Isolation and Identification of an Extracellular Subtilisin-Like Serine Protease Secreted by the Bat Pathogen Pseudogymnoascus destructans

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Isolation and Identification of an Extracellular Subtilisin-Like Serine Protease Secreted by the Bat Pathogen Pseudogymnoascus destructans

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Title:
Isolation and Identification of an Extracellular Subtilisin-Like Serine Protease Secreted by the Bat Pathogen Pseudogymnoascus destructans
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PLOS One
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Pannkuk, Evan L.
Risch, Thomas S.
Savary, Brett J.
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Bats ( lcsh )
Serine proteinases -- Inhibitors ( lcsh )
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serial ( sobekcm )

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White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction..

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RESEARCHARTICLEIsolationandIdentificationofan ExtracellularSubtilisin-LikeSerineProtease SecretedbytheBatPathogen PseudogymnoascusdestructansEvanL.Pannkuk1 ¤* ,ThomasS.Risch2,BrettJ.Savary3*1 GraduateProgramofEnvironmentalScience,ArkansasStateUniversity,Jonesboro,Arkansas,United StatesofAmerica, 2 DepartmentofBiologicalSciences,ArkansasStateUniversity,Jonesboro,Arkansas, UnitedStatesofAmerica, 3 ArkansasBiosciencesInstitute,ArkansasStateUniversity,Jonesboro, Arkansas,UnitedStatesofAmerica ¤ CurrentAddress:DepartmentofBiochemistryandMolecular&CellularBiology,GeorgetownUniversity, WashingtonDC,UnitedStatesofAmerica bsavary@astate.edu (BJS); elp44@georgetown.edu (ELP)AbstractWhitenosesyndrome(WNS)isacutaneousfungaldiseaseofbats.WNSisresponsiblefor unprecedentedmortalitiesinNorthAmericancavebatpopulations.Therehavebeenfew descriptionsofenzymeactivitiesthatmayfunctioninWNShost/pathogeninteractions, whilenostudyhasisolatedanddescribedsecretedproteases.Toaddressthehypothesis that Pseudogymnoascusdestructans secretesextracellularproteasesthatfunctioninwing necrosisduringWNSinfection,theobjectofthisstudywastoculture P destructans onvariousmedia,thenisolateandstructurallyidentifythoseproteasesaccumulatedstablyinthe culturemedium.Wefoundasingledominantproteaseactivityonminimalnutrientbrothenrichedwithproteinsubstrates,whichwasstronglyinhibitedbyphenylmethylsulfonylfluoride.This P destructans serineprotease(PdSP1)wasisolatedbypreparativeisoelectric focusingandconcanavalinAlectinaffinitychromatography.PdSP1showedamolecular weight27,900(estimatedbySDS-PAGE),broadpHoptimum6-8,andtemperatureoptimum60C.StructuralcharacterizationofPdSP1byMALDI-TOFMS,OrbitrapMS/MS,and Edmanamino-terminalpeptidesequencingmatcheditdirectlytoahypotheticalproteinaccessionfromthesequenced P destructans genomethatisfurtheridentifiedasaMEROPS familyS8Asubtilisin-likeserinepeptidase.Twoadditionalisoforms,PdSP2andPdSP3, wereidentifiedinthe P destructans genomewith90%and53%homology,respectively. P destructans S8Aserineproteasesshowedclosersequenceconservationto P pannorum andplantpathogenicfungithantohumanpathogenicdermatophytes.Peptide-specificpolyclonalantibodiesdevelopedfromthePdSP1sequencedetectedtheproteininwestern blots.Thesesubtilisin-likeserineproteasesarecandidatesforfurtherfunctionalstudiesin WNShost-pathogeninteraction. PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 1/18 OPENACCESS Citation: PannkukEL,RischTS,SavaryBJ(2015) IsolationandIdentificationofanExtracellular Subtilisin-LikeSerineProteaseSecretedbytheBat Pathogen Pseudogymnoascusdestructans .PLoS ONE10(3):e0120508.doi:10.1371/journal. pone.0120508 AcademicEditor: GotthardKunze,IPK,GERMANY Received: November15,2014 Accepted: January23,2015 Published: March18,2015 Copyright: 2015Pannkuketal.Thisisanopen accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense ,whichpermits unrestricteduse,distribution,andreproductioninany medium,providedtheoriginalauthorandsourceare credited. DataAvailabilityStatement: Allrelevantdataare withinthepaperanditsSupportingInformationfiles. Funding: ThisstudywasfundedbyU.S.Fishand WildlifeService2012StateWildlifeGrant(Arkansas GameandFishCommission,ART-40-R-1),the NationalSpeleologicalSociety,andTheCenterfor NorthAmericanBatResearchandConservationat IndianaStateUniversity.TheMALDI-TOFMS instrumentwasprovidedthroughtheNSFEPSCoR, RII:ArkansasASSETInitiativeP3Center(EPS0701890)intheArkansasBiosciencesInstituteat ArkansasStateUniversity.Thefundershadnorolein

