Direct Detection of Fungal Siderophores on Bats with White-Nose Syndrome via Fluorescence Microscopy-Guided Ambient Ionization Mass Spectrometry

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Direct Detection of Fungal Siderophores on Bats with White-Nose Syndrome via Fluorescence Microscopy-Guided Ambient Ionization Mass Spectrometry

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Title:
Direct Detection of Fungal Siderophores on Bats with White-Nose Syndrome via Fluorescence Microscopy-Guided Ambient Ionization Mass Spectrometry
Abbreviated Title:
PLOS One
Creator:
Mascuch, Samantha J.
Moree, Wilna J.
Hsu, Cheng-Chih
Turner, Gregory G.
Cheng, Tina L.
Blehert, David S.
Kilpatrick, A. Marm
Frick, Winifred F.
Dorrestein, Pieter C.
Gerwick, Lena
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PLOS one
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English
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White-nose syndrome ( lcsh )
Fluorescence microscopy ( lcsh )
Bats ( lcsh )
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serial ( sobekcm )

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Abstract:
The genus Troglocladius Andersen, Baranov et Hagenlund, gen. n. is erected based on T. hajdi Andersen, Baranov et Hagenlund, sp. n. collected at 980 m depth in the Lukina jama—Trojama cave system in Croatia. Morphological features such as pale color, strongly reduced eyes and very long legs make it a typical cave animal. Surprisingly, it has also retained large wings and appears to be capable of flight which would make T. hajdi the first flying troglobiont worldwide, disproving previous beliefs that bats are the only animals capable of flying in complete darkness. Morphologically the new species does not readily fit within any described genus, but shares characteristics with genera both in the tribes “Metriocnemini” and “Orthocladiini”. Bayesian molecular phylogenetic analysis using the markers COI, 18S rDNAs, 28S rDNA, CADI, and CADIV groups it with the genera Tvetenia, Cardiocladius and Eukiefferiella in the tribe “Metriocnemini”. Troglocladius hajdi may be parthenogenetic, as only females were collected. The discovery confirms the position of the Dinaric arch as a highly important hotspot of subterranean biodiversity.

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RESEARCHARTICLEDirectDetectionofFungalSiderophoreson BatswithWhite-NoseSyndromevia FluorescenceMicroscopy-GuidedAmbient IonizationMassSpectrometrySamanthaJ.Mascuch1,WilnaJ.Moree3,Cheng-ChihHsu2,3,GregoryG.Turner4,Tina L.Cheng5,DavidS.Blehert6,A.MarmKilpatrick5,WinifredF.Frick5,MichaelJ.Meehan2,3, PieterC.Dorrestein1,2,3,LenaGerwick1*1 CenterforMarineBiotechnologyandBiomedicine,ScrippsInstitutionofOceanography,Universityof CaliforniaSanDiego,LaJolla,CA92093,UnitedStatesofAmerica, 2 DepartmentofChemistryand Biochemistry,UniversityofCaliforniaSanDiego,LaJolla,CA,UnitedStatesofAmerica, 3 SkaggsSchoolof PharmacyandPharmaceuticalSciences,UniversityofCaliforniaSanDiego,LaJolla,CA,UnitedStatesof America, 4 PennsylvaniaGameCommission,2001ElemertonAve.,Harrisburg,PA17110,UnitedStatesof America, 5 DepartmentofEcologyandEvolutionaryBiology,UniversityofCaliforniaSantaCruz,Santa Cruz,CA,UnitedStatesofAmerica, 6 UnitedStatesGeologicalSurvey,NationalWildlifeHealthCenter, 6006SchroederRoad,Madison,WI53711,UnitedStatesofAmerica lgerwick@ucsd.eduAbstractWhite-nosesyndrome(WNS)causedbythepathogenicfungus Pseudogymnoascus destructans isdecimatingthepopulationsofseveralhibernatingNorthAmericanbatspecies.Littleisknownaboutthemolecularinterplaybetweenpathogenandhostinthisdisease.Fluorescencemicroscopyambientionizationmassspectrometrywasusedto generatemetabolicprofilesfromthewingsofbothhealthyanddiseasedbatsofthegenus Myotis .Fungalsiderophores,moleculesthatscavengeironfromtheenvironment,weredetectedonthewingsofbatswithWNS,butnotonhealthybats.Thisworkisamongthefirst examplesinwhichmicrobialmoleculesaredirectlydetectedfromaninfectedhostandhighlightstheabilityofatmosphericionizationmethodologiestoprovidedirectmolecularinsight intoinfection.IntroductionFungaldiseasesofanimalsandplantshavelongbeenafeatureofnaturalecosystems,butevidencesuggeststhatincidencesofmycosesareincreasinginfrequencyandseverityandthat theseemerginginfectiousdiseasesposeathreatintermsoflossofbiodiversityandfoodsecurity[ 1 ].Mycoseshavegarneredalargeamountofattentionrecentlyduetotheirdetrimentalimpactsonpopulationsoforganismsasvariedasseafans,turtles,bees,corals,frogs,crayfish,and bats[ 1 – 7 ].ThefungalinfectionaffectinghibernatingNorthAmericanbats,white-nosesyndrome(WNS),haskilledanestimated5.7 – 6.7millionbatssinceitwasfirstdescribedin2006 PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 1/12 a11111 OPENACCESS Citation: MascuchSJ,MoreeWJ,HsuC-C,Turner GG,ChengTL,BlehertDS,etal.(2015)Direct DetectionofFungalSiderophoresonBatswithWhiteNoseSyndromeviaFluorescenceMicroscopyGuidedAmbientIonizationMassSpectrometry.PLoS ONE10(3):e0119668.doi:10.1371/journal. pone.0119668 AcademicEditor: JacobLawrenceKerby,University ofSouthDakota,UNITEDSTATES Received: July17,2014 Accepted: January7,2015 Published: March17,2015 Copyright: Thisisanopenaccessarticle,freeofall copyright,andmaybefreelyreproduced,distributed, transmitted,modified,builtupon,orotherwiseused byanyoneforanylawfulpurpose.Theworkismade availableunderthe CreativeCommonsCC0 public domaindedication. DataAvailabilityStatement: Allrelevantdataare withinthepaperanditsSupportingInformationfiles. Funding: ResearchinthelaboratoryofPCDforthe developmentofreal-timeMSwassupportedby NationalInstitutesofHealthGrantGM094802( http:// www.nih.gov/ ).ResearchinthelaboratoryofAMK wassupportedbyNationalScienceFoundationgrant DEB-1115895( http://www.nsf.gov/ ).WorkbySJM waspartiallysupportedbyaP.E.O.fellowship( http:// www.peointernational.org/ ).Thefundershadnorole

