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BASIS FOR SELECTIVITY OF CATIONICANTIMICROBIAL PEPTIDES FOR BACTERIAL VS.
MAMMALIAN MEMBRANESEvgenia Glukhov1, Margareta Stark1,3, Lori L. Burrows2, and Charles
M. Deber1*Divisions of 1Structural Biology & Biochemistry, and 2Infection, Immunity, Injury &
Repair, Research Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8; andDepartments of 1Biochemistry and 2Surgery, University of Toronto, Toronto, Ontario
M5S 1A8, Canada3Present address: Department of Molecular Biosciences, Swedish University ofAgricultural Sciences, Biomedical Centre, Box 575. S-75123 Uppsala, Sweden.
Correspondence should be addressed to: Charles M. Deber, e-mail:[email protected]; FAX: 416-813-5005
Running head: Membrane interactions of cationic antimicrobial peptides
Novel cationic antimicrobialpeptides (CAPs) typified by structuressuch as KKKKKKAAXAAWAAXAA-NH2, where X= Phe/Trp, and several oftheir analogues – display high activityagainst a variety of bacteria, but exhibitno hemolytic activity even at high doselevels in mammalian erythrocytes. Toelucidate their mechanism of action andsource of selectivity for bacterialmembranes, phospholipid mixturesmimicking the compositions of naturalbacterial membranes (containing anioniclipids) and mammalian membranes(containing zwitterionic lipids +cholesterol) were challenged with thepeptides. We found that peptides readilyinserted into bacterial lipid mixtures,while no insertion was detected in model“mammalian” membranes. Depth ofpeptide insertion into model bacterialmembranes was estimated by Trpfluorescence quenching using doxylgroups variably positioned along thephospholipid acyl chains. Peptideantimicrobial activity generally increasedwith increasing depth of peptideinsertion. The overall results, in
conjunction with molecular modeling,support an initial electrostatic interactionstep in which bacterial membranesattract and bind peptide dimers onto thebacterial surface, followed by the“sinking” of the hydrophobic coresegment to a peptide sequence-dependentdepth of ca. 2.5 - 8 Å into the membrane,largely parallel to the membrane surface.Antimicrobial activity was likelyenhanced by the fact that the peptidesequences contain AxxxA sequencemotifs, which promote their dimerization– and possibly higher oligomerization - asassessed by SDS-PAGE gel analysis andFRET experiments. The high selectivityof these peptides for non-mammalianmembranes, combined with their activitytoward a wide spectrum of gram-negativeand gram-positive bacteria and yeast -while retaining water solubility -represent significant advantages of thisclass of peptides.
Natural antimicrobial peptides arepart of the innate immunity of a wide rangeof species ranging from insects andamphibians to mammals - including humans- defending against infections from bacteria,
JBC Papers in Press. Published on July 25, 2005 as Manuscript M507042200
Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
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fungi, parasites and enveloped viruses, withsome peptides also effective against tumorcells [1,2]. Currently, databases report over800 sequences for natural antimicrobialpeptides and proteins from animals andplants (http://www.bbcm.univ.trieste.it),while several thousand others have beendesigned de novo and produced synthetically[3]. Several classes of peptides haveemerged ,including: (i) linear peptides freeof cysteines and often with an amphipathicsequence (e.g., magainins; (ii) peptides withdisulfide bonds that can produce a flatdimeric β-sheet structure (e.g., HBD-2 );and (iii) peptides with an unusual biastoward certain amino acids, such as proline,arginine, tryptophan or histidine (e.g. ,indolicidin) [4-7]. Many antimicrobialpeptides are highly positively-charged, andexist predominantly as monomers withrandom coil structure in solution [8]. Whilethey differ widely in sequence and structure,cationic antimicrobial peptides (or CAPs)generally consist of 12-50 residues,approximately 50% of which arehydrophobic [9], and accordingly have thepotential to form an amphipathic α-helicalstructure when bound to membranes.
The increasing prevalence ofantibiotic resistance necessitates thedevelopment of new ways to combatbacterial infection. While someantimicrobial peptides are already in clinicaland commercial use, future design of novelantimicrobial peptides will necessitate theoptimization of multiple parameters, notablyreduction of toxicity against eukaryoticcells, and of susceptibility to proteolyticdegradation. A key problem in clinicaldevelopment of CAPs is the degree ofselectivity between microbial and host cells.Peptides likely make this differentiationbased on variations in the composition ofeach particular cell membrane [1]. Thus, foreukaryotic cells, the primary membranefeatures are the presence of cholesterol (up
t o 2 5 % ) , p r e d o m i n a n c e o fphosphatidylcholine lipids, and anessentially neutral (zwitterionic) outer leaflet[10]. In contrast, bacterial cells lackcholesterol, have phosphatidylethanolamineas their most common zwitterionic lipid, butalso contain 20-25% of negatively-chargedlipids in their outer membranes, includingphosphatidylglycerol and cardiolipin.Cationic antimicrobial peptides are active inthe low-medium micromolar range andshow little target or L- vs. D-residuespecificity (their D-enantiomers exhibitsimilar activity to their L-counterparts),indicating that they interact with achiralcomponents of the cell membrane [11,12]through a mechanism of physical disruption.Accordingly, bacteria may not easilydevelop resistance.
Many cationic peptides studied to-date have some toxicity, as measured bytheir tendency to induce lysis of erythrocytesat higher concentrations (e.g., gramicidins[13], pardaxin [14], mellitins (100% at 10µM) [15,16], mastorpans (90% at 25 µg/mL)[17], tachyplesins II [18], protegrin I ([19],indolicidin [7], and cathelicidins [20]).Examples of relatively non-toxic peptidesinclude mammalian defensins [21];dermaseptins [22]; spinegirin (no hemolysisat 100 µM) [23]); and magainin (hemolysisonly at the relatively high concentration of100 µg/mL) [24]).
