Báo cáo khoa học: Salt-resistant homodimeric bactenecin, a cathelicidin-derived antimicrobial peptide potx

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Salt-resistant homodimeric bactenecin, acathelicidin-derived antimicrobial peptideJu Y. Lee1, Sung-Tae Yang1,3, Seung K. Lee1, Hyun H. Jung1, Song Y. Shin2, Kyung-Soo Hahm2and Jae I. Kim11 Department of Life Science, BioImaging Research Center, Gwangju Institute of Science and Technology, Korea2 Department of Bio-Materials, Graduate School and Research Center for Proteineous Materials, Chosun University, Gwangju, Korea3 Section on Membrane Biology, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and HumanDevelopment, National Institutes of Health, Bethesda, MD, USAOver the course of evolution, endogenous antimicro-bial peptides have assumed the role of providing a firstline of defense against pathogenic infections in bothmammalian and nonmammalian species [1]. Amongthese host defense peptides, the cathelicidins are char-acterized by conserved cathelin-like domains (theproregion) and highly variable C-terminal antimicro-bial domains [2] that enable them to be classified intothree structural classes: amphipathic a-helical peptides,b-hairpin peptides stabilized by disulfide bridges, andlinear Trp-rich, Pro-rich peptides [3,4]. Bactenecin, acathelicidin purified from the granules of bovineneutrophils, is a b-hairpin monomer with one intra-molecular disulfide bond, and has been shown to haveKeywordsantimicrobial peptides; bactenecin;dimerization; peptide–membrane interaction;salt resistanceCorrespondenceJ. I. Kim, Department of Life Science,Gwangju Institute of Science andTechnology, Gwangju 500-712, KoreaFax: +82 62 970 2484Tel: +82 62 970 2494E-mail: jikim@gist.ac.kr(Received 19 July 2007, revised 28 April2008, accepted 4 June 2008)doi:10.1111/j.1742-4658.2008.06536.xThe cathelicidin antimicrobial peptide bactenecin is a b-hairpin moleculewith a single disulfide bond and broad antimicrobial activity. The proformof bactenecin exists as a dimer, however, and it has been proposed thatbactenecin is released as a dimer in vivo, although there has been littlestudy of the dimeric form of bactenecin. To investigate the effect of bacten-ecin dimerization on its biological activity, we characterized the dimer’seffect on phospholipid membranes, the kinetics of its bactericidal activity,and its salt sensitivity. We initially synthesized two bactenecin dimers (anti-parallel and parallel) and two monomers (b-hairpin and linear). Under oxi-dative folding conditions, reduced linear bactenecin preferentially foldedinto a dimer forming a ladder-like structure via intermolecular disulfidebonding. As compared to the monomer, the dimer had a greater ability toinduce lysis of lipid bilayers and was more rapidly bactericidal. Interest-ingly, the dimer retained antimicrobial activity at physiological salt concen-trations (150 mm NaCl), although the monomer was inactivated. This saltresistance was also seen with bactenecin dimer containing one intermole-cular disulfide bond, and the bactenecin dimer appears to undergo multi-meric oligomerization at high salt concentrations. Overall, dimericbactenecin shows potent and rapid antimicrobial activity, and resists salt-induced inactivation under physiological conditions through condensationand oligomerization. These characteristics shed light on the features that apeptide would need to serve as an effective therapeutic agent.AbbreviationsABD, antiparallel dimer bactenecin; Acm, acetamidomethyl; CDB, C-terminal dimeric bactenecin; CFU, colony-forming unit; hRBC, human redblood cell; KCTC, Korean Collection for Type Cultures; MIC, minimal inhibitory concentration; MTB, monomeric turn bactenecin; NDB,N-terminal dimeric bactenecin; PDB, parallel dimer bactenecin; POPC, 1-palmitoyl-2-oleoyl-phosphatidylcholine; POPG, 1-palmitoyl-2-oleoyl-phosphatidylglycerol; SLB, Ser-substituted linear bactenecin.FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS 3911antibacterial activity against both Gram-negative andcertain Gram-positive bacteria [5]. In addition, two lin-ear variants of bactenecin, Bac2S and Bac2A, showsimilar activities against Gram-negative bacteria andstronger activities against Gram-positive bacteria [6],and Bac2A also acts as a potent chemoattractant,inducing chemotaxis in undifferentiated THP-1 cells[7]. However, although bactenecin has largely beenstudied as a monomeric molecule, its proform report-edly exists as a dimer formed by intermolecular disul-fide bridges in the C-terminal antimicrobial domain.Moreover, it is known that the synthetic cyclic peptideis mainly active against Gram-negative bacteria [6,8],whereas the isolated native peptide showed activityagainst both Escherichia coli and Staphylococcus aureus[5]. This suggests that it may be necessary to recon-sider the structure of the mature native bactenecinin