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andhasspreadto25statesandfiveCanadianprovinces[ 8 – 11 ].ThepsychrophilicfungusidentifiedasthecausativeagentofWNS, Pseudogymnoascusdestructans ,isbelievedtobeanexotic pathogenofEuropeanoriginthatwasintroducedtotheU.S.throughhumanactivity[ 12 – 17 ]. ThemechanismthroughwhichWNSinducesmortalityremainstobefullyelucidated.Itis hypothesizedthatcutaneousinfectionofbatwingskinwith P destructans disruptselectrolyte balance[ 18 – 19 ].Thisimbalanceorsomeothermechanismcausesincreasesinthefrequency withwhichinfectedbatsarousefromtorporduringwinter[ 19 ].Frequentarousalsmayinturn depletefatstoresandleadtodeathbystarvation[ 20 ]. Identifyingmoleculesthatfungalpathogensusetointeractwithahostprovidesinsightinto themechanismoftheirpathogenicity.Betterunderstandingthesemechanismsmayleadtoimprovedmanagementstrategiesthataccountforthebiologyofthepathogen.Wethereforeset outtodirectlyprofilethemetabolicmilieuonthewingsofbatswithWNSwithafocusonfungalmolecules.Weusedfluorescentmicroscopy-guidedambientmassspectrometrytometabolicallyprofilethewingsofbatsofthegenus Myotis thatwerehealthy(n=5)orshowedsignsof WNSinfection(n=11).Thiswillenableustobegintoestablisha ‘ molecularsignature ’ for WNSandtodetermineiftherearemoleculesthatthefungusmayusetofacilitateinfection.MaterialsandMethods AnimalCollection&TissueSamplePreparationFivehealthy Myotislucifugus ,10 M lucifugus withWNS,andone Myotisseptentrionalis with WNSwereincludedintheanalysis( Table1 ).Allbatswerefounddeceasedduringroutine monitoringofmaternityorhibernationroostsbeingconductedbystateagencybiologists.In Pennsylvania,personnelofthePennsylvaniaGameCommissioncollectedthespecimensin compliancewithPennsylvaniaStatuteTitle34,Section322.DeceasedbatscollectedinWest VirginiawerecollectedbypersonneloftheWestVirginiaDepartmentofNaturalResources andnopermitswererequired.Healthybatswerecollectedfrom2004to2008inPennsylvania. AllbutoneofthesebatswerecollectedpriortotheemergenceofWNSinNorthAmericaand thebatcollectedafterWNSemergencehadnoobvioussignsof P destructans infection(e.g. cuppingerosions)whenevaluatedmicroscopically.WNSbatswerecollectedin2011inPennsylvaniaandinWestVirginiaduringaWNS-associatedmassmortalityevent.Nobatswereeuthanizedforthisstudy.Two6mmtissuepuncheswerecollectedfromthewingsofeachofthe deceasedbats,affixedto1x3inchmicroscopeglassslides,andstoredat-80Cpriortomass spectrometricanalysis.MicroscopyAmbientIonizationMassSpectrometryofBatWingsThemassspectrometricinterrogationofbatwingskinwasperformedusingahybridmicroscopy/ionizationtechniquewhichcombinesanambientnanospraydesorptionelectrosprayionization(nanoDESI)sourceandaninvertedmicroscopeasdescribed[ 21 ]( Fig.1A ).Unlikeprevious usageofthesetools,ouranalysisincorporatedtheuseoffluorescence.ThisallowedustoilluminatethewingswithUVlightandvisualizeareasoffluorescencethatcorrelatewiththedevelopmentofcuppingerosionsduetoinfectionwith P destructans [ 22 ]( Fig.1B ).Wecouldthen directlytargettheseareaswiththenanoDESIprobeforMSanalysis( Fig.1C ).Thesampleslide withwingpuncheswasplacedonthestageofaNikonDIAPHOT300microscopeandbright fieldandfluorescentimagesofthetissuewerecapturedusingaCCDcamera(NikonD40DSLR) toconfirmthepresenceorabsenceoffungalinfection.Thestagewasthenmanipulatedtomove thetissuesampletothedesiredpositionunderthemicrometer-sizedliquidjunctionformedby thetwoflame-pulledfusedsilicacapillariesofthenanoDESI.Thecapillarytubeswereflamedpulledfromoriginal150/50 m(O.D./I.D.)to~60 mO.D.andavoltageof2.2kVwasapplied MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 2/12 instudydesign,datacollectionandanalysis,decision topublish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclared thatnocompetinginterestsexist.