As derived from an earlier series of25-residue CAPs of prototypic sequenceKKAAAXAAAAAXAAWAAXAAAKKKK-NH2, where X = each of the 20commonly-occurring amino acids [25], aclass of novel synthetic 17-residue CAPs hasbeen developed in our laboratory [26]. Thelatter series of peptides contain ‘guest’ X-residues embedded in an Ala-rich sequence,generally containing a Trp residue asfluorescent probe (Table 1); several of thesepeptides have been previously studied inMIC and hemolysis assays [26]. Key
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features of the peptides are the consecutivenon-amphipathic hydrophobic core of 11residues, and the multi-positively chargedLys/Arg in either segregated (all basicresidues at the N- or C-terminus), orseparated forms (basic residues at bothtermini). The Lys or Arg tags solubilize thehydrophobic peptides in aqueous media[25,27]. Several of these peptides havebeen found to be highly effective against aseries of E. coli strains (MICs in the range of4-128 µM or 8-256 µg/mL) [26], and a widevariety of organisms, including P .aeruginosa - an opportunistic pathogen incystic fibrosis lung infections. In someinstances, D-enantiomers of these sequencesshow somewhat higher activity vs. their L-counterparts. Most of these same peptidesdisplay no hemolytic activity against rabbitor human red blood cells up to relativelyhigh concentrations (325 µM or 650 µg/mL)[26]. However, the detailed mechanism oftheir selective antimicrobial action has notbeen elucidated. Through studying thisseries of CAPs with a variety of biophysicaltechniques, including fluorescence and spin-labeling studies in phospholipid vesicles, wereport here an assessment of thechemical/structural factors that arepredominantly responsible for their highselectivity for non-mammalian membranes.
MATERIALS AND METHODS
Materials - Reagents for peptide synthesis,cleavage, and purification included 9-fluorenylmethoxy carbonyl (Fmoc)-protected amino acids (Novabiochem);dansyl- and dabcyl-chlorides (Molecularprobes, Inc. Eugene, OR); Fmoc-PAL-PEG-PS-resin, piperidine (Applied Biosystems,F o s t e r C i t y , C a l i f . ) ; N,N-dimethylformamide (DMF), methanol,diethyl ether, acetonitrile (CaledonLaboratories Ltd., Georgetown, Ontario,Canada); N, N-diisopropylethylamine
(DIEA; Aldrich); O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU; GL BiochemLtd., Shanghai, China); (1,2-dipalmitoyl-sn-3-[phospho-rac-(1-glycerol)] (DPPG), (1,2-dipalmitoyl-sn-3-[phospho-rac-(1-choline)](DPPC), the nitric spin probes 1-palmitoyl-2-stearoyl (n-doxyl)-sn-glycero-3-phosphocholine (n-doxyl SPPC, where n =the position of doxyl group in the stearoylchain; in the present series, n = 5, 10, 12,and 16), 1,2-dimyristoyl-sn-glycero-3-(phospho-L-serine) (DMPS), 1-palmitoyl-2-oleyl-glycero-3-phosphoethanolamine(POPE), cholesterol, and cardiolipin (AvantiPolar Lipids, Alabaster, AL). All chemicalswere used without further purification.Buffer was prepared using Tris sodium saltin doubly distilled water with adjustment topH =7.0 by HCl.Peptide synthesis - P e p t i d e s w e r esynthesized by standard solid-phaseprotocols using Fmoc chemistry on aPerSeptive Biosystems Pioneer peptidesynthesizer as described [25,26]. A lowload (> 0.15 mmol/g) of PAL-PEG-PS resinwas used to incorporate an amide function atthe peptide C-terminus. HATU couplingreagent (0.45 M in DMF) and DIEA base(1.0 M in DMF) were used with a four-foldexcess of protected amino acids.Fluorescent probes (either dabcyl (4-dimethylaminophenylazobenzoyl) chloride( 1 0 m g ) o r d a n s y l ( N - ( 5 -dimethylaminophthalene-1-sulfonyl)chloride (10 mg)) were coupled manuallyovernight after completion of solid phasesynthesis using 100 mg of peptidyl-resineach in 1 mL DMF with 50 µL of DIEA.Following a standard cleavage procedure(89% TFA, 4.5% phenol, 4.5% TDW, 2%triisopropylsilane), crude peptides wereprecipitated in cold ethyl ether. Deprotectedpeptides were purified on a reverse-phaseC18 h igh per fo rmance l iqu idchromatography column (Vydac, 250 × 21.5