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IntroductionPseudogymnoascus (basionym: Geomyces ) destructans isthefungusresponsibleforwhitenose syndrome(WNS)inbats[ 1 – 3 ].WNShascausedunprecedentedmortalitiesinNorthAmericancavedwellingbatstothepointofpossibleregionalextinctions[ 4 5 ].Aclinicalsignin WNS-infectedbatsisnecrosisofthewingmembrane,whichmayleadtoinfarctionandelectrolyteimbalances[ 6 – 12 ].Inadditiontolocomotionfunction,batwingsplayimportantroles intheirecologyrangingfrommicrobialprotectiontofecundity[ 13 14 ].Todate,therehas beennocausalevidenceprovidedforbatwinglesions.Onehypothesisisthatasbatsexhibit frequentarousalfromtorpor,they “ scratch ” theirwingstoremoveirritatingfungalhyphae andphysicallydamagetissues.Anotherhypothesisis P destructans secretesproteasesduring infection,allowingmycelialpenetrationintounderlyingtissues.Inadditiontothesepossible rolesinwingnecrosis,immunereconstitutioninflammatorysyndrome(IRIS)inpost-hibernatingbatsmayexacerbatedamageintensity[ 15 ]. Wingmembranesconsistofathinloadbearingportionandathicker “ scaffold ” connectivetissuetrabeculaeportionthatservestoreinforcethewingstructure[ 16 ].Thethinportion(epidermis)consistsofthinepithelialcellsandthekeratin-richstratumcorneum.Thedermisisthinand indistinguishablefromthehypodermis.Elastin/collagenfibers,nerves,bloodvessels,andmuscle fibersarefoundthroughout.Thethicker “ scaffold ” regioncontainshigherconcentrationsofelastin/collagenfibersandsebaceousglands.Elastin ’ scompositionisrichinhydrophobicamino acidsincludingglycine,valine,alanine,andproline.Structurally,collagenisatriplehelixcomposedofaminoacidtripletmotifsGly-Pro-XorGly-X-Hyp[ 17 ].Thecombinationofelastin/ collagenfibernetworkistheprimarycontributortotissueelasticity;however,thecontributionof eachfibertypetooverallelasticityisdebatable[ 18 ].Thecornifiedcellsofthestratumcorneum arehighlyenrichedinkeratins,whicharehydrophobicproteinswithhighamountsofdisulfide cross-links[ 19 ].Thecombinationofthesefibrousstructuralproteinscreatesanintegumentary protectivematrixaidingininnateimmunityasaphysicalbarriertopathogenicmicrobes. Fungisecretedepolymerizingenzymestodigestcomplexsubstratesintheirenvironment fornutritionalrequirements.Extracellularproteaseshydrolyzepeptidebondsinproteincatabolismtoyieldaminoacidsforassimilation[ 20 ].Classicalproteasenomenclaturegroupedthese enzymesbasedsolelyoncatalyticmechanism,producingfourgroups:serine,metal,thiol,and acidproteases[ 21 ].Currently,sevenclassesarerecognized:serine,metallo-,cysteine,aspartic, threonine,glutamic,andasparagineproteases,withotherproteaseswithunknownormixed functions[ 20 ].Proteaseclassificationnowincludesnotonlycatalyticmechanism,butalsoaccordingtothepolypeptidepositioncleaved,primaryaminoacidsequencehomology,and structure.Proteasesaregroupedintofamiliesbyprimarysequencehomologiesandfurther clusteredinclansbasedoncommontertiarystructures[ 22 ].Twoclassesfrequentlyimplicated infungalpathogenesisincludesecretedmetalloproteasesandserineproteases[ 23 ]. Becauseextracellularproteasessecretedbyfungimayfunctionasvirulencefactors,wehypothesizedthat P destructans producesextracellularproteasestoenablehyphalpenetration intochiropteranintegument.SuchsecretedenzymesmayplayacentralroleinpathogenestablishmentandbatwingnecrosisduringWNS.Ourobjectivewastoisolateandidentifyextracellularlysecretedproteaseactivitiesproducedin P destructans cultures.Weseparatedadominantproteinaccumulatedstablyinculturemediumandidentifiedittobeasubtilisin-like serineprotease.This P destructans serineprotease(PdSP1)produced invitro isacandidatefor furtherfunctionalstudiesinhost/pathogeninteractionstoestablishitsputativeroleinWNS. Whileotherstudieshavereportedgeneralscreeningforsecretedenzymeactivitiesin P destructans cultures[ 24 – 26 ],thisisthefirstisolationanddescriptionofa P destructans serineproteaseanditsassociationtoaspecificgenesequence. PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 2/18 studydesign,datacollectionandanalysis,decisionto publish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclared thatnocompetinginterestsexist.