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tothemthroughouttheexperiment.Thecapillarieswerealignedina “ V ” configurationsothat theyabuttedoneanotheratthebottomofthe “ V ” andthenangled45awayfromthepointof contactineitherdirection.Asyringepumpwasusedtocontinuouslydeliverthesolvent(either acetonitrileinwater(65/35,vol/vol)with0.2%formicacid,methanolinwater(50/50,vol/vol) with0.2%formicacid,ormethanol,acetonitrile,andtoluene(50/35/15,vol/vol/vol)with0.2% formicacid)atarateof ~ 1.0 L/minthrough300/100 m(O.D./I.D.)fusedsilicacapillarytubingtotheprimaryflame-pulledcapillary.Thissolventwasthenaspiratedbythesecondarycapillaryresultingintheformationofadynamicliquiddropletofapproximately100 mindiameter atthejunctionofthetwocapillaries.Thesamplestagewasraiseduntilthetissuecontactedthe liquiddropletandacontinuousstreamofsolventcontaininganalytesdesorbedfromthetissue wasdeliveredtotheinletofthemassspectrometer(hybrid6.4-TLTQ-FT;ThermoElectron, NorthAmerica)viaelectrosprayionizationgeneratedattheterminalendofthesecondarycapillary.Multiplespotsweresampledalongeachtissueslide.FungalExtractionforMSAnalysisReplicatesof P destructans weregrownonISP2agarplates(4g/literyeastextract,10g/litermalt extract,4g/literdextrose,and1.5to2%agar).Smallplugsofagarandfungalbiomasswerecollectedandcombinedwith100 lof n -butanolinmicrocentrifugetubes.Extractionswereallowed toproceedatroomtemperatureforonehour.Theorganiclayerswerecentrifugedandthesupernatantwassubsequentlyanalyzed.ATriversananomate-electrosprayionizationsourcewas usedtointroducetheextracts(dilutedinmethanolinwater(50:50)with1%formicacid)directly intotheMSinlet.Thiswasaccomplishedusingasprayvoltagesettingof1.3 – 1.45kVandapressureof0.35 – 0.5psiassetwithChipsoftsoftwareversion7.2.0.DatawerecollectedusingXcalibursoftwareversion1.4SR1runningthedata-dependentmethoddescribedbelow.MassSpectrometricAnalysisA6.4TFinniganLTQ-FT-ICRMS(ThermoElectron,NorthAmerica)wasusedtoanalyzetissuesamplesandfungalextracts.Theinstrumentwastunedto816 m/z usingbovine Table1.Specimeninformationforthe Myotis usedinthestudy. WNSStatusSpeciesSexCollectionDateCollectionLocation Pre-WNS Myotislucifugus unknown11/12/08BartonCave,FayetteCounty,PA Pre-WNS Myotislucifugus female8/15/05Shaver ’ sCreek,HuntingtonCounty,PA Pre-WNS Myotislucifugus male9/13/04HarlansburgCaveGate,LawrenceCounty,PA Pre-WNS Myotislucifugus female4/16/05CanoeCreekMine,BlairCounty,PA Pre-WNS Myotislucifugus female4/16/05CanoeCreekMine,BlairCounty,PA WNS Myotislucifugus female2/26/11HellholeCave,PendeltonCounty,WV WNS Myotislucifugus female2/26/11HellholeCave,PendeltonCounty,WV WNS Myotislucifugus male2/26/11HellholeCave,PendeltonCounty,WV WNS Myotislucifugus male2/26/11HellholeCave,PendeltonCounty,WV WNS Myotislucifugus male2/26/11HellholeCave,PendeltonCounty,WV WNS Myotislucifugus female2/26/11HellholeCave,PendeltonCounty,WV WNS Myotislucifugus female2/26/11HellholeCave,PendeltonCounty,WV WNS Myotislucifugus male2/26/11HellholeCave,PendeltonCounty,WV WNS Myotislucifugus male2/26/11HellholeCave,PendeltonCounty,WV WNS Myotislucifugus male2/26/11HellholeCave,PendeltonCounty,WV WNS Myotisseptentrionalis unknown1/7/11LincolnCavern,HuntingtonCounty,PA doi:10.1371/journal.pone.0119668.t001 MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 3/12

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cytochrome c (chargestate15,SigmaAldrich)ina65/35(vol/vol)acetonitrile/0.2%formic acidaqueoussolutionwithTunePlussoftwareversion1.0.Ionspectrafrom150 – 2,000 m/z werecollectedinpositiveionmode.Incaseswheretherewasahighbackground,theionspectraobservationrangewasreduced.Theautomatedinstrumentscancycleconsistedoftwosegments.ThefirstsegmenthadtwoprofilemodeMS1scans:onefullscanintheITmodewith 200msmaxfilltime;onefullscanintheFTcell(50,000resolution)with8secmaxinjecttime. ThiswasfollowedbyanMS1-dependenttandemmass(MS/MS)acquisitionwhichconsistedof 9scansinacyclewitha m/z =3isolationwindow.Thetop9mostabundantpeaksintheMS1scanweresequentiallyisolatedandfragmentedbyapplyingcollisioninduceddissociation (CID)energyusingnitrogenasthecollisiongas.Ionpeaksthathadbeenselectedtwicewere excludedandnotfragmentedagaininsubsequentcyclesinordertocoverasmanyindividual ionsaspossible.Thesedata-dependentMS/MSscans(ITmode,profilespectra)consistedofa maximum500msfilltime,35%normalizedcollisionenergy,0.25activationQ,and0.05s Fig1.Microscopyambientionizationmassspectrometryofbatwings. Tissuesamplesfromthewingsofhealthybatsandindividualswithwhite-nose syndromeweresubjectedtomicroscopyambientionizationmassspectrometry(A). Pseudogymnoascusdestructans infectionwasconfirmedbythe presenceoffluorescentlesionswhenthetissuewasexcitedwithUVlight(B).AnanoDESIsourcewasusedtodesorbanalytesfromfluorescenttissue regionsforMSanalysis(capillaryjunctionmakingcontactwiththetissuesurface,C). doi:10.1371/journal.pone.0119668.g001 MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 4/12