vivo [9].Although b-hairpin bactenecin and its analogs havebeen the subjects of numerous studies, little is knownabout the antimicrobial activity of the dimeric form,or the way in which it interacts with the bacterialmembrane. That said, earlier studies suggest thatdimerization of antimicrobial peptides leads to theappearance of a more diverse spectrum of antimicro-bial activity than is exhibited by monomers. Forinstance, Tencza et al. reported that dimeric LLP1,which is a Cys-containing peptide derived from a lenti-virus envelope protein that spontaneously formsdisulfide-linked dimers, possesses much greater antimi-crobial activity against S. aureus than monomericLLP1 [10]. In addition, disulfide-dimerized magainin 2[(mag-N22C)2] induces membrane permeabilization atlower concentrations than the monomeric form [11]. Inthe case of the channel-forming peptide alamethicin,channels formed by covalent dimers displayed lifetimesat a particular conductance that were up to 170-foldlonger than those of monomers [12]. Consistent withthat finding, in many cases dimerization was closelyconnected to enhanced antimicrobial activity mediatedby the formation of pores or channels in the lipidmembrane [13,14].For effective use in clinical pharmacotherapy, anti-microbial peptides need to remain active in the pres-ence of physiological levels of salt (120–150 mmNaCl), and structural constraints such as dimerizationor Cys-knot formation are also related to the salt sen-sitivity of antimicrobial peptides. For instance, defen-sins, a group of b-form antimicrobial peptides, aregenerally degraded under high-salt conditions [15], butoxidized b-defensin (Defr-1), which contains five Cysresidues that associate to produce dimers through for-mation of various intramolecular and intermoleculardisulfide bridges, exhibits potent and broad-spectrumantimicrobial activity that is not suppressed at highsalt concentrations [16]. In addition, the study ofprotegrin-1 and rhesus theta defensin-1, which haveb-strand and cyclic structures, respectively, has shownthat structural rigidity resulting from Cys-stabilizationenables the peptides to retain activity against mostbacteria in high-salt environments [17].It was previously reported that bactenecin is toosmall to disrupt the bacterial membrane unless a multi-mer is involved in forming pores or channels [8], andthat the native peptide may occur in both monomericand dimeric forms [9,18]. To test that idea, in the pres-ent study we chemically synthesized two dimers thatadopt parallel and antiparallel conformations and twomonomers that adopt b-hairpin and linear confor-mations, and investigated their biological activities.Results and DiscussionPeptide folding and its characterizationTo investigate the effect of dimerization on the antimi-crobial activity of bactenecin, we designed four bacten-ecin derivatives with differing chemical ⁄ physicalproperties reflecting the interactions among their Cysresidues (Fig. 1). Under most oxidative folding condi-tions, reduced linear bactenecin folded into a specificform (yield, 70–80%) that trypsin digestion experi-ments revealed to be an antiparallel dimer [antiparalleldimer bactenecin (ADB)] (supplementary Figs S1 andS2). Because the majority of reduced linear bactenecinspontaneously dimerizes, even at very low oxidativefolding concentrations (e.g. 10 lm), we attempted tosynthesize monomeric turn bactenecin (MTB) by utiliz-ing an iodine oxidation strategy often used for oxida-tive cyclization of Cys-containing peptides having afree Cys residue and to remove protective S-acetami-domethyl (Acm) groups, although in this case therewas no S-Acm group [19]. Under these conditions,dimerization was completely blocked, and MTB wasobtained with a yield of about 90%. Interestingly, wefailed to produce any parallel dimer bactenecin (PDB)when the oxidative folding condition was applied tounprotected ADB or MTB peptide, suggesting thatADB is thermodynamically more favorable than PDBin an air oxidative folding pathway. As ADB andPDB differ only in the orientations of their twostrands with respect to one another, we suggest thatmainly unfavorable terminal charge repulsion inhibitsPDB formation. By adding one protective S-Acm toreduced linear bactenecin (Fig. 1), we were able toutilize an iodine oxidation strategy to synthesize PDBSalt-resistant homodimeric bactenecin J. Y. Lee et al.3912 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBSand successfully harvest the dimer after a sequentialtwo-step reaction leading to disulfide formation (yield,80%). The position of the Cys residue carrying theS-Acm group was alternated because the amino acidcompositions near the two Cys residues were similar toone another. Finally, we synthesized Ser-substitutedlinear bactenecin (SLB) to investigate the structuraland ⁄ or functional role of the disulfide bond.Conformational studiesWe used