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mm) using a linear gradient 20-30% B(Buffer A: 0.1% TFA, 95% TDW, 5%AcCN; Buffer B: 0.1% TFA, 95% AcCN,5% TDW) for the first 10 minutes, then 30-50% B for another 30 minutes. Absorbancewas monitored at 215 nm. Molecularmasses were confirmed by MALDI massspectrometry. Concentrations of peptideswere determined in triplicate by standardBCA assay. Peptide solutions were stored at-20 °C.Circular dichroism (CD) - Circulardichroism spectra were collected at differenttemperatures using a Jasco J-720spectropolarimeter. Spectral scans wereperformed from 250 to 195 nm, with stepresolution of 0.1 nm and bandwidth of 1.0nm at speed 50 nm/min [28]. A 1 mm-path-length quartz cuvette was used for themeasurements and values from 7 scans wereaveraged per sample. Freshly preparedsamples were measured at 20-60 µM range.The aqueous buffer contained 10 mM bufferof Tris-HCl, 10 mM NaCl at pH = 7.0 wasused as a solvent for measurements and itsbackground (in presence of appropriatedetergents were needed) was subtracted foreach sample. 25 mM sodium dodecylsulfate(SDS) solutions were generally prepared bydissolving the desired amount of reagentsinto this buffer. The molar ellipticity [29]was calculated following Chen et al. [30] as:
[θn]222 = -39500*(1-2.57/n)where n = number of residues.SDS-PAGE gel electrophoresis - Peptidesamples were subjected to SDS-PAGE,using NuPage pre-cast 12% Bis-Tris gels(1.0 mm× 10 well) and buffers: NuPAGELDS sample buffer (Novex, San Diego,CA), NuPAGE MES SDS running buffer.Peptides were dissolved in differentconcentrations in sample buffer and heatedat 85 °C for 10 min prior to electrophoreticseparation at 125 mV. The values ofMWexp.:MWtheor. ratios were calculated fromeach stained gel (either Coomassie Blue or
Silver staining for peptides (10-140 µM )using See blue and Mark12 markers and theNIH 1.62 Image Program, softwarea v a i l a b l e a t h t t p : / / w w w -cellbio.med.unc.edu/henson_mrm/pages/NIH.html) [31].Preparation of phospholipid vesicles. - Thedesired mixtures of phospholipids,cholesterol, cardiolipin were dried in glasstubes first under nitrogen and thenlyophilized overnight to obtain lipid films.Then, dry lipid films were suspended for 1 hon water-bath at 40-50 °C in Tris-HClbuffer, pH = 7.0 (10 mM Tris, 10 mMNaCl), and sealed with parafilm undernitrogen. The mixtures were vortexedoccasionally to disperse the lipids. Vesicleswere kept at 44 °C to maintain the lipidsabove the gel-to-liquid crystallilne phasetransition, and used at the same day. Theappropriate aliquots of the desired peptideswere added.Small unilamellar vesicles (SUVs) wereprepared using standard procedure asdescribed previously [28], but with nopeptides present. Sonication of lipiddispersions was performed in 5 ml glasstubes in a bath-type sonicator (G112SP1G,Laboratory Supplies Co., Hicksville, NY)for 5 minutes in cold water (until clear). Toavoid degradation of unsaturated lipids,sonication was performed at ∼10 °C under anitrogen atmosphere.Large unilamellar vesicles (LUVs) wereprepared as described [29] by freeze-thawing the desired lipid suspension fivetimes under nitrogen to produce largemultilamellar vesicles. The suspension wasextruded 11 times through polycarbonatemembranes with 0.1 mm diameter pores(Nuclepore Corp., Pleasanton, CA) on anAvanti mini-extruder apparatus.Fluorescence measurements - Fluorescenceemission spectra of peptide Trp residueswere recorded on a Hitachi F-400 PhotonTechnology International C-60 fluorescence
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spectrometer equipped with water bathcirculator for regulating the temperature.Semimicro quartz cuvettes of 0.5 mL (10mm excitation path length and 4 mmemission path length) and 2 ml (10 mm × 10mm, in titrations) (Hellma, Concord, ON)were used. Excitation wavelength was 280nm, and emission spectra were recordedfrom 305 to 365 nm. Spectra were collectedwith a step size of 1 nm with averaging ofthree cycles. Excitation and emission slitwidths were 2 and 6 nm (2 nm and 6 nmband pass, 1 and 3 turns of the slitmicrometers ) , respec t ive ly . Al lmeasurements were corrected for lightscattering effects of vesicles by subtractionof background and by the correctionfunction of the manufacture software.Background samples (blank suspensions ofbuffer, or lipids in buffer) were preparedusing the same protocol except pure waterinstead of peptide solution was added. Blueshifts were calculated as the difference inwavelength of the maxima in emissionspectra of lipid-peptide and aqueous peptidesamples. Peptides were used in 1 or 4 mMof detergent, such that lipid-to-peptide ratiosof either 250 or 1000 were established at aconstant peptide concentration of 4 µM inall experiments except titrations. In thelatter case, data were corrected for dilutionof the peptides during the course of theexperiment.
