Recognition of Membrane Sterols by Polyene Antifungals Amphotericin B and Natamycin, A 13C ...

13 Pages·2017·3.58 MB·English

by  Christine Middleton

ORIGINALRESEARCH
published:17June2016
doi:10.3389/fcell.2016.00057
Recognition of Membrane Sterols by
Polyene Antifungals Amphotericin B
13
and Natamycin, A C MAS NMR
Study
FilipCiesielski1†,DavidC.Griffin1,JessicaLoraine1†,MichaelRittig1,
JossDelves-Broughton2andBoyanB.Bonev1*
1SchoolofLifeSciences,Queen’sMedicalCentre,UniversityofNottingham,Nottingham,UK,2HealthandNutrition,DuPont
UKLtd,Beaminster,UK
Editedby:
MichaelaWenzel, The molecular action of polyene macrolides with antifungal activity, amphotericin B
UniversityofAmsterdam,Netherlands
and natamycin, involves recognition of sterols in membranes. Physicochemical and
Reviewedby:
functional studies have contributed details to understanding the interactions between
NorelleDaly,
JamesCookUniversity,Australia amphotericinBandergosteroland,toalesserextent,withcholesterol.Fewermolecular
AxelHollmann, detailsareavailableoninteractionsbetweennatamycinwithsterols.Weusesolidstate
NationalScientificandTechnical
13C MAS NMR to characterize the impact of amphotericin B and natamycin on mixed
ResearchCouncil,Argentina
lipid membranes of DOPC/cholesterol or DOPC/ergosterol. In cholesterol-containing
*Correspondence:
BoyanB.Bonev membranes, amphotericin B addition resulted in marked increase in both DOPC and
[email protected] cholesterol 13C MAS NMR linewidth, reflecting membrane insertion and cooperative
†PresentAddress:
perturbationofthebilayer.Bycontrast,natamycinaffectslittleeitherDOPCorcholesterol
FilipCiesielski,
ISISNeutronandMuonSource, linewidthbutattenuatescholesterolresonanceintensitypreferentiallyforsterolcorewith
ScienceandTechnologyFacilities lesserimpactonthechain.Ergosterolresonances,attenuatedbyamphotericinB,reveal
Council,Oxford,UK;
specific interactions in the sterol core and chain base. Natamycin addition selectively
JessicaLoraine,
DepartmentofBiology,Universityof augmentedergosterolresonancesfromsterolcoreringoneand,atthesametime,from
York,York,UK
the end of the chain. This puts forward an interaction model similar to the head-to-tail
model for amphotericin B/ergosterol pairing but with docking on opposite sterol faces.
Specialtysection:
Thisarticlewassubmittedto Low toxicityof natamycinis attributedtoselective,non-cooperative sterol engagement
MembranePhysiologyandMembrane comparedtocooperativemembraneperturbationbyamphotericinB.
Biophysics,
asectionofthejournal Keywords:antimicrobials,antifungals,membranes,cholesterol,ergosterol,13CsolidstateMASNMR,receptor
FrontiersinCellandDevelopmental
recognition
Biology
Received:28April2016
Accepted:30May2016 INTRODUCTION
Published:17June2016
Citation: Amphotericin B and natamycin belong to the class of macrolide polyene antibiotics, which are
CiesielskiF,GriffinDC,LoraineJ, used to prevent and treating opportunistic fungal infections, as well as in the control of food
RittigM,Delves-BroughtonJand spoilage.Bothantimycoticcompoundsaremembrane-activeandhavebeenshowntointeractwith
BonevBB(2016)Recognitionof ergosterol (Gabrielska et al., 2006; Matsuoka et al., 2006). While amphotericin B uses ergosterol
MembraneSterolsbyPolyene
asareceptorformembranedisruption,natamycindoesnotpermeabilisemembranesdirectlybut
AntifungalsAmphotericinBand
Natamycin,A13CMASNMRStudy. underminesthefunctionofergosterol-dependentmembranetransport.