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MaterialsandMethods FungalCultivationandSecretedProteinProductionP destructans wasobtainedfromDr.KevinKeel(SoutheastCooperativeWildlifeDisease Study,CollegeofVeterinaryMedicine,TheUniversityofGA,USA)andculturedintheEnvironmentalPathogenLabatArkansasStateUniversity[permits:CDC(#2009 – 09 – 136), USFWS(#LE227131 – 0),IBC(SOP#AMP-001 – 111009andSOP#AMP-002 – 111009)].Stock culturesweremaintainedonmaltagarat8C(Difco,BectonDickinson,Sparks,MD,USA).To prepareinoculaforprotein-productioncultures, P destructans wasgrownonmaltbrothfor twomonths.Mycelialplugs(4ofca.2cmdia.)wereinoculatedinliquidculturemedia(100ml in2LFernbachflaskforhighsurfaceareatovolumeratio)andgrownstaticallyinthedarkat 8C.Totalproteaseproductionwasdeterminedincomplexculturemedia(Difco):tryptone peptone,brain/heartinfusion,proteosepeptone,trypticsoybroth(TSB),andcomparedwith minimalnutrientbroth(MNB)(NH4Cl9.34mM,NaCl8.55mM,K2HPO41.72mM,KH2PO42.92mM,MgCl2 .6H2O0.49mM,FeSO4 .7H2O0.49mM,0.01%SDS,and0.01%yeastextract) supplementedwith0.5%protein:gelatin(G),keratin(K),casein(C),orelastin(E). Culturesweregrownfor6 – 8weeksthenharvestedwhenmyceliacoveredtheentireliquid surface.Culturesupernatantswererecoveredbycentrifugationat10,000xgfor30min,filtered througha0.45 mmembrane,andthenconcentratedbylyophilizationorultrafiltration.Concentratedmediawereexchangedintodeionizedwater(containing0.02%w/vNaN3)with Econo-Pac10DGdesaltingcolumns(Biorad#732 – 2010).PreparativeIsoelectricFocusingPreparativeisoelectricfocusing(IEF)wasperformedforsixhoursat12WwithaBioRadRotoforcellusing1%BioRadpH3 – 10ampholytesaddedtosalt-freeproteinsamplesfollowing manufacturer ’ sinstructions.ThepHofeachfractionwasrecordedandadjustedtopH7.0with HCl/NaOH.FractionswerebroughttoanequalvolumewithultrapureH2O(18m O ),andproteinandproteaseactivity(measuredatpH7.0)wasdeterminedforeach.LectinAffinityChromatographyGlycosylatedproteinswereseparatedbylectinaffinitychromatographywithconcanavalinA Sepharose(ConA;Sigma#C7911)followingmanufacturer ’ srecommendation.Briefly,ConA (ca.10ml)packedinsmallcolumns(1.5cmIDx12cm)waswashedwithfivebedvolumesof bindingbuffer(20mMTris,0.5MNaCl,pH8),andthensamplewasappliedtwice.Following washingtheConAcolumnwithfivebedvolumesofbindingbuffer,theglycoproteinswere elutedwithfivebedvolumesofelutionbuffer(20mM -D-methylglucoside,0.5MNaCl, 20mMTris,pH4).TheelutedfractionwasneutralizedwithTrisbuffer(pH9).ConAsamples wereexchangedintoultrapureH2OwithBio-RadEconopak10DGcolumnpriortofurther analysisbyproteinandenzymeassayorgelelectrophoresis.Allsampleswerestoredwith 0.02%(w/v)NaN3at4C.ProteinDeterminationandElectrophoreticAnalysesThetotalproteinconcentrationwasdeterminedusingBradford ’ sCoomassieBrilliantBluereagentandBSAasastandard[ 27 ].Sampleswerepreparedforgelelectrophoresisusingtrichloroaceticacid(TCA)precipitation.TCApelletswerewashedwithice-coldacetone,dried in vacuo andreconstitutedin50 lSDSLaemmlisamplebuffer.ProteinsinculturemediumsampleswereseparatedusingtheBioRadMini-PROTEANTCX12%pre-castgelswiththe LaemmliTris/glycinebuffersystemandstainingwithCoomassieBrilliantBluereagent PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 3/18