For depth quenching measurements,10% (mole percent) of n-doxyl SPPC intodifferent types of vesicles was incorporated[32]. The appropriate aliquots of the desiredpeptides were added to 1 mM of lipidvesicles in Tris-HCl buffer, pH = 7.0 (10mM Tris, 10 mM NaCl) 10 minutes prior tomeasurements. Each experiment wasrepeated at least twice, usually four times.The fluorescent intensities of the emissionmaxima were taken in the presence and inthe absence of n-doxyl quenchers invesicles. Parallax analysis was performed
according to published procedures [33]. Thedistance of the Trp residue from the bilayercentre (ZCF) was calculated by:
ZCF=LC5+[-ln(F5/F16)/πC-L5:162]/2 L5:16 ]
(Eq. 1)
where LC5 represents the distance from thebilayer center to the shallow-placed n-doxylquencher (n = 5), which is 10.8 Å for LUV-RBC(outer) and LUV-bact vesicles, and 9.9Å for SUV-I & SUV-II vesicles (Table 2); Cis the mole fraction of quencher (0.1 in allcases) divided by the lipid area (70 Å) [34];F5 and F16 are the relative fluorescenceintensities of the shallower and deeperquenchers, respectively; and L5:16 is thedifference in the depth of this twoquenchers. For depth measurements, we use0.9 Å per methylene group CH2. The indolering insertion depth (d) was calculated asone-half the bilayer thickness (= 28.8 Å forSUV-I & SUV-II vesicles, or 30.6 Å forLUV-RBC (outer) and LUV-bact vesicles)minus ZCF.FRET measurements - Labeling of peptideswith dansyl (donor) and dabcyl (acceptor)was achieved by coupling of dansyl ordabcyl chlorides to the N-terminal Lys [35].Steady-state fluorescent spectra wererecorded on the Photon spectrometer.Samples were examined at constant stirringin a stoppered 10 mm ×10 mm disposablecuvette by removing and adding newportions of mixtures. Peptide stocksolutions were diluted in buffer (10 mMTris-HCl, 10 mM NaCl, pH = 7.0) witheither 25 mM SDS detergent at roomtemperature or 1 mM of SUV-bact or SUV-RBC (outer) (Table 2) at 44 °C. Theconcentration of dansyl labeled peptides(donor) was kept constant at 1 µM, and thetotal concentration of dansyl-, dabcyl- andunlabeled peptides was kept constant at 5µM in SDS mixtures, or at 0.5 µM and 2.5
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µM in experiments with vesicles. Eachmixture was allowed to equilibrate for 3min. Emission spectra were collected from450 nm to 650 nm at room temperature inSDS micelles, and at 44 oC in SUV-bact. λex
= 341 nm, 0.5 sec/nm, band pass was 2 nmfor excitation and 4 nm for emission. Eachspectrum is the average of two or three runs.Computational modeling - Energy-minimized models of the interactionbetween two idealized helices wereproduced using a global conformationsearch program CHI as described [36]. Theprogram CHI identifies structures withenergetically favorable packed interfacesbetween paired helices, analyzes all possibleinteractions, and returns sets of probablestructures.
RESULTS
The disruption mechanism ofbacterial membranes by the present familyof synthetic CAPs was examined in cellmembrane mimic conditions by circulardichroism, fluorescence, and electrophoreticmethods. The majority of these peptides(Table 1) have average core values of +1.4 -1.5 on the Liu-Deber scale [25], which areabove the threshold for spontaneous cellmembrane insertion. We also studied onesignificantly more hydrophobic peptide (AllD W17-6K-(4L)), which possesses anaverage core hydropathy value of ca. 3.1.Peptide secondary structure - TM Finderdata analysis [37] predicts that inmembrane-mimetic environments, segmentsof these CAPs will adopt an α-helicalsecondary structure. In case of Lys-segregated peptides (such as F17-6K), thissegment is predicted to consist of residues 4-10 of the hydrophobic core, while for Lys-separated peptides (such as F17), residues 3-9 from the corresponding core are likely tobe involved. Across this series, no morethan seven residues (about 40% of total
peptide residues) are predicted to beinvolved in formation of α -helix duringmembrane insertion. Circular dichroismdata are consistent with these predictions.As typified by F17, the present CAPpeptides are largely in random coilconformations in aqueous buffer (Fig. 1a).In SDS micelles, transition of the CDspectrum to a helical-type pattern isobserved; however, as derived from theexperimental molar ellipticity values, only5-8 residues are involved in helix formationin agreement with TM Finder analysis. Noinfluence of concentration (20 - 60 µM),temperature (25 - 75 oC), or pH (in the rangeof 7-11) was found on the spectra in the caseof F17 (not shown). Qualitatively-similareffects were observed for the natural CAPmagainin II (Table 1, Fig. 1b), with theincreased helical content in SDS micelles vs.F17 arising from its greater length andlonger helical segment(s). Similar to certainother CAPs (16, 38), no correlation hasgenerally been observed between degree ofhelicity and antimicrobial activity; highhelicity often correlates with high hemolyticproperties, albeit not with antimicrobialactivity [16].Peptide location within the bilayer - Thefirst indication of bacterial membraneinsertion - and evidence of peptideselectivity for bacterial vs. mammalian cellmembranes - was obtained by exposure ofthe peptides to anionic and zwitterionicunilamellar lipid vesicles. In theseexperiments, lipid/peptide (L/P) ratios weremaintained high enough (250 and above) atlow peptide concentration (4 µM, below thelowest MIC values) in order to mimic theinitial steps of peptide-mediated membranedisruption. Measurements of fluorescenceemission intensity and shifts in the λmax ofthe Trp probe incorporated into thehydrophobic core of peptides in the library(Table 1) were particularly useful foranalysis of the position of a given peptide
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within the membrane. Exposure of suitablepeptides at 4 µM to freshly prepared anionicLUV-bact vesicles containing a lipidmixture corresponding to a typical bacterialmembrane (Table 2; Fig. 2), caused a blueshift in Trp λmax values, accompanied byintensity enhancement. These resultsprovide evidence of insertion of peptidesinto the bilayer [39] and effective removalof the peptides from the bulk aqueousenvironment. The significant blue shift of∆λmax = 17-23 nm indicates a membrane-buried Trp in negatively-charged LUVscontaining 25% anionic lipids (AL),corresponding to “bacterial vesicles” (LUV-bact). Bilayer insertion of peptides wassimilarly apparent for 10% AL (LUV-RBC)and 90% AL (SUV-I) as well (data notshown). In contrast, virtually no shifts ineither maximum wavelength or fluorescentintensity were seen even for the mosthydrophobic peptide (All-D W17-6K-(4L))with LUVs formed from a mixture of purezwitterionic lipids and cholesterol,mimicking the outer leaflet of red blood cellmembranes [LUV-RBC(outer) (0% AL,25% cholesterol)] (Table 2, Fig. 2). In theabsence of cholesterol, the zwitterionicvesicles (LUV-Zwit, Table 2) similarlyshow evidence of lack of peptide insertion;however, partial insertion was observed forAll-D W17-6K-(4L) (Fig. 2).Depth of membrane penetration by syntheticCAPs - To estimate the penetration depth ofTrp and correspondingly, of the peptidesinto model cell membranes, a dual quencherassay (parallax analysis) was employed (seeMaterials and Methods section). In ourexperiments, 10% of spin-labeled PC with anitroxide (doxyl) group, covalently attachedto the methylene carbon at either the 5 or 16position of the acyl chains, was incorporatedinto phospholipid vesicles (≈30 nm (SUVs)and ≈100 nm (LUVs) diameter). Theresulting quenching by the doxyl groupduring Trp fluorescence measurements
provides an accurate probe for estimatingthe penetration depth of this residue into thelipid bilayer.
