasi-elastic Light Scattering (QUELS)—To estimate the average size of the LPS particles, QUELS measurements were performed at 38 °C using a BIC 200SM/TurboCorr light scattering instrument (Brookhaven Instruments Corp, Holtsville, NY) measuring the scattered light at 90°. Samples of LPS O26:B6 collected from the calorimeter cell after titration with temporins, as well as from the original LPS suspension, were measured. Measurement of QUELS with LPS O111:B4 were conducted in samples diluted in HEPES buffer, pH 7.4, containing 0.14 m NaCl, in absence of EDTA. Size distributions obtained by QUELS were analyzed using the Contin method, as provided by the manufacturer. Synergism between Temporins Toward Bacteria—To investigate the role of the size of the polysaccharide chain of LPS in the molecular mechanism underlying the synergistic effect of temporins A (FLPLIGRVLSGIL-NH2) and B (LLPIVGNLLKSLL-NH2) when each is combined with temporin L (FVQWFSKFLGRIL-NH2) against Gram-negative bacteria, we tested the ability of all combinations of these peptides to inhibit the growth of E. coli O111:B4 and E. coli O26:B6. The latter strain is known to have short LPS polysaccharide chains, as indicated both by its low molecular weight bands in SDS-PAGE (Fig. 1) and by its low critical micelle concentration, which is considered to be directly proportional to the polysaccharide chain length of LPS (48Aurell C.A. Wistrom A.O. Biochem. Biophys. Res. Commun. 1998; 253: 119-123Crossref PubMed Scopus (110) Google Scholar). The antimicrobial activity of temporins was also assayed against three cell wall-defective mutant strains of E. coli D21, which have lower amounts of sugar residues in their LPS backbone. The first is E. coli D21 e7, which is devoid of the O-antigen domain and has a shorter outer oligosaccharide core. The second is E. coli D21 f1, lacking the O-antigen and the outer core region, and the third is E. coli D21 f2, carrying the only KDO-lipid A complex (40Boman H.G. Monner D.A. J. Bacteriol. 1975; 121: 455-464Crossref PubMed Google Scholar, 41Farnaud S. Spiller C. Moriarty L.C. Patel A. Gant V. Odell E.W. Evans R.W. FEMS Microbiol. Lett. 2004; 233: 193-199Crossref PubMed Google Scholar, 42Marvin H.J. ter Beest M.B. Witholt B. J. Bacteriol. 1989; 171: 5262-5267Crossref PubMed Google Scholar) (Fig. 2). The antimicrobial activity and FIC indices of temporins are illustrated in Table 1.TABLE 1MICs of temporins and FIC indices of their combination against several E. coli strainsBacterial strainsMICaMICs are presented as average values from three independent measurements.FIC indexbFIC indices were interpreted as follows: FIC ≤ 0.5, synergism; <0.5 FIC <1, additivity; ≤1 FIC <4, indifference; FIC ≥ 4.0, antagonism.Temp ATemp BTemp LTemp A + LTemp B + LTemp A + BμmE. coli O111:B410050100.480.500.56E. coli O26:B65050100.560.610.56E. coli D21 e725252.50.560.580.56E. coli D21 f112.5252.50.560.570.57E. coli D21 f26.2512.52.50.660.900.56a MICs are presented as average values from three independent measurements.b FIC indices were interpreted as follows: FIC ≤ 0.5, synergism; <0.5 FIC <1, additivity; ≤1 FIC <4, indifference; FIC ≥ 4.0, antagonism. Open table in a new tab The data revealed that A+L and B+L pairs exhibited a relatively greater synergistic effect on E. coli O111:B4 (FIC indices of 0.48 and 0.5, respectively) (39Lewis R.E. Diekema D.J. Messer S.A. Pfaller M.A. Klepser M.E. J. Antimicrob. Chemother. 2002; 49: 345-351Crossref PubMed Scopus (176) Google Scholar, 49Odds F.C. J. Antimicrob. Chemother. 2003; 52: 1Crossref PubMed Scopus (1681) Google Scholar, 50Pag U. Oedenkoven M. Papo N. Oren Z. Shai Y. Sahl H.G. J. Antimicrob. Chemother. 2004; 53: 230-239Crossref PubMed Scopus (68) Google Scholar) compared with temporins A+B. In contrast, FIC values ranging from 0.56 to 0.9 were found when the same peptide combinations were tested on E. coli O26:B6 and the other bacterial strains. The highest FIC values were obtained toward E. coli D21 f2, indicating the disappearance of a synergistic effect, in parallel to a shortening of the LPS carbohydrate domain. The Effect of LPS on the Organization of Temporins—To ascertain whether differences in the antimicrobial activity of temporins A+L or B+L against E. coli O111:B4 and E. coli O26:B6 reflected a different organization of the peptides when in contact with LPS with a different length of the polysaccharide region, we studied the effect of purified LPS O26:B6 (with short carbohydrate chains) on temporins that were labeled at their N terminus with rhodamine. Rhodamine is a fluorescent probe that is only slightly sensitive to the polarity of its environment and does not interfere with the antibacterial activity of the peptide (data not shown). When rhodamine-labeled monomers are self-associated, and the rhodamine probes are in proximity, the result is self-quenching of the fluorescence emission. However, a significant increase in the fluorescence intensity of the peptide, after treatment with a proteolytic enzyme, indicates that the peptide is self-associated (51Ghosh 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). Here, the fluorescence of the rho-peptides was measured before and after adding different concentrations of LPS. Fig. 3, A, B, and C show the results with rho-temporin A, rho-temporin B, and rho-temporin L, respectively. The addition of increasing concentrations of LPS O26:B6 (arrow at time 0 in Fig. 3) to labeled temporins A or B quickly decreased peptide fluorescence, in a dose-dependent manner, suggesting oligomerization of the two peptides. The strongest effect was achieved when the peptide:LPS ratio was 1:4. The addition of proteinase K (right arrow in Fig. 3) to LPS-treated rho-temporins A and B caused a rapid enhancement of fluorescence up to the initial level measured prior to the addition of LPS, thus confirming the induction of peptide assembly by LPS O26:B6. In contrast, the addition of LPS to rho-temporin L did not significantly affect its fluorescence, but raised it when the peptide: LPS molar ratio increased to 1:4 (Fig. 3C), highlighting a partial disaggregation of this peptide after contact with the endotoxin. Accordingly, proteolytic digestion of LPS-treated rho-temporin L induced an additional increase of fluorescence. Altogether, the overall results with LPS O26:B6 were quite similar to those previously reported for each of these temporins with LPS O111:B4 (15Rosenfeld Y. Barra D. Simmaco M. Shai Y. Mangoni M.L. J. Biol. Chem. 2006; 281: 28565-28574Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The Effect of Unlabeled Temporins on the Fluorescence of Rhodamine-labeled Peptides—To understand the reason for the lack of synergism in the activity of temporins A+L and B+L on E. coli O26:B6, we investigated the effect of temporin L on the oligomerization of A and B in the presence of LPS O26:B6 (Fig. 4) and compared it with that of LPS O111:B4 (15Rosenfe
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