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activationtime.Eachscancontained4microscansandwasrecordedinaverage.Datawereacquiredfor10minutes.MolecularNetworkingThestructuralrelationshipsbetweendifferentmassesinMS1scans(precursorions)were mappedinCytoscapeversion2.8.3basedonthesimilarityoftheirtandemMSfragmentation patternsasassessedthroughmolecularnetworkingusingtheweb-basedGlobalNaturalProductsSocialMolecularNetworkingtool(GNPS; http://gnps.ucsd.edu/ )[ 23 – 25 ].Acosinecutoff of0.6wasselectedforgenerationofthenetwork.Backgroundsignalsarisingfromsolventsor agarweresubtractedduringthenetworkprocessingstage.Thenetworkwasthenimported intoCytoscapeforvisualization.Precursorionswererepresentedbynodesandthesimilarity betweentwoprecursorionsasdeterminedbycomparisonoftheirfragments/cosinescorewas representedasanedgebetweentwonodes.Themagnitudeofthecosinescorewasrepresented byedgethicknesswithpairsofcompoundswithhighscoreshavingthickerlines.Asearchof thepubliclycuratedGNPSstandardslibrarywasalsoperformedtodetermineifanyofthe massesrepresentedinthenetworkmatchedtoknowncompoundsthatwehadnotconsidered inouranalysis.AlldatafilesusedinthegenerationofthenetworkweredepositedintheGNPS MassIVEdatarepositoryandarepubliclyavailable(MassIVEIDMSV000078620).SeedingtheNetworkwithDesferrichrome,Ferrichrome,and TriacetylfusarinineCStandardsIron-freeferrichrome(SantaCruzBiotechnology),ferrichrome(Iron-freeferrichromewiththe additionofiron(III)chloridehexahydrate),andtriacetylfusarinineC(EMCmicrocollections GmbH)wereusedasstandardstoverifythemolecularassignmentofthesiderophoresinthe tissuesamplesthroughaprocessknownasdereplication[ 25 ].Thesolubilizedstandardswere introduceddirectlyintotheMSinletwithaTriversananomate-electrosprayionizationsource andanalyzedusingthedata-dependentmethoddescribedabove.Amolecularnetworkwas thengeneratedusingthedatafilesfromthestandards,fungalextracts,andtissuesamples.The nodescorrelatedwiththestandarddatafileswereexaminedtoestablishifthey(1)clustered withexperimentalnodesor(2)wereincorporatedintoconsensusnodesthatalsoincludedexperimentalfiles.Followingthisneighborhoodanalysis,thespectraweremanuallyexamined andthefragmentationoftherawspectraevaluatedforsimilarity.Throughthisanalysisitwas possibletodefinitivelydeterminewhetherornotdesferrichrome,ferrichromeandtriacetylfusarinineCweredetectedfromthewingsurfacesand/orfromfungalcultures.ResultsandDiscussion Detectionoffungalmetabolitesonbatwingsbyfluorescence microscopyambientionizationmassspectrometryandmolecular networkingFluorescentmicroscopyambientmassspectrometryofbatwings,extractsfromcultured P destructans ,andcommercialstandardsgeneratedalargenumberofMS/MSspectra.These spectraweresubjectedtomolecularnetworking[ 23 25 ]givingrisetoanetworkcomprisedof 1,503nodesrepresentingprecursorionsderivedfromintactchemicalcompoundsand2,463 edgesrepresentingtheirstructuralrelationships( Fig.2A ; S1Fig .).Eachnodewascoloredwith respecttothesampletype(s)fromwhichitderived.Theresultingmolecularnetworkprovides avisualpictureofthediversityandoriginofMS/MSspectrathatwerecollected. MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 5/12

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Thenetworkhighlightedanumberofmoleculesthatwerepresentonthewingsofbatswith WNSthatwerenotdetectedfromhealthybatsand viceversa .ThesedistinctchemicalsignatureswerefoundtobeduetothepresenceoffungalmetabolitesontheWNSwings.Individual variationandahostresponsetoinfectionlikelyalsocontributetheobserveddifference.Several oftheMS/MSspectrafromtheexperimentalsamplesmatchedthoseofknownfungalmetabolitesandthesenodeswereinspectedinfurtherdetail. Inthefirstnodeweinspected,spectrafromfungalextractsmatchedtoacompoundinthe GNPSstandardslibrary,themycotoxincitrinin,indicatingthatP.destructansmayhavethecapacitytoproducecitrininoracitrinin-likemolecule( S2A,S2BFig .).Thisskin-permeable nephrotoxinisknowntobeproducedbyfungiofthegenera Penicillium Monascus ,and Aspergillus [ 26 – 27 ].Aproteinblastofthe P destructans genomeusingthecitrininpolyketide Fig2.Detectionofsiderophoresonthewingsofbatswithwhite-nosesyndromeviamolecularnetworking. MolecularnetworkingoftheMS/MSdata wasusedtodeterminethestructuralrelationshipsbetweenthemetabolitesdetectedfromwingsurfaces,cultured Pseudogymnoascusdestructans ,and commercialsiderophorestandards.Thesiderophoresdesferrichrome( m/z 710.324)andferrichrome( m/z 763.230)wereobservedfromthewingsofbats withwhite-nosesyndromeandfromcultured P destructans andformedconsensusnodeswithcommercialstandards(A,B).TriacetylfusarinineC( m/z 928.344)wasalsopresentonWNSwingsandinthe P destructans colony(C).Noneofthethreesiderophoresweredetectedonthewingsofhealthybats.In AandCinsetsonlythenodewiththe m/z ofinterestanditsfirstandsecondneighborshavebeendisplayedfromtheoverallclusterforsimplicity. doi:10.1371/journal.pone.0119668.g002 MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 6/12