Negatively-charged LUV-bactvesicles (25% AL, no cholesterol, Table 2)were employed in Trp parallax methodexperiments to estimate depth of insertion(d) as a function of peptide concentrationfrom 1.5 to 6 µM for one of the moreeffective CAPs (F17-6K), and for one of theless active peptides (F17); above 6 µM, themixture with F17-6K loses transparency. While the depth of Trp penetrationincreased with increasing peptideconcentration in the case of F17-6K to aplateau around 6.5 Å near 3 µM, no depthvariation was found for F17 over the sameconcentration range (depth = 1.5 – 2 Å).Both peptides were shown to be closer to thebilayer surface then to its centre, but themore active peptide was found to penetrateabout 3 Å deeper into the bilayer at thisrange of concentrations.Membrane penetration depth vs.antimicrobial activity - Values of Trp indoledepth after spontaneous insertion intofreshly prepared LUV-bact vesicles werecalculated in a similar manner for sixselected peptides from Table 1 at 4 µM (Fig.3). The amount of quenching varied for eachpeptide as a function of the doxyl moietyposition from 35% to 80%. The depth of Trppenetration into “bacterial” vesicles wasfound to range between 2.4 - 7.9 Å.Peptides sorted in decreasing order of theirTrp depth vs. their MIC values, as shown forthree bacterial strains A-C in Fig. 3.Overall, we found that depth penetration andMIC values were highly correlated: as agiven peptide penetrates deeper into thebilayer, it has lower MIC values (i.e., higheractivity) against various bacterial strains invivo. Similar experiments performed forthree peptides (F17, F17-6K and F17-6R)using LUV-RBC(outer) vesicles gave no
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quenching and hence no indication ofpeptide insertion (not shown).Effect of cholesterol on depth of peptideinsertion - We investigated a proposedspecific role of cholesterol – which ispresent in mammalian cells, but absent inbacterial membranes – as a “protectant”against penetration by the peptides. Usinganionic vesicles as a model, we found thatthe depth of the Trp residue from All-D F17-6K-(2L) was essentially unaffected by theincorporation of 25 mol% cholesterol (SUV-I vs. SUV-II) into the bilayer, as determinedby parallax analysis with 10% of either 5-,10-, 12- or 16- doxyl labeled DPPC (Fig. 4).These results are discussed further below.Peptide oligomerization state(s) - Althoughthe high content of Ala residues hadoriginally been introduced into the peptidedesign process to serve as “background” or“template” residues of mid-rangehydrophobicity [25], an emerging body ofwork on helix-helix interaction motifs inmembrane-based peptides has suggested thattwo ‘small’ residues separated by threeresidues (termed GxxxG or AxxxA motifs)are important mediators of helix-helixdimerization in membranes [40, 41].Consistent with this situation, all peptidesfrom Table 1 studied at concentrationsbetween 10 µM to 100 µM in SDS-PAGEassays on NuPAGE gels at pH = 7.3 gaveindication of formation of discrete SDS-resistant dimers (Fig. 5). We observedMWexp./MWtheor. values around 1.8 - 2.7(indicative of dimers) for peptides with one(at the hydrophobic core centre), two(central and N-terminal), and three (bothends and centre of the core) sequentialAxxxA motifs. However, under the sameconditions, MWexp./MWtheor. values of 1.1 -1.3. typical for monomers, were observedfor the natural antimicrobial peptidesmagainin II and cecropin P2 (Table 1) up toloading concentrations of 140 µM (notshown). No correlation was found between
helicity levels in CD spectra and values ofthe MWexp.:MWtheor ratio.FRET measurements of peptide self-association - Given the observation of SDS-resistant peptide dimers, we usedfluorescence resonance energy transfer(FRET) to further confirm dimerization ofthe peptides upon membrane insertion.Specific labeling of peptides was achievedduring synthesis by attaching either dansyl-or dabcyl chloride to the N-terminal Lysresidue. Labeled peptides were cleavedfrom the resin and purified using the sameprotocols as for unlabeled peptides. CDspectra and SDS-PAGE analysis of dansyl-labeled and dabcyl-labeled All-D F17-6K-(2L) revealed that the helical content andrelative mobility were the same for bothlabeled and unlabeled forms of the peptide(not shown). Quenching of donor (N-terminal dansyl labeled peptide)fluorescence as a function of the acceptor(N-terminal dabcyl labeled peptide) fractioncan be used to determine the stoichiometryof association in a peptide oligomer [35,42];in the case of monomers, no quenchingshould be observed. If oligomerizationoccurs, the relative quantum yield of thedonor decreases linearly in the case ofdimerization, or according to a morecomplex function upon higher orderoligomer formation [43,44]. FRETexperiments performed at pH = 7.0 inanionic bacterial-membrane vesicles (SUV-bact with 25% AL (2.5 µM of totalpeptides)) (Fig. 6) confirmed the hightendency of the peptides towarddimerization. SUVs were used here forreduction of light scattering effects. As seenin Fig. 6, where quenching vs. the molefraction of dabcyl acceptor is plotted for theAll-D F21-10K and All-D F17-6K peptides,essentially linear relationships are obtained.These two most active peptides exhibited asimilar level of quenching in all membraneenvironments examined. Thus, these
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peptides form discrete dimers in themembrane-mimetic environment of SUV-bact bilayers. Similar results were obtainedin comparable experiments performed inSDS micelles (not shown).