CD spectroscopy to estimate the secondarystructure of the bactenecin derivatives in buffer and ina membrane-mimicking environment achieved with theaddition of SDS (Fig. 2). Consistent with previouslyreported CD spectra [8], those for MTB showed a typi-cal type I b-turn structure with a negative band in thevicinity of 205 nm in both environments [20]. For thetwo dimers, ADB and PDB, a spectrum exhibiting anegative band at 210 nm was observed in buffer,whereas an ordered b-strand structure with amaximum near 200 nm and a minimum at 220 nm wasobtained in the presence of SDS micelles. This waswell fitted to typical b-strand globular proteins, whichshow a strong positive band near 200 nm and a nega-tive band below 220 nm [21,22]. A more ordered struc-ture indicated by the red-shift from 210 to 220 nm, aswell as the presence of a positive band at 200 nm, maybe caused not only by the interaction of b-strandswithin a given dimer, but also by the interaction ofb-strands between dimers. Interestingly, SLB showed adisordered structure in buffer but, upon interactionwith SDS micelles, the CD spectrum changed to onesimilar to those of the dimers. The spectral behaviorobserved for SLB suggests that linear bactenecin has astrong propensity to form a b-structure in a membraneenvironment, and may be indicative of the importanceof the dimeric structure for specific interactions withthe bacterial membrane.Antimicrobial and hemolytic activitiesThe peptides’ antimicrobial activities against selectedGram-positive and Gram-negative bacteria, as well astheir hemolytic activities, are summarized in Table 1.As previously reported, MTB was more potent againstGram-negative bacteria [minimal inhibitory concen-tration (MIC) = 2–4 lm] than against Gram-positivebacteria (MIC = 4–8 lm), and was without hemolyticactivity. ADB and PDB displayed activity similar tothat of MTB against Gram-negative bacteria, with somehemolytic activity (10–20% hemolysis at 100 lm), butexhibited about four times greater potency (MIC =1–2 lm) against Gram-positive bacteria. These resultsare consistent with those obtained with the isolatednative peptide, which displayed broad-spectrum antimi-crobial activity against Gram-positive and Gram-nega-tive bacteria, and suggests to us that it is probable thatnative dimeric forms are also active in vivo.It was previously reported that Bac2A, in which aCys residue was substituted with Ala, had somewhatbetter activity against Gram-positive bacteria thanMTB [6]. Like Bac2A, SLB also showed slightly betterantimicrobial activity against Gram-positive bacteriathan MTB, with no hemolytic activity. Taken togetherwith the results of the CD analysis, these findingssuggest that in a membrane environment, ADB, PDBand SLB take on a common b-structure that enablesbetter interaction with Gram-positive bacteria.Dye leakage from liposomesIt is well known that dimerization can cause a signifi-cant change in a peptide’s interaction with the bacte-MTBSLBH2N-RLCRIVVIRVCR-CO2HRLCRIVVIRVCRH2N-RLCRIVVIRVCR-CO2HH2N--CO2HAcmSHPDBADBH2N-RLSRIVVIRVSR-CO2HH2N-RLCRIVVIRVCR-CO2HH2N-RLCRIVVIRVCR-CO2HAcmSHAcmH2N-RLCRIVVIRVCR-CO2HH2N-RLCRIVVIRVCR-CO2HH2N-RLCRIVVIRVCR-CO2HAcmHO2C-RCVRIVVIRCLR-NH2H2N-RLCRIVVIRVCR-CO2HH2N-RLCRIVVIRVCR-CO2HFig. 1. Scheme employed for the synthesis of bactenecin and itsderivatives through formation of disulfide bridges. (A) ADB wasfolded in 2M acetic acid ⁄ H2O ⁄ dimethylsulfoxide (1 : 2 : 1, v ⁄ v ⁄ v)solution for 24 h at room temperature. (B) MTB was oxidized inacetic acid ⁄ H2O (4 : 1, v ⁄ v) solution, after which iodine was added(10 equivalents to the number of disulfide bonds). (C) PDB wasprepared in two steps: air oxidation in distilled water at 47 °C wascarried out for 5 days, after which the partially oxidized peptideswere dissolved in acetic acid ⁄ H2O (4 : 1, v ⁄ v) solution, and iodinewas added (10 equivalents to the number of disulfide bonds) for2h.J. Y. Lee et al. Salt-resistant homodimeric bactenecinFEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS 3913rial membrane, whether or not it enhances the antimi-crobial activity of the peptide [10–14]. To assess theeffect of dimerization on peptide-induced membranedisruption leading to microbial cell death, we examinedthe capacity of peptides to release calcein fromliposomes composed of 1-palmitoyl-2-oleoyl-phosphat-idylglycerol (POPG) ⁄ 1-palmitoyl-2-oleoyl-phosphati-dylcholine (POPC) (1 : 1), which served as a model ofthe bacterial membrane (Fig. 3). At a molar pep-tide ⁄ liposome ratio of 1 : 10, PDB and ADB inducedleakage in about 90% and 70% of liposomes, respec-tively. By contrast, both MTB and SLB displayed onlyweak membrane lytic activity, with about 20% of lipo-somes showing leakage. In terms of the structure–activity relationships, it is noteworthy that ADB, PDBand SLB all assume a common b-structure in a mem-brane environment, despite the significant