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synthasefrom Monascuspurpureus asthequeryrevealedahomologoussequencewith44% identity.( S2CFig .).Itmaybeinterestingtoevaluatetheoverallabilityofthefungustoproduce mycotoxinsinthefutureandtoevaluatethecontributionsofanysuchmolecules topathogenicity. Anothernodewasamatchforthefungalsiderophoredesferrichrome( m/z 710.324, Fig.2A ).Siderophoresaremicrobialiron-chelatingmoleculesthatareeitherretainedintracellularlyasameansofironstorageorareofadiffusibletypethatgatherferricironfromtheextracellularenvironmentandarethenretrievedbytheorganism[ 28 ].Thenodeincorporated scansfromWNSwings,thecultured P destructans colony,andthedesferrichromestandard; scansfromthewingsofhealthybatswerenotincluded( Fig.2A ).Manualinspectionofthe MS/MSfragmentationdatafortheseprecursorsconfirmedthatthepatternobservedfromthe WNSwingsandthefungalcolonymatchedthatofthecommercialdesferrichromestandard ( S3Fig .).Thisindicatesthatthemassesobservedfromtheculturedfungusandonthewing skinofinfectedbatsderivefromdesferrichromeandthat,presumably, P destructans isproducingthissiderophoreduringitscolonizationofbats.Whenthesignalforthesiderophore wasanalyzedinatargetedfashion,byperformingMS/MSdirectlyontheisolatedparention duringmicroscopynanoDESIanalysis,desferrichromewasdetectedon10ofthe11WNSbats andwasvirtuallyundetectableinallofthesamplesfromhealthybatwings(ANOVA, p = 0.025; Fig.3 ; S4Fig .).Inaddition,theiron-chelatedformofthemolecule,ferrichrome,wasdetected( m/z 763.230, Fig.2B ; S5Fig .).Thisindicatesthatthereissufficientironpresentonbat wingstobechelatedbythesiderophore. Asecondsiderophore,triacetylfusarinineC,wasalsodetectedbymolecularnetworking ( Fig.2C ).Anodewith m/z 928.344incorporatedscansfromaWNSwingandthecultured P destructans colonyandclusteredwithtriacetylfusarinineCstandardnodes.Manualinspectionofthetandemmassspectraconfirmedthatthenodederivedfromthefragmentationof triacetylfusarinineC,althoughitfailedtoformaconsensusnodewiththestandardunderthe molecularnetworkingparametersselected( S6Fig .).Thisindicatesthatthissiderophoreisalso producedby P destructans duringinfection. Interrogationofthe P destructans genomeforsecondarymetaboliteenzymesinvolvedin siderophorebiosynthesisandimagingmassspectrometryofthecultured P destructans colony furthersupporttheproductionofferrichromeandtriacetylfuarinineCbythefungus ( S1SupportingInformation ; S7Fig .; S1Table ).Intheabsenceofacontrolledlaboratoryexperimentinwhichbatsareanalyzedbeforeandafterinfectionwith P destructans ,itcannotbeunequivocallystatedthattheobservedmetabolitesareproducedby P destructans sinceother fungialsohavetheabilitytoformthesesiderophores.However,thisseemsunlikelygiventhe markeddifferenceweobservebetweenWNSandhealthywingsandwehypothesizethat P destructansrequiresironinordertogrowandmountinfectionandthereforeproducessiderophoresduringitscolonizationofbats. Microbesrequireironasacofactorinredoxreactionsinvolvedinavarietyofcellularprocesses.Consequently,alargepartofthehostresponsetoinfectioncentersonlimitingthe amountofironavailabletoinvadingmicrobes[ 29 – 32 ].Becauseofthis,theamountofavailable ironis,ingeneral,sub-optimalformicrobialgrowth[ 30 33 ].Whetherthisisthecaseforhibernatingbatsisunknown. High-affinitysiderophoresareimportantforsatisfyingmicrobialirondemandinthefaceof hostdefensesandanumberofinvestigationshavedemonstratedthattheyareessentialfor growthandvirulence[ 34 – 38 ].Inaddition,productionofhydroxamatesiderophoresisafeatureofthedermatophyticpathogenicfungi Microsporum and Trichophyton andferrichrometypesiderophores,specifically,havebeendemonstratedtobenecessaryforepithelialinvasion by C albicans inan invitro modeloforalcandidiasis[ 39 – 40 ].Ourdetectionoffungal MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 7/12

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siderophoresonwingsofbatswithWNSsuggeststhatthesemoleculesmayhavearoleininfectionand/ortissueinvasion. Becauseoftheimportanceofironinmicrobialinfections,anumberofantimicrobialstrategiesthattargetsiderophoreshaverecentlyemerged.Theseincludeeffortsthatlinkantibiotics orantifungalstosiderophorestomediatetheiruptake,inundationwithirrelevantsiderophores thattheorganismcannotuseoranaloguesthatcannotbindiron,additionofironchelators suchasEDTA,anddisruptionofendogenoussiderophorepathwaysthroughtheblockingof biosyntheticenzymesortransporters[ 41 – 44 ].Wecontendthatsuchmethodsofirondeprivationshouldbeinvestigatedfortheireffectivenessagainst P destructans infection.ConclusionHereweusedfluorescencemicroscopyambientmassspectrometryincombinationwithmolecularnetworkingtodetectmetabolitesdirectlyfromthewingskinofbatswithWNS.Theapproachestablishedthepresenceoffungalsiderophoresoninfectedbatwings.Siderophoresare importantforfungalgrowthandvirulenceandmethodsthatinterferewiththeirbiosynthesis oractivitymayproveeffectiveinlimiting P destructans infection. Fig3.Statisticalanalysisofdesferrichromedaughterionintensityamonghealthyanddiseasedbat wings. Analysisofvariance(ANOVA)oftheabsoluteintensitiesofthedesferrichromedaughterion, m/z 650 (neutrallossofC2H4O2),observedonWNSandhealthybatwings(Seealso S4Fig .).A 3 m/z ionwindow wasallowedwhenselectingtheprecursor, m/z 710.Differencesintheabsoluteintensitiesofthedaughter ionsamong10ofthe11WNSwingsand5healthywingswerestatisticallysignificant( p =0.025).Onthe eleventhWNSwing,noionswithinthe m/z 710 3 m/z rangewasselectedforfragmentationbythe automaticdata-dependentmethodduetolowionintensityandthesamplecouldthereforenotbeincludedin theplot. doi:10.1371/journal.pone.0119668.g003 MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 8/12