Front.CellDev.Biol.4:57. Amphotericin B is produced by Streptomyces nodosus and kills fungal cells by forming
doi:10.3389/fcell.2016.00057 membrane pore complexes with ergosterol. The molecule is approximately 23 Å long, which is
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Ciesielskietal. SterolRecognitionbyPolyeneAntifungals
slightlylessthanthetypicalmembranethicknessanditsactivity toAmphoteracinB,natamycinhasbeenlesswell-characterized.
is sensitive to lipid chain length (Matsuoka et al., 2006). Solid Previous views that natamycin shares mode of action with
state NMR has been used to show that amphotericin B can amphotericinB,i.e.,poreformationanddissipationofionsand
form a single ring spanning the lipid bilayer when the drug is celldeathhavebeenoverturnedbyobservationsthatnatamycin
deliveredfromonesideofamembrane(Gabrielskaetal.,2006) does not form membrane pores but interacts specifically
or a double length ring when it is present on both sides of a with ergosterol excluding it from membrane functionality (te
membrane(Matsuokaetal.,2005).Treatmentwithamphotericin Welscher et al., 2008) and preventing vacuolar trafficking (te
Bisrestrictedtoaminimumduetoitsseveresideeffects,such Welscher et al., 2010) and endocytosis (Van Leeuwen et al.,
as nephrotoxicity. However, due to lack of alternatives, for the 2009). Recent studies develop a clearer mechanistic picture,
past50yearsitremainsoneofthemostcommonantimycotics. in which natamycin/ergosterol interactions downregulate
There is a great interest in understanding its mechanism of ergosterol-dependentmembranetransportproteins(teWelscher
action and factors which affect the specificity toward fungal etal.,2012).
cells as this knowledge could allow designing more efficient WhileinteractionsofamphotericinBand,toalesserextentof
and specific drugs. Studies with red blood cells showed that natamycinwithergosterolhavealreadybeenstudiedindetailin
amphotericin B can target cholesterol-containing membranes thepast(Fournieretal.,1998,2008;Gagosetal.,2005;Matsumori
(Kotler-Brajtburgetal.,1974),whichisthoughttobetheprimary et al., 2006; Kasai et al., 2008; Gruszecki et al., 2009; Foglia
cause of its toxicity. However, experiments also revealed that etal.,2012;GagosandArczewska,2012;Umegawaetal.,2012),
amphotericinBhasgreateraffinitytoergosterolthancholesterol less is known about their interactions with cholesterol. In this
(Readio and Bittman, 1982), partially explaining the specificity study we use solid state magic angel spinning (MAS) NMR to
of the drug. However, because the channel formation involves investigatetheeffectsofamphotericinBandnatamycinonmixed
eightamphotericinBmoleculesandeightsterols,Baginskigroup lipidmembranescontainingDOPCandcholesterolorergosterol.
(Baginski et al., 2002) have speculated that small differences Natural abundance 13C is used in MAS NMR to observe the
in sterol affinity cumulate and become significant enough to effects of each antifungal on lipid and sterol dynamics and the
trigger specificity. Their molecular simulations showed that differentialeffectofamphotericinBandatamycinonmembranes
channels formed with ergosterol are bigger than those formed containingcholesterolorergosterol.
withcholesterol,greatlyaffectingtheionpermeability.
A great deal of attention has been also focused on the two MATERIALS AND METHODS
sterols. Both are known to induce liquid ordered phases on
lipidbilayerswheretrans-gaucheisomerizationsvanish(Endress Sample Preparation
et al., 2002; Mouritsen and Zuckermann, 2004; Rog et al., Lipidswerepurchasedandusedwithoutfurtherpurification,1,2-
2009), however, the degree of ordering depends on the lipid di-oleoylphosphatidylcholine(DOPC)fromAvantiPolarLipids;
type. For example ergosterol has a stronger ordering effect on cholesterol from BDH chemicals Ltd Poole England; and, 98%
the saturated lipid chains than cholesterol, but the opposite Ergosterol(nitrogenfixed)fromAcrosOrganics.Amphotericin
is true for unsaturated phospholipid bilayers (Urbina et al., BwaspurchasedfromMPBiomedicalsandusedwithoutfurther
1998). Furthermore, quantum calculations showed significant purification.NatamaxwaskindlydonatedbyDuPontandlactose
differencesinelectricpotentialsdistributionsbetweenergosterol wasremovedviamultiplewashesindistilledwatertoyieldpure
and cholesterol. Highly negative regions were predicted in the natamycin.