differencesin their membrane lytic activities. In that regard, atwo-step mechanism for membrane disruption leadingto leakage has been suggested [23]. The peptide first1060MTB SLB–100 02040MTB SLB–30–20–40–200m 2 · dmol–1)–40–601015PDB15 20ADB0 3 (deg·c m –10–5050 5 10 [ ] X1 –25–20–15–20–15–10–5190 200 210 220 230 240 250190 200 210 220 230 240 250Wavelength (nm)Fig. 2. CD spectra for MTB, SLB, ADB andPDB. Spectra were recorded at 25 °Cin10 mM sodium phosphate buffer (pH 7.4)(d)orin30mM SDS micelles ( ). Eachpeptide was used at a concentration of25 lM.Table 1. MIC (lM) values and hemolytic activities of the peptides.Results indicate the ranges of three independent experiments,each performed in triplicate. The hemolytic activity was determinedusing 100 lM peptide, and the results represent the means ofduplicate measurements from three independent assays.MIC (lM)MTB SLB ADB PDBBacterial strainS. aureus 4–8 2–4 1–2 1–2S. epidermidis 2–4 2 1–2 2B. subtilis 4 2 1–2 2E. coli 1–2 2–4 1–2 2–4P. aeruginosa 2–4 4–8 2–4 4Sa. typhimurium 4–8 4–8 2–4 4% Hemolysis 0 0 10 20100608020400.001 0.01 0.1 1 10Calcein release (%)0[Peptide]/[Lipid]Fig. 3. Calcein release from liposomes was measured as a functionof the molar peptide ⁄ lipid ratio. Peptide concentrations were 5 lMfor POPC ⁄ POPG (1 : 1) liposomes. Fluorescence from liposomeslysed with Triton X-100 was used as an indicator of 100% leakage.s, MTB; d, SLB; ,, ADB; ., PDB. Results represent the meansof three independent experiments.Salt-resistant homodimeric bactenecin J. Y. Lee et al.3914 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBSbinds to the membrane (the membrane affinity of thepeptide), after which it elicits membrane disruption(the membrane-perturbing activity). It is thus likelythat even though all three peptides exhibit a similarstructural transition upon binding to membrane sur-faces, only the dimers show enhanced membrane lyticactivity, perhaps due to induction of oligomerizationof the dimeric peptides by the hydrophobic membraneenvironment.Kinetics of the bactericidal activityTo further study the antibacterial activity of ADB andPDB, the kinetics of their bactericidal activity againstboth Gram-positive (S. aureus; Fig. 4A) and Gram-negative (E. coli; Fig. 4B) bacteria were investigated,with magainin 2 serving as a control. The time neededfor PDB and ADB to induce 100% cell death was aslittle as 5 min for Gram-positive bacteria, and bothpeptides showed the same kinetics. About 30 min ormore were needed to kill 100% of Gram-negative bac-teria, with PDB acting more rapidly than ADB. Thekinetics of MTB’s bactericidal activity were similar tothose of ADB for Gram-negative bacteria, but werevery slow for Gram-positive bacteria, with about 20%of cells remaining viable even after exposure for60 min. SLB acted almost as rapidly as ADB or PDBagainst Gram-positive bacteria, but acted more slowlythan the other three peptides against Gram-negativebacteria. Although, overall, PDB and ADB showedonly slightly greater antimicrobial activity than MTBand SLB, we suggest that the capacity of the dimers tokill bacteria quickly enough to prevent replicationgives them a greater ability to control bacterial expan-sion, thereby reducing the likelihood that resistancewill develop [24]. In other words, the rapidity withwhich bacteria are killed may be an important factorwhen evaluating the activity of antimicrobial peptidesin vivo and when assessing their potential for clinicaluse [25].Effect of saltStudies of cationic antimicrobial peptides have shownthat the salt concentration can affect their activity,even at less than physiological levels [26]. To determinewhether dimerization affects salt sensitivity, S. aureusand E. coli were exposed to 8 lm peptide in the pres-ence or absence of 150 mm NaCl. In the absence ofsalt, all four peptides killed 100% of the bacteria. Inits presence, the two dimers exhibited generally unal-tered activity against both Gram-positive (S. aureus;Fig. 5A) and Gram-negative (E. coli; Fig. 5B) bacteria.By contrast, the antimicrobial activity of MTB againstS. aureus was completely lost in the presence of150 mm NaCl, and the activity against E. coli wasreduced by > 60%. Although in a membrane environ-ment SLB showed a potency and CD pattern that weresimilar to those of the dimers, in the presence of150 mm NaCl, it killed only about 75% of S. aureusand was completely inactive against E. coli. It thusappears that Cys-derived dimerization enables thepeptides to retain potent bactericidal activity in thepresence of physiological levels of salt.Similarly, it was previously reported that the antimi-crobial activity of the guinea pig 11 kDa polypeptide,which is a homodimer joined by intermolecular disul-fide bonds, was unaffected by the presence of NaCl,whereas the activities of the guinea pig 5 kDa peptideand various defensins, which all contain intramoleculardisulfide