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accordingtomanufacturer ’ sinstructions[ 28 ].ProteinsizewasestimatedwithPrecisionPlus ProteinAllBluestandards(BioRad#161 – 0373)usingU-SCAN-ITGraphDigitizingSoftware (v5.1,SilkScientificCorp,USA).PercentageproteincompositionstainedbandswerealsoestimatedwithU-SCAN-ITsoftware. SamplesweredesaltedwithEcono-Pac10DGdesaltingcolumns.Extracellularproteins wereseparatedby10%ReadyGelZymogramGel(gelatin;BioRad#161 – 1113)following themanufacturer ’ sinstructions.Sampleswereconcentratedforelectrophoresiswithasingle acetoneprecipitation,runinnon-reducingcon ditions,incubatedfor30mininrenaturation buffer(BioRad#161 – 0765),thenincubatedfor16hatroomtemperatureindevelopment buffer(BioRad#161 – 0766).Gelswerestainedwith30%methanol,10%aceticacid, 0.5%CoomassieBrilliantBlueR-250(30min)anddestainedinthemethanol/acetic acidsolution.EnzymeActivityAssaysProteaseactivityinculturemediawasinitiallydeterminedwithfluoresceinisothiocyanate (FITC)caseinaccordingtothemanufacturer ’ sinstructionsandcomparedtoatrypsinstandard (ThermoFisherScientific,kit#23266).Valuesareexpressedasunits/ml,whereoneunitisdefinedas1.0mg/1.0mltrypsinstandard.FordeterminingoptimalpH,proteaseactivitieswere determinedwithazocoll(Sigma#A4341)addedin5.0mgportionsto1.0ml100mMpotassiumphosphatebuffer,pH7.0at37C.Affinity-purifiedproteinsampleswereaddedin200 l aliquots,thereactionwascontinuedat37Cfor30minwithgentleshaking,andabsorbance wasmeasuredat520nm[ 29 ]. Serineproteaseactivityofaffinity-purifiedproteinswasquantifiedusingapeptidep NA (Suc-Ala-Ala-Pro-Phe-NHPhNO2)colorimetricsubstrate(Sigma#S7388;0.1mM).ProteinaseK(E.C.3.4.21.64;Sigma#P6556)wasusedasapositivecontroltodeterminerelativeserine proteaseactivity.Substratewaspreparedin0.1MTris-HCl,0.01MCaCl2bufferatpH7.5. Theproteinsamples(100 l)weremixedwithpeptide-pNAsubstrate(600 l)andincubated for10minatroomtemperature,thenproductabsorbancewasdeterminedat410nmandcomparedtoa4-nitroanilinestandardcurve.Oneunitserineproteaseactivitywasdefinedas 1 mol4-nitroanilinereleasedperminuteat25C.BiochemicalCharacterizationThecontributionsofproteaseclassesinmixedproteinsampleswasassessedbyaddingvarious reagentsandsaltsfollowedwithanactivityassaywithFITCcaseinsubstrate:[phenylmethylsulfonylfluoride(PMSF,1mM,MeOH),iodoacetamide(IAA,10mM,H2O),sodiumdodecyl sulfate(SDS,0.5%),dithiothreitol(DTT,0.1%),andCa2+,Mg2+,andZn2+,(allmetalions 5mM)].ThepHoptimaofcrudemediumandaffinity-purifiedproteinweredeterminedwith azocollasasubstrate.Amino -TerminalSequenceDeterminationAmino-terminalproteinsequencesforindividualproteinsblottedtoPVDFmembranewere determinedbyEdmansequencingchemistrywithanABProcise494instrumentattheProtein CoreFacilityoftheIowaStateUniversity( www.protein.iastate.edu/nsequence494 ).Individual proteinsresolvedbySDS-PAGEwereelectroblottedto0.45 mImmobilon-PPVDFmembranewith10mMCAPS,pH11,with10%methanol(v/v)transferbufferfor1hrat150V constantaspreviouslydescribed[ 30 ]. PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 4/18