DISCUSSION
Since the majority of antimicrobialpeptides are positively charged atphysiological pH, a prevailing view is thatselectivity stems fundamentally fromelectrostatic attraction of the cationicpeptide to the anionic bacterial membranes[46]. While this view is consistent with thepresent findings, electrostatics cannot be theonly contributing factor, because asmentioned in the Introduction, manycationic peptides also disrupt neutrallycharged mammalian cells at higherconcentrations. Therefore, more subtleproperties of the peptide, including itshydrophobic moment [47], oligomerizationstate [48], and/or the specific type andorientation of larger residues as dictated bysequence, may also play a role indetermining the extent of peptide insertionand disruption of membrane integrity. Inone example, monomeric peptides werepractically devoid of antimicrobial activity,while their pentameric covalently attachedoligomers were highly active on humanerythrocytes [49].Peptide selectivity for bacterial membranes- Whether as monomers or higher oligomers,it is widely believed that a general non-receptor-mediated mechanism is responsiblefor peptide antimicrobial activity in mostcases, which appears to involvepermeabilization of phospholipid bilayermembranes via “barrel-stave”, “toroidalpore”, or “carpet detergent-like” formation[1,3,8]. Microbial cell membrane disruptionby the present family of CAPs can likely beascribed, at least in large measure, to one ofthese non-stereospecific mechanisms. Since
the CAPs studied here are relatively short,with a hydrophobic stretch of 11 residues,they would not be expected to form pores,which would require full membrane-spanning segments with α-helical structuresin bilayers. As well, strong electrostaticinteractions among several adjacentpositively-charged residues with negatively-charged lipid head groups may inhibitpeptide reorientation and thus the formationof well-defined pores [48]. In addition, theTrp depths of ca. 2 – 8 Å found in thepresent study are too shallow fortransmembrane insertion. Thus, this class ofpeptides appears unlikely to operate viaeither the barrel-stave or toroidal poremechanism of bacterial killing.
Within the category of the ‘carpetmodel’, one can consider three possiblemodes of peptide insertion into membranes:parallel (0°), perpendicular (90°) anddiagonal (at some intermediate angle) to themembrane surface. The experimentally-determined distances of 2.4 - 7.9 Å from thebilayer surface for Trp residues in a 28 - 30Å model membrane demonstrate relativelyshallow peptide penetration that most likelycorresponds to side chain insertion with thepeptide positioned at least partially parallelto – and within or slightly below – theeffective membrane surface region. Inaddition, perpendicular and/or diagonalinsertion is probably not the case for Lys-separated peptides such as F17 (d = 2.4 ±0.9 Å), as three sequential Lys residues areunlikely to be buried deeply inside thebilayer. Nevertheless, in the case of thesegregated peptides – except one withpositive charge on both ends [F17-6K C-term, which also has an N-terminal freeamino group (d = 2.8 ± 1.4 Å)] – all threegeometric modes of insertion remainpossible, because the depth of Trp insertionfor the perpendicular mode of membraneinsertion is calculated from models to bebetween 5.6 - 8.4 Å from the lipid-water
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in te r face , cons is ten t wi th ourexperimentally-determined values of 5.0 -7.9 Å. Calculation of magainin depth inbilayers gave similar values – 8-10 Å [50].Overall, our data regarding depth of Trpinsertion suggest that the peptide backboneper se probably resides partially toward theincreasingly hydrophobic region of thebilayer, with the precise depth of itspenetration a complex function of both thepercent/type of anionic lipids present inmembrane composition, as well as thepeptide sequence itself. Since all peptidesmust remain tethered to the membranesurface via electrostatic bonds betweenLys/Arg residues and membrane phosphategroups, this sinking motion mayconcomitantly place torsional stress on thebound phospholipid molecules, furthercontributing to bilayer disruption. Theinverse correlation observed between valuesof Trp depth in LUV-bact and peptide MICvalues (Fig. 3) suggests that bacterialmembranes could become increasinglysusceptible to lysis as a given peptidepenetrates deeply enough. Perhaps thesource of the most potent antimicrobialactivity of these CAPs resides in theircapacity to not only to insert, but to do so ina manner parallel to the bilayer surface,with resulting disruption of bilayer packing.Peptides penetrate bacterial lipidmembranes as dimers - Several peptides inthe present CAP series contain AxxxAmotifs in their sequences. These motifs –analogous to the well-known GxxxG motifin which the two ‘small’ residues promoteclose approach of helices in transmembranedimers [40,41] – suggest that these CAPshave the innate capacity to form dimers orhigher ol igomers in membraneenvironments. Indeed, SDS-PAGEelectrophoresis and FRET experimentsconfirmed the strong tendency of thesepeptides toward dimerization even at verylow concentrations (below MIC values)