bonds, were inactivated by NaCl [27].Together, these results strongly suggest that intermo-lecular disulfide connections contribute greatly toretention of a peptide’s antibacterial activity at highsalt concentrations.Peptide oligomerizationA high ionic strength may reduce the electrostaticinteraction between cationic peptides and anionic lipidhead groups through counterion screening, thereby80 100 A B 80 100 40 60 40 60 CFU·mL–1 (%)0 20 0 20 0 10 20 30 40 50 60 0 10 20 30 40 50 60Time (min)Fig. 4. Kinetics of the bactericidal activity ofthe four bactenecin derivatives againstS. aureus (A) and E. coli (B). Bacteria treatedwith the respective peptides (8 lM) werediluted at the indicated times and then pla-ted on LB agar. The CFUs were thencounted after 24 h of incubation at 37 °C.s, MTB; d, SLB; ,, ADB; ., PDB;, mag-ainin 2. Results represent the means of twoindependent experiments.J. Y. Lee et al. Salt-resistant homodimeric bactenecinFEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS 3915reinforcing the membrane surface region [23,28]. In thecase of defensin, it was reported that the number ofpositive charges in the molecule was directly propor-tional to its ability to retain antimicrobial activity athigher salt concentrations [28]. Consistent with thatrelationship, the bactenecin dimers used in the presentstudy have twice as many positively charged residuesas bactenecin monomer.On the other hand, the salt tolerance of dimeric bac-tenecins may reflect, to some degree, the structuralrigidity afforded by the two intermolecular disulfidebonds. To test that idea, we substituted the Cys resi-due at position 11 or 3 of bactenecin with a Ser andsynthesized two PDB derivatives containing a singledisulfide bond: N-terminal dimeric bactenecin (NDB)and C-terminal dimeric bactenecin (CDB) (Fig. 6A). Inthe presence of SDS micelles, the CD spectra of bothNDB and CDB showed b-structure patterns, similar tothose of ADB and PDB, but in buffer solution NDBshowed a b-structure with reduced molar ellipticity,whereas CDB showed a random-like conformation(Fig. 6B). In addition, both NDB and CDB exhibitedunexpectedly lower antimicrobial potency in theabsence of salt (MIC = 32 and 16 lm for S. aureusand E. coli, respectively), and just 30–40% of the activ-ity of MTB. In the presence of 150 mm NaCl, how-ever, NDB and CDB almost completely killed thetested Gram-positive and Gram-negative bacteria, andshowed a potency similar to that of ADB and PDB,which have two disulfide bonds (Fig. 6C). It is note-worthy that, in the absence of salt, both NDB andCDB exhibited much less antibacterial activity thanMTB, SLB, ADB or PDB, and they displayed verydifferent potencies in the presence or absence of salt.Finally, we compared the multimeric state ofbactenecin derivatives by carrying out an electrophore-sis experiment on Tricine–acrylamide gel. Four deriva-tives, SLB (linear bactenecin), MTB (bactenecin havingone intramolecular disulfide bond), NDB (bactenecinhaving one intermolecular disulfide bond) and PDB(bactenecin having two intermolecular disulfide bonds)were selected and exposed to an environment con-taining a high concentration of salt (300 mm NaCl).As shown in Fig. 7, the two monomers (SLB andMTB) migrated with apparent molecular masses of 1.5 kDa, whereas the two dimers (NDB and PDB)migrated with apparent molecular masses of 6.5 kDa or more in both the presence and theabsence of salt. In addition, both SLB and MTBshowed somewhat fainter bands in the presence of salt,but PDB exhibited a strong band whether salt waspresent or not. This suggests that both SLB and MTBare monomers and that PDB is a dimer in both thepresence and the absence of salt. Interestingly, NDBshowed a weak band at around  6.5 kDa in theabsence of salt, but a strong band at around 14.2 kDa at the presence of salt, implying that NDBundergoes multimeric oligomerization in the presenceof 300 mm NaCl. Thus, although the same amount ofeach peptide was loaded, these peptides exhibitedsignificantly different band densities and mobilities ona Tricine–acrylamide gel, which provides a clue as towhy bactenecin dimers retain their potent antibacterialactivity at high salt concentrations.In conclusion, our findings are noteworthy in partbecause they confirm the potential importance ofdimeric forms of antimicrobial peptides in vivo, andbecause the ladder-like structure of homodimericantimicrobial peptides makes them relatively easy to100 100 A B 80 80 40 60 40 60 0 20 % Bacteria killed % Bacteria killed0 20 MTB SLB ADB PDB MTB SLB ADB PDB Fig. 5. Salt sensitivity of the antimicrobial activity of the four bac-tenecin derivatives against S. aureus (A) and E. coli (B). To deter-mine the effect of salt on the antimicrobial activity of the peptides,each peptide (8 lM) was incubated with bacteria for 3 h in theabsence (gray bars) or presence (black bars) of 150 mM