ergosterol,whichcouldaccountforitshigheraffinitytowardthe DOPCwasmixedwitheithercholesterolorergosterolin7:3
polar macrolide structures within a hydrophobic environment ratios in chloroform/methanol and re-suspended in a working
ofalipidbilayer(Baginskietal.,1997a).Inaddition,molecular buffer 10mM HEPES pH 7.0, 10mM NaCl, 1mM EDTA (the
dynamics simulations have showed that ergosterol tail chain samebufferwasusedforallsamples).Natamycinwasdissolvedin
is stiffer and more elongated than that of cholesterol, making methanolandaddedtotheDOPC/sterollipidfilm(finalratioof
the former more stable and providing stronger van der Waals DOPC/sterol/drugwas7:3:1.5),andaftersolventremovalalsore-
contactswiththedrugs. suspendedintheworkingbuffer.AmphotericinBwasdissolved
Other studies, on the other hand, suggest that cholesterol is indi-methylformamide(DMF)andaddedtoDOPC/Cholesterol
notrequiredfortheamphotericinB-drivenformationofpores. lipidfilm(finalratio7:3:1.5)andDMFwasremovedbyrepeated
Cotero et al. (1998) found that ionic current can be detected 7×dilutionwithdistilledwaterandcentrifugationat20,000rpm
◦
on amphotericin B addition without the presence of sterols in at 20 C (Beckman coulter Avanti J25) for 30min. Residual
a membrane. They hypothesized that the role of a sterol in the DMFwasremovedunderhighvacuumandthesamplewasre-
channelformationisviatheeffectithasonthemembraneitself, suspendedintheworkingbuffer.Allsampleswerethenfreeze-
insteadofdirectlyaffectingthechannelformation.Furthermore, thawed a minimum of 5 times each, pelleted at 16,060 g for
molecular simulations also backed this view, showing that 15min (Biofuge pico, Heraeus) and the pellets were loaded in
cholesteroldoesnoteveninteractwithamphotericinBatallin 4mmMASNMRrotors.
anyspecificway(Baginskietal.,1997b).
NatamycinisproducedbyStreptomycesnatalensisandisused Solid State NMR Spectroscopy
onacommercialscaleinthefoodindustryaspreservativeE235 Allsolid-stateNMRexperimentswereperformedonaVarian400
andalsoasanadditivetotopicalantifungalcreams.Incontrast MHz(VNMRS)spectrometerusinga4mmT3MASNMRprobe
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(Varian,PaloAltoCA,USA).Cross-polarization,CP,(Hartmann B (middle) or natamycin (top). Amphotericin has a profound
and Hahn, 1962) with proton ramp (Metz et al., 1994) was effect on membrane dynamics and all lipid and cholesterol
usedtoprepareobservable13Cmagnetization.A96kHzproton resonances appear broadened, indicating that amphotericin B
excitation pulse was followed by 3.5ms of 45kHz Hartmann- doesinsertinthemembraneandthatinsertionhasadisordering
Hahn contact. MAS NMR spectra were acquired using 60kHz effect leading to increased librational freedom for both DOPC
SPINALdecoupling(Fungetal.,2000)tosuppressheteronuclear and cholesterol. The linewidth is likely related to motional
1H−13C dipolar interactions or using 96kHz FSLG decoupling interference with the proton decoupling, as the lipid CO
(Bieleckietal.,1989).Acquisitionswererepeatedeach3.5swith resonances, which have no directly bonded protons appear less
150ms acquisition times under SPINAL and 75ms duration affects. Despite this line broadening, the relative intensity of
underFSLG.Toobtainheteronuclear-decoupledhigh-resolution cholesterol resonances compared to DOPC does not appear
spectra, 1024 transients were averaged, while 8192 transients significantly altered, which indicates no preferential interaction
were added when homonuclear decoupling was used. Sample ofamphotericinBwithcholesteroloverDOPC.