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SupportingInformationS1Table.Comparisonofknownsiderophorebiosyntheticenzymestoputative Pseudogymnoascusdestructans proteins. (XLSX) S1Fig.Molecularnetworkcomposition. Themolecularnetworkwascomposedofnodesthat incorporatedMS/MSscansfromonlyasinglesampletypeaswellasconsensusnodesthatincorporatedspectrafromdifferentexperimentalsampletypes.ThenumberofnodesineachcategoryandtheirrelationshipstoeachotherareconveyedinaVenndiagram. (TIF) S2Fig.Potentialforproductionofthemycotoxincitrininby Pseudogymnoascusdestructans AcomparisonofthedatatoalibraryofstandardsfromtheGlobalNaturalProductsSocialMolecularNetworkingdatabaseresultedinaputativematch,citrinin,tothe P destructans cultureextract(A,B).Aproteinblastofthe P destructans genomeusingthepolyketide synthaseresponsibleforcitrininbiosynthesisasthequeryreturnedahomologousaminoacid sequence(C). (TIF) S3Fig.Comparisonofdesferrichromefragmentationamongstandardsandsamples. MS/ MSofthedesferrichromestandard m/z 710(A).TheMS/MSfragmentationpatternsfromthe P destructans colony(B)andWNSwing(C)matchedthefragmentationofthe standardprecursor. (TIF) S4Fig.Variationintheintensityofdesferrichromedaughterion m/z 650detectedfrom white-nosesyndromewingsamples. Incaseswheredesferrichromeprecursorionsdisplay highintensitiesrelativetoothermetabolites,theirfragmentationpatternswillbelesscomplicatedandtheywillhaveagreatercosinecorrelationwiththestandard(A).Whentheintensitiesofthedesferrichromeionsarelessintense,backgroundnoiseorpeaksfromother compoundsofsimilarmassmaybefragmentedalongwiththemresultinginmorecomplicated MS/MSspectraandalowercosinecorrelationwiththestandardeventhoughthemoleculeis present(B). (TIF) S5Fig.Comparisonofferrichromefragmentationamongstandardsandsamples. FT-MS/ MSofferrichromeFe3+complexstandard m/z 763(A).TheIT-MS/MSfragmentationpatterns for m/z 763fromthe P destructans colony(B)andWNSwings(C)matchedthefragmentation ofthestandardprecursor. (TIF) S6Fig.ComparisonoftriacetylfusarinineCfragmentationamongstandardsandsamples. FT-MS/MSoftriacetylfusarinineCFe3+complexstandard m/z 928(A).TheIT-MS/MSfragmentationpatternsfor m/z 928fromthe P destructans colony(C)andWNSwings(D) matchedthefragmentationofthestandardprecursor. (TIF) S7Fig.Imagingmassspectrometryof Pseudogymnoascusdestructans colonies. P destructans wasgrowndirectlyonISP2agar(Ai)orontopofapermeablemembraneoverlaidonagar (Aii)whichwaslaterremovedforIMSanalysis.Coloniesandagarwereexcisedandplacedon aMALDItargetplatealongwithaportionofplainagarwhichservedasanegativecontrol(B). Aerialhyphaewereremovedfromthe P destructans colonygrowndirectlyontheagar,and MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 9/12

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themembraneand P destructans colonywereremovedfromthesecondsampletorevealthe agarunderneath(C).Severalmetaboliteswereobservedtobeassociatedwiththefungalcolony (Di)orsecretedintotheagarunderneathit(Dii).Amongthemetabolitesforwhichionswere observedwerethesiderophoresdesferrichromeandtriacetylfusarinineC. (TIF) S1SupportingInformation.SupportingMethodsandResultsforImagingMassSpectrometryandGenomicAnalysis. (PDF)AcknowledgmentsTheauthorswouldliketothankDr.ChrisHoeflerandtheDr.PaulStraightLaboratoryforthe giftofcellophanemembranes,Dr.CraigStihlerforcollectionofspecimensfromWestVirginia, Dr.LauraSanchezandPaulD.Boudreauforclipartandassistancewithfigures,andDr.Laura Sanchez,DonNguyen,andDr.VanessaV.Phelanfortrainingandassistancewithmassspectrometryandmolecularnetworking.WealsothankDr.KevinTidgewellforreviewingthe manuscriptandprovidinghelpfulinsights.Theuseoftrade,productorfirmnamesisfordescriptivepurposesonlyanddoesnotimplyendorsementbytheU.S.government.AuthorContributionsConceivedanddesignedtheexperiments:SJMWJMCCHPCDTLC.Performedtheexperiments:SJMWJMCCHMJM.Analyzedthedata:SJMWJMCCH.Contributedreagents/materials/analysistools:TLCGGTDSBAMKWFFLGPCD.Wrotethepaper:SJMWJMCCH TLCGGTDSBAMKWFFLGPCD.References1. FisherMC,HenkDA,BriggsCJ,BrownsteinJS,MadoffLC,McCrawSL,etal.Emergingfungalthreats toanimal,plantandecosystemhealth.Nature.2012;484:186 – 194.doi: 10.1038/nature10947 PMID: 22498624 2. KimK,HarvellCD.Theriseandfallofasix-yearcoral-fungalepizootic.AmNat.2004;164:S52 – S63. PMID: 15540141 3. Sarmiento-RamirezJM,AbellaE,MartinMP,TelleriaMT,Lopez-JuradoLF,MarcoA,etal. Fusarium solani isresponsibleformassmortalitiesinnestsofloggerheadseaturtle, Carettacaretta ,inBoavista, CapeVerde.FemsMicrobiolLett.2010;312:192 – 200.doi: 10.1111/j.1574-6968.2010.02116.x PMID: 20875054 4. CameronSA,LozierJD,StrangeJP,KochJB,CordesN,SolterLF,etal.PatternsofwidespreaddeclineinNorthAmericanbumblebees.ProcNatlAcadSci USA .(2011);108:662 – 667.doi: 10.1073/ pnas.1014743108 PMID: 21199943 5. HarvellCD,KimK,BurkholderJM,ColwellRR,EpsteinPR,GrimesDJ,etal.Emergingmarinediseases — Climatelinksandanthropogenicfactors.Science.1999;285:1505 – 1510.PMID: 10498537 6. HoldichDM,ReynoldsJD,Souty-GrossetC,SibleyPJ.AreviewoftheeverincreasingthreattoEuropeancrayfishfromnon-indigenouscrayfishspecies.KnowlManagAquatEc.2009;394 – 395:11. 7. BlehertDS,HicksAC,BehrM,MeteyerCU,Berlowski-ZierBM,BucklesEL,etal.BatWhite-NoseSyndrome:AnEmergingFungalPathogen?Science.2009;323:227 – 227.doi: 10.1126/science.1163874 PMID: 18974316 8. FrickWF,PollockJF,HicksAC,LangwigKE,ReynoldsDS,TurnerGG,etal.AnEmergingDisease CausesRegionalPopulationCollapseofaCommonNorthAmericanBatSpecies.Science.2010;329: 679 – 682.doi: 10.1126/science.1188594 PMID: 20689016 9. LangwigKE,FrickWF,BriedJT,HicksAC,KunzTH,KilpatrickAM.Sociality,density-dependenceand microclimatesdeterminethepersistenceofpopulationssufferingfromanovelfungaldisease,whitenosesyndrome.EcologyLetters.2012;15:1050 – 1057.doi: 10.1111/j.1461-0248.2012.01829.x PMID: 22747672 MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 10/12