(Figs. 5 and 6). With the present category ofpeptides, such strong dimer formation mightserve as a key determinant of the highantimicrobial activity, i.e., oligomerizationwithin the membrane surface region isexpected to create substantially largerdisturbances in the bilayer once insertionoccurs. It should be noted that in CAPswhich act through barrel-stave and toroidalpore mechanisms, peptides oligomerizebefore or during bilayer insertion (3). Aswell, synthetically-produced dimers ofantimicrobial peptides, e.g., by formation ofintermolecular disulfide bridges, showincreased activity [51].Role of cholesterol in CAP/lipid interactions- Our overall experimental data suggest thatthe presence or absence of cholesterol per segenerally plays a secondary role in CAPinteractions with various lipid mixtures. Thepresent findings demonstrate that cholesteroldoes not influence insertion of peptides in asignificant manner when studied in anionicbilayers without or with added cholesterol(Fig. 4). With respect to mammalianmembranes, we found that this series ofwater-soluble peptides, containing highlycharged Lys or Arg tags, ultimately cannotleave the bulk water for attachment/insertioninto erythrocyte-like bilayers until theaverage hydrophobicity of the peptide coresequence begins to approach sufficientlyhigh levels (as in the case where thesequence contains four Leu residues); andthat the peptides can use this latter propertyas a mode of insertion only when cholesterolis absent (Fig. 2). Since a given peptidebecomes antimicrobially active once theaverage hydropathy of its core sequenceexceeds the minimal ‘hydrophobicitythreshold’ for insertion into zwitterionicmicellar membranes [26], it appears thatthere may additionally exist an upperhydrophobicity limit – creating, in effect, awindow of hydrophobicity – that, ifexceeded, becomes deleterious to peptide
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bioactivity. This latter phenomenon mayalso be associated with a tendency towardonset of hemolytic character for increasinglyhydrophobic peptides. Conceptually-related observations in systematic studies ofthe cyclic peptide antibiotic gramicidin Sindicate that a defined range of peptidehydrophob ic i t y and s equen t i a lamphiphilicity are key requirements fortherapeutic effectiveness [52,53]. It shouldbe emphasized that certain of the lipidmixtures created for study here – a bacteriallipid mixture with cholesterol (SUV-II) anda mammalian lipid mixture w i t h o u tcholesterol (LUV-Zwit) - would in any casenot be encountered by antimicrobialpeptides in vivo. Therefore, the presence ofnegative charge on the outer cell membranesurface of bacterial cells remains theprincipal reason generally for peptideselectivity.Mechanism of bacterial membranedisruption by CAPs - Data reported herereinforce the essentials of a “grip and dip”process [26], now embellished by thepresent experimental measurements ofpeptide penetration depth and peptidedimerization. Thus, antimicrobial activityappears to devolve from peptides actingbroadly analogously to a “carpet model”mechanism by (i) attachment of anti-parallelpeptide dimer species to anionic (but notzwitterionic) membrane surfaces viaelectrostatic attractions through Lys/Argside chains; (ii) insertion of the peptidehydrophobic core segment by several Å in asequence-dependent manner, largely parallelto the membrane surface; and (iii)consequential membrane disruption/lysis,where the parallel-inserted peptides act as a‘submarine-like’ species to force the chainsof the bacterial bilayer apart, in essence“unzipping” the membrane. Paradoxically,since native membrane proteins are seen toreside in bilayers with TM helices aligned tothe major lipid axes (i.e., perpendicular to
the membrane surface) – ostensibly amanner least disruptive to lipid packing -one can speculate that CAPs may potentiallyinflict the greatest harm to microbial cellswhen inserted parallel to the membranesurface, as found in the present work.Peptide dimerization would be expected tocatalyze such destruction of the bacterialmembrane by, in effect, forming largehydrophobic particles of damagingdimensions – likely enhanced by clusteringof large, aromatic residues.
Molecular modeling supports theinvolvement of AxxxA motifs in peptide-peptide packing interactions. An energy-minimized dimer of the putative helicalregions F17-6K (Fig. 7A) indicates key Alaresidues at the interface between the twopeptide molecules. The model displays theanti-parallel dimer of F17-6K, as thisarrangement maximally separates the polarLys-tag regions. If depicted against amembrane background the peptide dimer inFig. 7B projects its large Phe and Trp sidechains downward toward the bilayer interior,largely within one sector of the dimercircumference, a situation that couldpotentially act as a locus of local disruptionof lipid packing. These latter considerationsrepresent a manifestation of where thepeptide sequence is likely to play a subtlerole in CAP activity. Qualitative structuralanalysis suggests that placement of thehelical backbone of the model in Fig. 7Bnear a membrane surface region wouldposition the Trp side chain in the region 5 –8 Å below the surface, consistent with thevalues deduced from doxyl-labeled lipidexperiments.
CONCLUSION
The exceptional advantages inactivity and selectivity of our modelpeptides can thus be explained by theirfavorable combination of geometrical and
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physical properties. The fact that thepeptides studied here show no affinity forzwitterionic lipid vesicles in the presence ofbiologically-relevant contents of cholesterolis consistent with their lack of hemolyticactivity in erythrocytes [26]. Whenevaluated in the context of magainin andother natural CAPs which do show somehemolysis, we suspect that the grouping ofsequentially-consecutive positive charges onone or both termini of the present CAPs mayconstitute an untenable locus for adsorptionto zwitterionic membranes (vs. the wider
distribution of polar residues in theamphipathic natural CAPs), and thusrepresent the main source of the extremelyhigh level of selectivity for bacterialmembranes and the low toxicity of thesepeptides in mammalian membranes. Theseproperties, in combination with low MICvalues toward a wide spectrum of gram-negative and gram-positive bacteria - whileretaining water solubility - representsignificant advantages of this class ofpeptides.
ACKNOWLEDGMENTS
This work was supported, in part, by grants to L.L.B. from the Canadian InfectiousDiseases Society, and to C.M.D. from the Canadian Institutes of Health Research (CIHR)and the Natural Sciences and Engineering Research Council of Canada (NSERC). E.G.holds a post-doctoral award from the CIHR Strategic Training Program in StructuralBiology of Membrane Proteins Linked to Disease. M .S. held a Sweden-AmericaFoundation Award in 2001-2002.