temperature was regulated using balanced heated/vortex tube- While fast lipid dynamics is dominated by cooperative
cooled gas flow (Ciesielski et al., 2009) and MAS/RF-induced axial rotation, changes in segmental mobility in response to
temperature elevation of 4◦C was taken into consideration antifungals can still be observed by 13C solid state NMR and
by indirect reference to lipid main transition suppression in segmental dynamics in sterols is even more informative. In the
DMPCandDPPCliposomes.Samplerotationwassetat5kHz, presence of amphotericin B cholesterol core residues c1–6, c9,
which is sufficient to produce well-averaged high-resolution c11,c14,c15–17aresignificantlyattenuated,indicatingreduced
spectrainmobilehydratedlipidsystems.Spectralpredictionand core mobility (Figure2 middle). Curiously, c12 is attenuated
processingwasdoneusingADCLabs(Toronto,Canada). butstillobservedandmethylsc18,c19,andc21,whichlinethe
sameregionofthecholesterolmoleculedonotappearaffected.
Amphotericin has little effect on chain residues c20–c25, as
RESULTS well. However, resonance intensity from chain terminal methyl
groupsc26andc27areenhanced,whichpointstoparticipation
Molecular Interactions of Amphotericin B
of the chain end in the interaction between amphotericin and
and Natamycin with Membrane Sterols cholesterol.Chainterminalcouplinghasbeenreportedbetween
High-resolution13CMASNMRspectra,acquiredunderSPINAL ergosterolterminalmethylgroupse27,e28andamphotericinB
◦
decouplingfromDOPCliposomescontaining30%sterolat20 C intheirmembranecomplex(Umegawaetal.,2012).Together,the
yielded well-resolved resonances. Cholesterol and ergosterol dataputforwardaninteractionmodel,inwhichthecholesterol
structures are shown in Figure1 alongside atom numbering ringcoreplaystheleadingroleininteractionswithamphotericin
usedinresonanceassignments(Zorinetal.,2010).PureDOPC B, chiefly on the c4, c6, c15 ridge and with engagement of the
membraneswerecharacterizedforreference.Samplesmadewith chainendc26,c27.
cholesterol produced well-resolved lipid and sterol resonances The impact of natamycin on the spectrum from
(Figure2), which were assigned following (Forbes et al., 1988; DOPC/cholesterol membrane is quite different (Figure2
Zorin et al., 2010). DOPC/ergosterol samples produced well- top).Overall,resonanceshaveretainedtheirwidthofferinggood
resolved lipid peaks but broad and low-intensity ergosterol spectral resolution. The impact of natamycin on cholesterol
resonances in most cases (Figure3), due to intermediate mobility indicates the formation of a molecular complex,
mobility of this sterol in DOPC membranes, which interferes which bears resemblance to that between amphotericin B and
with cross-polarization and homonuclear dipolar decoupling. cholesterol showing ring core and chain-terminal interactions
Resolved ergosterol peaks were assigned according to $Soubias with natamycin. However, distinct differences in segmental
et al. (Soubias and Gawrisch, 2005) and used to monitor the dynamicsrevealmobilityrestrictionsonallringmethylgroups,
membrane response to antifungals. The same set of samples aswellasonringA(c1–5,c10)andthebaseofthechain.This
was studied using 13C MAS NMR spectroscopy with FSLG points to natamycin engagement of the cholesterol molecule
decoupling during signal acquisition, which removes proton ontheoppositesidetothatwithamphotericinBandinvolving
degeneracy and provides J-resolved 13C spectra for sufficiently cholesterolring1(c1–c5)andthechainend(c25–c27),however,
mobile molecular segments. Well-resolved couplings were alsowithcontactonmethylsc18,c18andnotdirectlywiththe
measuredforDOPCresonancesonly,duetothepoorresolution otherrings.