NaCl, afterwhich 50 lL aliquots of the suspension were plated on LB agar forcolony counts. Results represent the means of two independentexperiments.Salt-resistant homodimeric bactenecin J. Y. Lee et al.3916 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBSsynthesize. Although the two dimers studied, ADB andPDB, had similar activities, synthesis of PDB was com-plex. By contrast, ADB is easily folded under mostfolding conditions. Interestingly, bactenecin dimersundergo multimeric oligomerization at high saltconcentrations. Further studies on the structuralchanges in PDB and NDB that occur at the mem-brane are in progress so as to better understand themechanism by which each dimer interacts with themembrane.Experimental proceduresPeptide synthesis, disulfide formation andcharacterizationAll peptides were synthesized using the solid-phase peptidesynthesis method performed manually with Fmoc chemis-try. The peptides were cleaved from the resin using trifluo-H2N-RLCRIVVIRVSR-CO2HH2N-RLCRIVVIRVSR-CO2HH2N-RLCRIVVIRVSR-CO2HNDBBACH2N-RLSRIVVIRVCR-CO2HH2N-RLSRIVVIRVCR-CO2HH2N-RLSRIVVIRVCR-CO2HCDB30NDB CDB1020m2·dmol–1)NDB CDB-10003(deg·cm30-20[ ] X 10190 200 210 220 230 240 250190 200 210 220 230 240 250-30301020-100-20-30Wavelength (nm)lled8010080100S. aureus E. coliacteria ki40604060% Ba020020MTB NDB CDBMTB NDBCDB0Fig. 6. Synthesis, secondary structure andsalt sensitivity of NDB and CDB, twobactenecin derivatives containing a singledisulfide bond. (A) NDB and CDB werecompletely folded in 2M aceticacid ⁄ H2O ⁄ dimethylsulfoxide (1 : 2 : 1,v ⁄ v ⁄ v) solution for 36 h with gentle stirringat room temperature. (B) CD spectra wererecorded at 25 °Cin10mM sodium phos-phate buffer (pH 7.4) (d) and in 30 mM SDSmicelles (). (C) Each peptide (8 lM) wasincubated with bacteria for 3 h in theabsence (gray bars) or presence (black bars)of 150 mM NaCl, after which 50 lL aliquotsof the suspension were plated on LB agarfor colony counts. Results represent themeans of two independent experiments.No salt 300 mM saltMKNDB SLB PDB MTB NDB SLB PDB MTB26.61714.26.53.5Fig. 7. Coomassie-stained 15% Tricine gel of bactenecin and itsderivatives without salt and with 300 mM NaCl. Fifteen microgramsof each peptide were loaded. Mass markers in kDa are shown onthe left.J. Y. Lee et al. Salt-resistant homodimeric bactenecinFEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS 3917roacetic acid containing various scavengers and purified bypreparative RP-HPLC (Shimadzu, Tokyo, Japan). The pur-ity of peptides was verified by analytical RP-HPLC, andcorrect peptide masses were confirmed by MALDI-TOF MS (Shimadzu).Dissolving reduced linear bactenecin to a concentrationof 1 mm in buffer solution containing 2 m aceticacid ⁄ H2O ⁄ dimethylsulfoxide (1 : 2 : 1) at room temperaturefor 24 h with gentle stirring effectively yielded ADB. MTBexhibiting a b-hairpin conformation was oxidized in aceticacid ⁄ H2O (4 : 1), and this was followed by addition ofiodine (10 equivalents to the number of disulfide bonds). Atwo-step method for disulfide bond formation was used toprepare PDB. Briefly, partially protected peptides werejoined using Fmoc solid-phase chemistry on Wang resin.The free thiol groups of the peptides were bonded by airoxidation in distilled water at 47 °C, while the course of thereaction was monitored using HPLC. Peptides linked bysingle disulfide bonds were obtained after 5 days at a yieldof > 90%. The second procedure was initiated by dissolv-ing the peptide in acetic acid ⁄ H2O (4 : 1) and adding iodine(10 equivalents to the number of disulfide bonds), afterwhich stirring was continued for an additional 2 h to effectremoval of the Acm groups and conversion to PDB with ayield of 80%. ADB and PDB were confirmed by enzymaticdigestion with trypsin (supplementary Figs S1 and S2). Alinear peptide SLB, in which Cys was substituted with Ser,was also synthesized.Trypsin digestionA trypsin digestion was carried out to distinguish betweenPDB and ADB (supplementary Fig. S1). Samples of PDB(100 lg) and ADB (100 lg) were dissolved in 0.2 mL of50 mm Tris ⁄ HCl buffer (pH 8), after which modified tryp-sin (5 lg) was added to a final protease ⁄ protein ratio of1 : 20 (w ⁄ w), and the mixture was incubated at 37 °C for6 h. Analytical RP-HPLC analysis of the reaction mixturewas then carried out (supplementary Fig. S2), andMALDI-TOF MS was used to analyze the mass of eachpeptide.CD analysisThe CD spectra of the peptides were recorded using aJasco J-710 CD spectrophotometer (Jasco, Tokyo, Japan)with a 1 mm path-length cell. Wavelengths were measuredfrom 190 to 250 nm (bandwidth, 1 nm; step resolution,0.1 nm; speed, 50 nmÆmin)1; response time, 0.5 s). The col-lected CD spectra for the peptides were averaged over 16scans in 0.5 mm POPC ⁄ POPG (1 : 1) liposomes and overfour scans in 10 mm sodium phosphate buffer (pH 7.4) or30 mm SDS micelles at 25 °C. The spectra are expressed