PAGE 11

10. NewsRelease:NorthAmericanbatdeathtollexceeds5.5millionfromwhite-nosesyndrome.Topof FormUnitedStatesFishandWildlifeService.2012.Available: http://www.fws.gov/northeast/feature_ archive/Feature.cfm?id=794592078 11. NewsRelease:Whereisitnow?Whitenosesyndrome.org.2014.Available: http://www. whitenosesyndrome.org/about/where-is-it-now 12. GargasA,TrestMT,ChristensenM,VolkTJ,BlehertD. Geomycesdestructans sp.nov.,associated withBatWhite-NoseSyndrome.Mycotaxon.2009;108:147 – 154. 13. MinnisAM,LindnerDL.Phylogeneticevaluationof Geomyces andalliesrevealsnocloserelativesof Pseudogymnoascusdestructans ,comb.nov.,inbathibernaculaofeasternNorthAmerica.Fungal Biol.2013;117:638 – 649.doi: 10.1016/j.funbio.2013.07.001 PMID: 24012303 14. LorchJM,MeteyerCU,BehrMJ,BoylesJG,CryanPM,HicksAC,etal.Experimentalinfectionofbats with Geomycesdestructans causeswhite-nosesyndrome.Nature.2011;480:376 – 378.doi: 10.1038/ nature10590 PMID: 22031324 15. PuechmailleSJ,FrickWF,KunzTH,RaceyPA,VoigtCC,WibbeltG,etal.White-nosesyndrome:is thisemergingdiseaseathreattoEuropeanbats?TrendsEcolEvol.2011;26:570 – 576.doi: 10.1016/j. tree.2011.06.013 PMID: 21835492 16. PuechmailleSJ,WibbeltG,KornV,FullerH,ForgetF,MuhldorferK,etal.Pan-EuropeanDistribution ofWhite-NoseSyndromeFungus( Geomycesdestructans )NotAssociatedwithMassMortality.PLoS ONE.2011;6(4):e19167.doi: 10.1371/journal.pone.0019167 PMID: 21556356 17. WarneckeL,TurnerJM,BollingerTK,LorchJM,MisraV,CryanPM,etal.InoculationofbatswithEuropean Geomycesdestructans supportsthenovelpathogenhypothesisfortheoriginofwhite-nosesyndrome.ProcNatlAcadSci USA .2012;109:6999 – 7003.doi: 10.1073/pnas.1200374109 PMID: 22493237 18. MeteyerCU,BucklesEL,BlehertDS,HicksAC,GreenDE,Shearn-BochslerV,etal.Histopathologic criteriatoconfirmwhite-nosesyndromeinbats.JVetDiagnInves.2009;21:411 – 414.PMID: 19564488 19. CryanPM,MeteyerCU,BlehertDS,LorchJM,ReederDM,TurnerGG,etal.Electrolytedepletionin white-nosesyndromebats.JWildlDis.2013;49:398 – 402.doi: 10.7589/2012-04-121 PMID: 23568916 20. ReederDM,FrankCL,TurnerGG,MeteyerCU,KurtaA,BritzkeER,etal.FrequentArousalfromHibernationLinkedtoSeverityofInfectionandMortalityinBatswithWhite-NoseSyndrome.PLoSONE. 2012;7(6):e38920.doi: 10.1371/journal.pone.0038920 PMID: 22745688 21. HsuCC,WhiteNM,HayashiM,LinEC,PoonT,BanerjeeI,etal.Microscopyambientionizationtopdownmassspectrometryrevealsdevelopmentalpatterning.ProcNatlAcadSci USA .2013;110: 14855 – 14860.doi:10.1073/pnas.1310618110 PMID: 23969833 22. TurnerGG,MeteyerCU,BartonH,GumbsJF,ReederDM.Non-LethalScreeningofbatwingskin usingUVfluorescencetodetectlesionsindicativeofwhite-nosesyndrome.JWildlDis.2014;50(3): 000 – 000. 23. WatrousJ,RoachP,AlexandrovT,HeathBS,YangJY,KerstenRD,etal.Massspectralmolecular networkingoflivingmicrobialcolonies.ProcNatlAcadSci USA .2012;109:E1743 – E1752.doi: 10. 1073/pnas.1203689109 PMID: 22586093 24. SmootME,OnoK,RuscheinskiJ,WangPL,IdekerT.Cytoscape2.8:newfeaturesfordataintegration andnetworkvisualization.Bioinformatics.2011;27(3):431 – 432.doi: 10.1093/bioinformatics/btq675 PMID: 21149340 25. YangJY,SanchezLM,RathCM,LiuX,BoudreauPD,BrunsN,etal.Molecularnetworkingasadereplicationstrategy.JNatProd.2013;76(9):1686 – 1699.doi: 10.1021/np400413s PMID: 24025162 26. FlajsD,PeraicaM.Toxicologicalpropertiesofcitrinin.ArhHigRadaToksikol.2010;60(4)457 – 464. doi: 10.2478/10004-1254-60-2009-1992 27. BoonenJ,MalyshevaSV,TaevernierL,diMavunguJD,deSaegerS,DeSpeigeleerB.Humanskin penetrationofselectedmodelmycotoxins.Toxicol.2012;301(1 – 3):21 – 32. 28. WinkelmannG.Ecologyofsiderophoreswithspecialreferencetothefungi.Biometals.2007;20: 379 – 392.PMID: 17235665 29. HoodHM,SkaarEP.Nutritionalimmunity:transitionmetalsatthepathogen-hostinterface.NatRev Microbiol.2012;10:525 – 537.doi: 10.1038/nrmicro2836 PMID: 22796883 30. ParrowNL,FlemingRE,MinnickMF.Sequestrationandscavengingofironininfection.InfectImmun. 2013;81(10):3503 – 3514.doi: 10.1128/IAI.00602-13 PMID: 23836822 31. TheurlI,FritscheG,LudwiczekS,GarimorthK,Bellmann-WeilerR,WeisG.Themacrophage:acellularfactoryattheinterphasebetweenironandimmunityforthecontrolofinfections.Biometals.2005; 18:359 – 367.PMID: 16158228 MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 11/12