ABBREVIATIONS
BCA, bis-cinchinonic acid analysis for total protein; MIC, minimum inhibitoryconcentration; RBC, red blood cell.
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Table 1. Sequences and designations of synthetic cationic antimicrobial peptidesemployed in the present work. Lower case letters in sequences indicate D-enantiomersof the amino acids. Polar residues (Lys, Arg, Glu) are shown in red; aromatic residuesare shown in green. MIC’s in selected gram-negative and gram positive bacteria, andhemolytic activities in rabbit and human erythrocytes, have been reported for peptidesF17, F17-6K, All-D F17-6K, F17-6R, and W17-6K [26].
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Table 2. Molar percentages of lipid composition and designations for vesiclepreparations used in the present work. Compositions for E. coli (minor componentsomitted) and erythrocyte membranes, are as given by Gennis [54]. SUV = smallunilamellar vesicles; LUV = large unilamellar vesicles; bact = bacterial membranecomposition; RBC = red blood cell membrane composition; RBC(outer) = outer leaflet ofRBC membrane composition; Zwit = zwitterionic.
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FIGURE LEGENDSFig. 1. Secondary structures of (a) the F17 antimicrobial peptide; and (b) the natural CAPmagainin II, as determined by circular dichroism spectroscopy. Spectra were recorded atroom temperature in buffer containing 10 mM Tris-HCl, 10 mM NaCl (pH = 7.0) in thepresence or absence of 25 mM SDS detergent as indicated on the diagram. Peptidesequences are as given in Table 1. Peptide concentration was 40 µM. The curvesreported are based on the average of three runs with background subtracted.
Fig. 2. Blue shifts in wavelength maxima emission (∆λmax) of Trp fluorescence uponexposure of selected peptides (4 µM) to freshly prepared vesicles (1 mM of lipids). Bluebars: LUV-bact [25% AL, 75% zwitterionic lipids – similar to the composition ofbacterial cell membranes]. Red bars: LUV-RBC(outer) [0% AL, 75% zwitterioniclipids, 25% cholesterol – similar to the outer leaflet of erythrocyte cell membranes].Purple bars: LUV-Zwit [0% AL, 100% zwitterionic lipids, no cholesterol]. See Table 2for details of lipid components. Temperature = 44 oC. Experiments were performed induplicate. Error bars are based on standard deviations derived from the measuredwavelength positions.
Fig. 3. Trp depth values (d, [Å]) of selected peptides (4 µM) in LUV-bact (1 mM) at 44oC, sorted by decreasing MIC values (= increasing antimicrobial activity) in variousbacterial cells: A – E. coli C498; B – S. epidermis C621; C – P. aeruginosa ATCC27853. Values of d given in the figure are the averages of 2-4 repetitions of eachexperiment. Error bars are based on standard deviations derived from the measuredfluorescence intensities.
Fig. 4. Relative fluorescence intensity of peptide All-D F17-6K-(2L) in negatively-charged SUVs (SUV-I & SUV-II) in the absence (labeled “no doxyl”) and in the presenceof 10% doxyl-labeled groups at 5-, 10-, 12- and 16- positions. Lipid preparations are:SUV-I - no cholesterol present; and SUV-II – 25% cholesterol. Experiments wereperformed in duplicate. Error bars are based on standard deviations derived from themeasured fluorescence intensities.
Fig. 5. SDS-PAGE (silver staining) analysis of CAPs. The lanes correspond to: A –F17; B – F17-6K; C – All-D F17-6K(2L); D – F15-4K; and E – All D F21-10K.Peptides were diluted in SDS-containing sample buffer at ∼10 µM (200 ng) and heatedfor ten minutes at 85 oC prior to electrophoresis using 12% Bis-Tris NuPAGE gel at125V of constant voltage. MWexp/MWtheor is the ratio of experimentally-determined toactual molecular weight of each CAP, where a value = 2 corresponds to dimer.Molecular weight standard (Mark 12) is given at the left of the diagram. Molecularweights of CAPs are given in Table 1.
Fig. 6. FRET efficiencies in experiments using dansyl-labeled donor peptides in thepresence of increasing mole fractions of the corresponding dabcyl-labeled acceptorpeptides. Peptides are the labeled forms of All-D F21-10K and All-D F17-6K, asindicated on the diagram. Peptides are studied at 0.5 µM in anionic SUV-bact vesicles.Total peptide concentration is 2.5 µM. Solvent buffer is 10 mM Tris-HCl, 10 mM NaCl,
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pH = 7.0. Curves are consistent with dimer formation in each case. Values shown arethe average of at least three repetitions of each measurement. Error bars are based onstandard deviations derived from the measured fluorescence intensities normalized to thevalue in the absence of acceptor.
Fig. 7. Energy-minimized dimer models showing the interaction surface betweenantiparallel helical segments from the hydrophobic core of the cationic antimicrobialpeptide F17-6K. (a) side view; (b) end view. The model was generated using the globalconformational search software CHI (see Materials and Methods section) with idealizedhelices corresponding to peptide residues 9-17 (FAAWAAFAA). A dielectric constant of1 was used in the calculation. See text for a further discussion.
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Figure 4
0
20
40
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no dox. 5 10 12 16
n-doxyl position
Flu
ore
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s
SUV-I (no cholesterol)
SUV-II (25% cholesterol)
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Evgenia Glukhov, Margareta Stark, Lori L. Burrows and Charles M. Debermembranes
Basis for selectivity of cationic antimicrobial peptides for bacterial vs. mammalian
published online July 25, 2005J. Biol. Chem.
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