asmolar ellipticity [h] versus wavelength.Antibacterial activityAntimicrobial activities of each peptide against six selectedorganisms, including three Gram-positive and three Gram-negative bacteria, were determined using broth microdilu-tion assays [29]. Six organisms obtained from the KoreanCollection for Type Cultures (KCTC), Korea ResearchInstitute of Bioscience and Biotechnology (Taejon, Korea)were used for the assays. The Gram-negative bacteria wereE. coli KCTC 1682, Salmonella typhimurium KCTC 1926,and Pseudomonas aeruginosa KCTC 1637. The threeGram-positive bacteria were Bacillus subtilis KCTC 3068,Staphylococcus epidermidis KCTC 1917, and S. aureusKCTC 1621. Briefly, single colonies of bacteria were inocu-lated into medium (LB broth) and cultured overnight at37 °C. An aliquot of the culture was then transferred to10 mL of fresh medium and incubated for an additional3–5 h at 37 °C until mid-logarithmic phase. A two-folddilution series of peptides in 1% peptone was prepared,after which serial dilutions (100 lL) were added to 100 lLof cells [2 · 105colony-forming units (CFU)ÆmL)1]in96-well microtiter plates (F96 microtiter plates; Nunc,Odense, Denmark) and incubated at 37 °C for 16 h. The low-est concentration of peptide that completely inhibited growthwas defined as the MIC. MIC values were acquired as aver-age or triplicate measurements in three independent assays.Hemolytic activityThe hemolytic activities of the peptides were determinedusing human red blood cells (hRBCs). After washing offresh hRBCs three times with NaCl ⁄ Pi(35 mm phosphatebuffer, 150 mm NaCl, pH 7.4), 100 lL of a 4% (v ⁄ v)hRBC suspension in NaCl ⁄ Piwas dispensed into sterilized96-well plates along with 100 lL of peptide solution. Theplates were then incubated for 1 h at 37 °C and centrifugedfor 5 min at 1000 g. Aliquots (100 lL) of supernatant weretransferred to 96-well plates, and hemoglobin release wasmonitored on the basis of the absorbance at 414 nm usingan ELISA plate reader (Molecular Devices, Sunnyvale, CA,USA). Percentage hemolysis was calculated using thefollowing formula: hemolysis (%) = [(A405 nmsample) A405 nmzero lysis) ⁄ (A405 nm100% lysis ) A405 nmzerolysis)] · 100. Zero and 100% hemolysis were determined inNaCl ⁄ Piand 0.1% Triton X-100, respectively. The recordedhemolysis (%) was the average of duplicate measurementsin three independent assays.Preparation of liposomesLarge unilamellar vesicles (average diameter, 100 nm) con-taining the fluorescent probe calcein were prepared byextrusion [30]. Briefly, phospholipids composed of POPG ⁄POPC (1 : 1) were dissolved in chloroform and then driedSalt-resistant homodimeric bactenecin J. Y. Lee et al.3918 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBSovernight under vacuum to make a thin lipid film. Thedried film was then hydrated with Tris ⁄ HCl buffer (10 mmTris, 150 mm NaCl, 1 mm EDTA, pH 7.4) containing70 mm calcein (pH adjusted to 7.4 with NaOH) and vortex-mixed. The suspensions were subjected to five freeze–thawcycles and then pressure-extruded through polycarbonatefilters (LiposoFast, 0.1 lm pore size, 20 times). Vesiclescontaining entrapped calcein were separated from freecalcein by gel filtration on Sephadex G-50 columns (Phar-macia, Uppsala, Sweden) equilibrated with Tris ⁄ HCl buffer.To prepare the small unilamellar vesicles used for CDspectroscopy, dried lipid film was hydrated with Tris ⁄ HClbuffer and then sonicated in an ice bath for 30 min using atitanium-tipped sonicator. The lipid concentration was0.5 mm.Calcein leakage studiesAs mentioned above, the fluorescent probe calcein wasencapsulated in large unilamellar vesicles at a self-quench-ing concentration of 70 mm. For leakage experiments, theindicated amounts of peptide were added to 3 mL of buffercontaining calcein-loaded liposomes. The fluorescence inten-sity of the calcein released from the liposomes, which wasmeasured with mixing after the addition of a peptide, wasmonitored at 520 nm (excited at 490 nm) in a Shima-dzu RF-5301 spectrofluorometer. Fluorescence fromliposomes lysed with Triton X-100 (20% in Tris buffer)was used as an indicator of 100% leakage.Kinetics of bactericidal activity and saltsensitivityThe kinetics of the peptides’ bactericidal activity wasassessed using E. coli KCTC 1682 and S. aureusKCTC 1621 at a peptide concentration of 8 lm, which wasthe highest MIC for any bactenecin derivative against thestrains used. The initial density of the cultures was approxi-mately 2 · 105CFUÆmL)1. After 0, 5, 10, 30 or 60 min ofexposure to the peptides at 37 °C, 50 lL aliquots of serial10-fold dilutions (up to 10)3) of the cultures were platedonto LB agar plates to obtain viability counts. Colonieswere counted after incubation for 24 h at 37 °C.To determine the salt sensitivity of the antimicrobialactivity, peptides were incubated at 37 ° Cin100lLof1%peptone solution also containing 