ofthesterolmultiplets. Ergosterol-containingmembranesundergomotionsnearthe
Molecular interactions in membranes can be followed using NMR timescale, which leads to some interference with proton
13C MAS NMR by following changes in segmental dynamics. decoupling and cross-polarization and erodes some ergosterol
Such changes impact longitudinal relaxation, which leads to and antibiotic resonances from the 13C MAS NMR spectra
loss of intensity in resonances from directly interacting groups (Figure3, bottom). Resolved resonances inform on the impact
(Sangheraetal.,2011)orintensityenhancementunderCP-MAS of antibiotic presence on segmental dynamics through changes
wherefastmolecularmobilityinterferingwithCPordecoupling inresonanceintensity(dependentonthelongitudinalrelaxation
is moderated in a complex and NMR signal is enhanced. timeT )bycomparisontotherepeatdelay.
1
Figure2shows13CMASNMRspectrafromDOPC/cholesterol The presence of amphotericin B in the DOPC/ergosterol
membranesalone(bottom)andinthepresenceofamphotericin membrane is associated with attenuation of ergosterol
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FIGURE1|Structuresofcholesterol(A),ergosterol(B),amphotericinB(C),andnatamycin(D).
resonances,whichisconsistentwithalteredmoleculardynamics e24, and e25 (Figure3, middle). The terminal methyls e27
duringcomplexformationbetweenergosterolandamphotericin and e28 are not attenuated but are shifted. This points to an
B.Thisattenuationismostnoticeableandspecificforresonances interactionpicture,whichinvolvesthee1-e9-e12edgewithe19
e1, e9, e12, and e19, which complement the interfacial ridge andthebeginningofthechaine20,e22,e24alongwithringDat
of rings A, B, and C in ergosterol along with methyl e19. The e15(Figure1).
opposite side of the molecule, e15, is also attenualted, which Additionofnatamycintoergosterol/DOPCmembranesyields
appearstolinktomobilityrestrictionsinthechainate20,e22, a better resolved spectrum, in which a number of natamycin
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FIGURE2|High-resolution13CMASNMRspectraofhydratedDOPC/cholesterolmembranes:(A)fullspectrumshowinglipidresonancesandsome
cholesterolresonances;membranealone(bottom);withamphotericinB(middle);andwithnatamycin(top);(B)aliphatic/ringregionexpanded,
showingcholesterolassignment.
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FIGURE3|High-resolution13CMASNMRspectraofhydratedDOPC/ergosterolmembranes:(A)fullspectrumshowinglipidresonancesandsome
ergosterolresonances;membranealone(bottom);withamphotericinB(middle);andwithnatamycin(top);(B)aliphatic/ringregionexpanded,
showingergosterolassignment.
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FIGURE4|ChangesinDOPCresonancelinewidthonadditionofamphotericinB(redsquares)ornatamycin(bluediamonds)withrespecttoDOPC
chemicalshiftsinDOPC/cholesterol(A)orDOPC/ergosterol(B)membranes,respectively.
residues are also observed (Figure3, top). The attenuation resonances e23, e24, and e25 attenuated, as well as terminal
of ergosterol signals in this spectrum is far more widespread methyls e27 and e28. Curiously, resonance e26 is significantly
involving the same ergosterol ridge e1, e10, e9, e11, as well enhanced,showingthattheendoftheergosterolchainundergoes
methyl e19 but also resonances e3 and e4 on ring A, e7 on the non-cooperative motions near the intermediate timescale that
opposite side of ring B, e13 and e15 on ring D. Interestingly, are affected profoundly on natamycin binding. In contrast to
akin to amphotericin, natamycin does not attenuate methyl amphotericinB,antamycinappearstoengagemethyle21atthe
e18.Theergosterolchainisalsomoreprofoundlyaffectedwith beginning of the chain (Figure1). The impact of natamycin
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FIGURE5|ChangesinDOPCresonanceisotropic13CchemicalshiftonadditionofamphotericinB(redsquares)ornatamycin(bluediamonds)with
respecttoDOPCchemicalshiftsinDOPC/cholesterol(A)orDOPC/ergosterol(B)membranes,respectively.