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32. FluckingerM,HaasH,MerschakP,GlasgowBJ,RedlB.Humantearlipocalinexhibitsantimicrobialactivitybyscavengingmicrobialsiderophores.AntimicrobAgentsandChemother.2004;48:3367 – 3372. PMID: 15328098 33. MiethkeM,MarahielMA.Siderophore-basedironacquisitionandpathogencontrol.MicrobiolMolBiol Rev.2007;71:413 – 451.PMID: 17804665 34. HissenAHT,WanANC,WarwasML,PintoLJ,MooreMM.The Aspergillusfumigatus siderophorebiosyntheticgenesidA,encodingL-ornithineN-5-oxygenase,isrequiredforvirulence.InfectImmun. 2005;73:5493 – 5503.PMID: 16113265 35. SchrettlM,BignellE,KraglC,JoechlC,RogersT,ArstHNJr,etal.Siderophorebiosynthesisbutnot reductiveironassimilationisessentialfor Aspergillusfumigates virulence.JExpMed.2004;200: 1213 – 1219.PMID: 15504822 36. SchrettlM,BignellE,KraglC,SabihaY,LossO,EisendleM,etal.DistinctRolesforIntra-andExtracellularSiderophoresduring Aspergillusfumigatus Infection.PLoSPathog.2007;3(9):e128.doi: 10. 1371/journal.ppat.0030128 37. JohnsonL.Ironandsiderophoresinfungal-hostinteractions.MycolRes.2008;112:170 – 183.doi: 10. 1016/j.mycres.2007.11.012 PMID: 18280720 38. EisendleM,SchrettlM,KraglC,MuellerD,IllmerP,HaasH.Theintracellularsiderophoreferricrocinis involvedinironstorage,oxidative-stressresistance,germination,andsexualdevelopment inAspergillusnidulans .EukaryoticCell.2006;5:1596 – 1603.PMID: 17030991 39. MorH,KashmanY,WinkelmannG,BarashI.Characterizationofsiderophoresproducedbydifferent speciesofthedermatophyticfungi Microsporum and Trichophyton .Biometals.1992;5:213 – 216. 40. HeymannP,GeradsM,SchallerM,DromerF,WinkelmannG,ErnstJF.Thesiderophoreirontransporterof Candidaalbicans (Sit1p/Arn1p)mediatesuptakeofferrichrome-typesiderophoresandisrequiredforepithelialinvasion.InfectImmun.2002;70:5246 – 5255.PMID: 12183576 41. HaasH,EisendleM,TurgeonBG.Siderophoresinfungalphysiologyandvirulence.AnnuRevPhytopathol.2008;46:149 – 187.doi: 10.1146/annurev.phyto.45.062806.094338 PMID: 18680426 42. MiethkeM,MarahielMA.Siderophore-basedironacquisitionandpathogencontrol.MicrobiolMolBiol Rev.2007;71:413 – 451.PMID: 17804665 43. SchrettlM,HaasH.Ironhomeostasis-Achilles ’ heelof Aspergillusfumigatus ?CurrOpinMicrobiol. 2011;14:400 – 405.doi: 10.1016/j.mib.2011.06.002 PMID: 21724450 44. MillerMJ,ZhuH,XuY,WuC,WalzAJ,VergneA,etal.Utilizationofmicrobialironassimilationpro-cessesforthedevelopmentofnewantibioticsandinspirationforthedesignofnewanticanceragents. Biometals.2009;22:61 – 75.doi: 10.1007/s10534-008-9185-0 PMID: 19130268 MetabolomicProfilingofBatswithWhite-NoseSyndrome PLOSONE|DOI:10.1371/journal.pone.0119668March17,2015 12/12


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