2 · 105CFUÆmL)1bacte-ria and 0 or 150 mm NaCl. After incubation for 3 h at37 °C, 50 lL of the suspension was plated on LB agar forcolony counts.Tricine gel electrophoresisElectrophoresis was performed with 15 lg samples of eachbactenecin derivative dissolved in 2· sample buffer(125 mm Tris ⁄ HCl, pH 6.8, 20% glycerol, 2% mercaptoeth-anol, 0.04% bromophenol blue, and 4% SDS). The entiresample was loaded onto a 15% Tricine gel, after which thegel was fixed and stained with Coomassie dye.AcknowledgementsThis study was supported by the SRC ⁄ ERC programof MOST ⁄ KOSEF (R11-2000-083-00000-0) and theBrain Research Center of the 21st Century FrontierResearch Program (M103KV010005-06K2201-00510).References1 Hoffmann JA, Kafatos FC, Janeway CA & EzekowitzRA (1999) Phylogenetic perspectives in innate immu-nity. Science 284, 1313–1318.2 Zelezetsky I, Pontillo A, Puzzi L, Antcheva N, Segat L,Pacor S, Crovella S & Tossi A (2006) Evolution of theprimate cathelicidin. Correlation between structuralvariations and antimicrobial activity. J Biol Chem 281,19861–19871.3 Zanetti M (2004) Cathelicidins, multifunctional peptidesof the innate immunity. J Leukoc Biol 75, 39–48.4 Bals R & Wilson JM (2003) Cathelicidins – a family ofmultifunctional antimicrobial peptides. 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FEBS J272, 4398–4406.19 Basak A & Lotfipour F (2005) Modulating furin activ-ity with designed mini-PDX peptides: synthesis andin vitro kinetic evaluation. FEBS Lett 579, 4813–4821.20 Perczel A & Hollosi M (1996) Circular Dichroism andthe Conformational Analysis of Biomolecules. PlenumPress, New York.21 Chang CT, Wu CS & Yang JT (1978) Circular dichroicanalysis of protein conformation: inclusion of the beta-turns. Anal Biochem 91, 13–31.22 Woody RW. (1995) Circular dichroism. MethodsEnzymol 246, 34–71.23 Matsuzaki K, Harada M, Funakoshi S, Fujii N &Miyajima K (1991) Physicochemical determinants forthe interactions of magainins 1 and 2 with acidic lipidbilayers. Biochim Biophys Acta 1063, 162–170.24 Hornef MW, Putsep K, Karlsson J, Refai E & Anders-son M (2004) Increased diversity of intestinal antimicro-bial peptides by covalent dimer formation. Nat Immunol5, 836–843.25 Travis SM, Anderson NN, Forsyth WR, Espiritu C,Conway BD, Greenberg EP, McCray PB, Lehrer RI,Welsh MJ & Tack BF (2000) Bactericidal activity ofmammalian cathelicidin-derived peptides. Infect Immun68, 2748–2755.26 Goldman MJ, Anderson GM, Stolzenberg ED, KariUP, Zasloff M & Wilson JM (1997) Human beta-defen-sin-1 is a salt-sensitive antibiotic in lung that is inacti-vated in cystic fibrosis. Cell 88, 553–560.27 Yomogida S, Nagaoka I & Yamashita T (1996) Purifi-cation of the 11- and 5-kDa antibacterial polypeptidesfrom guinea pig neutrophils. Arch Biochem Biophys 328,219–226.28 Wu Z, Hoover DM, Yang D, Boule`gue C, SantamariaF, Oppenheim JJ, Lubkowski J & Lu W (2003) Engi-neering disulfide bridges to dissect antimicrobial andchemotactic activities of human beta-defensin 3. ProcNatl Acad Sci USA 100, 8880.29 Yang ST, Lee JY, Kim HJ, Eu YJ, Shin SY, Hahm KS& Kim JI (2006) Contribution of a central proline inmodel amphipathic alpha-helical peptides to self-associ-ation, interaction with phospholipids, and antimicrobialmode of action. FEBS J 273, 4040–4054.30 Yang ST, Jeon JH, Kim Y, Shin SY, Hahm KS & KimJI (2006) Possible role of a PXXP central hinge in theantibacterial activity and membrane interaction ofPMAP-23, a member of cathelicidin family. Biochemis-try 45, 1775–1784.Supplementary materialThe following supplementary material is availableonline:Fig. S1. Trypsin cleavage sites and mass values of eachpeptide.Fig. S2. HPLC profiles of the peptide fragments aftertrypsin digestion.This material is available as part of the online articlefrom http://www.blackwell-synergy.comPlease note: Blackwell Publishing is not responsiblefor the content or functionality of any supplementarymaterials supplied by the authors. Any queries (otherthan missing material) should be directed to the corre-sponding author for the article.Salt-resistant homodimeric bactenecin J. Y. Lee et al.3920 FEBS Journal 275 (2008) 3911–3920 ª 2008 The Authors Journal compilation ª 2008 FEBS . bacteria [5]. In addition, two lin-ear variants of bactenecin, Bac2S and Bac 2A, showsimilar activities against Gram-negative bacteria andstronger activities. thatadopt parallel and antiparallel conformations and twomonomers that adopt b-hairpin and linear confor-mations, and investigated their biological activities.Results
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Xem thêm: Báo cáo khoa học: Salt-resistant homodimeric bactenecin, a cathelicidin-derived antimicrobial peptide potx, Báo cáo khoa học: Salt-resistant homodimeric bactenecin, a cathelicidin-derived antimicrobial peptide potx, Báo cáo khoa học: Salt-resistant homodimeric bactenecin, a cathelicidin-derived antimicrobial peptide potx