on mobility at e5 and e8 is difficult to assess due to overlap dynamics.Bycontrast,whiletheobservedspectroscopicchanges
with natamycin polyene chain signal. Similarly, e14 and e17 in amphotericin B/ergosterol spectra support the molecular
resonances are no longer resolved and are obscured by the model,proposedin(Umegawaetal.,2012),theyalsosuggestthat
epoxydecarbonsfromnatamycin(Figure3,top). theeffectisfarmorecooperativeaffectingthewholemembrane,
Thechangesinergosterolsegmentalmobilityinthepresence while the natamycin/ergosterol complexes appear to interact
of natamycin along with the persistent spectral resolution with each other and with the membrane bulk much less and,
indicate a more specific antibiotic/sterol molecular interaction, subsequently,perturbmembranelipidtomuchlesserextent.The
somewhatresemblingthatbetweenamphotericinandergosterol significance of this observation is that while amphotericin B at
(Umegawa et al., 2012). Such molecular complexes appear to a mechanistic level may affect the functionality of ergosterol-
have comparatively little effect on lipid and sterol cooperative dependentproteinsthroughsignificantmodulationofmembrane
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FIGURE6|Changesincholesterol13Cchemicalshiftdueinthepresenceofantifungalcompounds:(A)amphotericinB,(B)natamycin.Carbon
numbers,shownontheabscissa,correspondtothoseinFigures1,7androughlyfollowend-toenddirectiononthesterolmolecule.Trendlinesguidetheeyeto
approximateend-to-endchemicalshiftchangesalongeachsterolmolecule.
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FIGURE7|Structuresofcholesterol(A,C)andergosterol(B,D)showingsitesof13CNMRspectralattenuationamphotericnB(A,B)and
natamycin(C,D).
properties(teWelscheretal.,2008),natamycinappearstohavea of membrane active molecules or indirectly by changes in
veryspecificeffectonergosterol,whichisnottransmittedtothe molecularorderparametersandpacking(Bonevetal.,2001).
restofthemembrane. Lipidresonancefullwidthathalfheight(FWHH)isaffected
primarilybylateralmolecularlibrationsorrockingandincreases
in membranes with lower bending modulus, where membrane
Effects of Amphotericin B and Natamycin undulation is facilitated by lose chain packing (Ciesielski et al.,
on Membrane Phospholipid 2009). By contrast, the presence of cholesterol stiffens the
membrane and improves packing in the chain region without
Besides specific interactions with sterols, followed directly
from changes in 13C MAS NMR spectra from cholesterol impairingaxiallipidrotation,whichleadstosignificantreduction
and ergosterol, the addition of polyene antibiotics also affects oflinewidthandimprovedspectralresolution(Zorinetal.,2010).
membrane phospholipid. Within hydrated membranes, Resolution can also be improved under proton decoupling and
phospholipids undergo fast cooperative axial motions with mobilitythatinterfereswithdecouplingcanincreaselinewidth.
GHz rates and the full width at half height (FWHH) of lipid Thus,theFWHHisrelatedtothetransverserelaxationrateand
resonances is affected by the cooperative mobility, fast axial a good indicator of changes in librational mobility of lipids in
rotation, and slow angular librations, as well as by additional, responsetomembraneinteractions.
individual segmental motions. The presence of additional The changes in DOPC 13C linewidth in mixed membranes
membraneconstituentscanaffectanyormanyofthesemotions with cholesterol or ergosterol before and after addition
and the effects are seen in the high-resolution 13C MAS NMR of amphotericin B or natamycin are shown in Figure4
spectra from the membrane lipid (Ciesielski et al., 2009; Zorin alongside the carbon number. Addition of amphotericin B to
et al., 2010). In addition, the isotropic chemical shifts of each cholesterolcontainingDOPCmixedmembranesleadstomarked
carbon environment can be affected directly by the proximity increase in linewidth, particularly in chain region, indicative
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