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PeptideMassFingerprintbyMALDI-TOFMSStructure-basedproteinidentificationwasperformedbypeptidemassfingerprintingusing trypsindigestionsandseparationbyMALDI-TOFMS[ 31 ].Briefly,SDS-PAGE-resolvedproteinbandswereexcisedfromgels,processedwith0.1%w/vRapigest(Waters,Milford,MA, USA),anddigestedovernightwithTrypsin-Gold(Promega,Madison,WI,USA)accordingto manufacturer Â’ sinstructions.TrypticpeptideswererecoveredanddesaltedwithC18ZipTips. Peptidesweremixedwith -cyano-4-hydroxy-cinnamicacidbeforebeingspottedontoa MALDIplate.SpectrawereobtainedwithaWatersMALDIMicroMXMSinpositivereflectronmode(pulsevoltage,2,000V;reflectron,5,200V;source,15,000V)witha20HzN2laser at337nm.SpectraldatawereprocessedwithMassLynxv4.1andProteinLynxGlobalServer 2.5(Waters).Denovo PeptideSequencingbyOrbitrapMS/MSSequencedatafromtrypsindigestsfromaffinity-purifiedproteinswasobtainedfromtheUniversityofArkansasforMedicalSciencesProteomicsCoreFacility(tri.uams.edu/researchresources-services-directory/core-facilities-technical-services/proteomics-core/)usinga ThermoScientificLTQ-OrbitrapVelosmassspectrometer[ 32 ].Thefivemostabundantpeptideswereselectedforhigh-resolutiontandemMS,witha99.0%confidenceproteinthreshold and95.0%confidencepeptidethreshold.BioinformaticsThepeptideionlistgeneratedbyMALDI-TOFMSwasusedforputativefungalenzymeidentificationbytheMascotsearchengine( www.matrixscience.com ).ProcessedspectraweresubmittedtoMASCOT(FixedModification:Carbamidomethyl,VariableModification:Oxidation, Peptidetol.200ppm)forcomparisonto insilica digestedproteins.TheBLASTpprogramat NCBI( www.ncbi.nlm.nih.gov/blast/ )wasusedtodeterminegenesequenceidentityandcorrelateidentitytoknownfungalproteases[ 33 ].Putativeenzymefunctionwasdeterminedwiththe UniversalProteinResource(UniProt; www.uniprot.org )[ 34 ].Thepresenceofasignalpeptide wasdeterminedwiththeSignalP4.1server( www.cbs.dtu.dk/services/SignalP/ ).Phylogenetic analysisof P destructans enzymetodermatophyticandplantpathogenicfungiwasperformed by www.phylogeny.fr/ [ 35 36 ].A3-DstructureoftheproteinwasgeneratedbyFirstGlancein Jmol(bioinformatics.org/firstglance/fgij/).MultiplesequencealignmentsforthethreehomologousPdSPproteinswereperformedwithClustalOmegav1.2.0( www.ebi.ac.uk/Tools/msa/ clustalo/ ).PdSP1antiserumandWesternBlottingRabbitpolyclonalantibodiesrecognizingPdSP1-specificpeptideGSVDSTDTRASSSNwere generatedandaffinitypurifiedbytheGenScriptCorporation(Piscataway,NJ,USA)[ 37 ].Peptideswereselectedbysequencehydrophilicity,orientation,sequencelength,andhomologyto closelyrelatedproteinsidentifiedbyaBLASTpsearch.ProteinswereelectroblottedtoPVDF membraneinthemannerdescribedtopreparesamplesfor N -terminalsequencing.Proteinblottedmembraneswereblockedwith20%nonfatdrymilkandincubatedinPdSP1primary antibody(1:10,000)inTrisbufferedsaline(TBS)tweensolution(1.5MNaCl,0.5MTris-base, 1ml/LTween20,pH7.5).Proteinswereincubatedinsecondarygoatanti-rabbitalkalinephosphataseconjugateantibody(1:3,000)inTBS-Tweenandvisualizedwithnitro-bluetetrazolium chlorideand5-bromo-4-chloro-3 Â’ -indolyphosphate p -toluidinesaltinalkalinephosphatase buffer(150mMNaCl,1mMMgCl2,100mMTris,pH9). PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 5/18

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Results FungalcultivationandenzymeproductionRepresentative P destructans culturescomparinggrowthonMNB(minimalnutrientbroth withsupplementedprotein, e g .,MNB+G)media,oncomplexfungalculturemedia( e g .,TSB), andextracellularproteinprofilesareshownin Fig.1 .Morediffuseandslowerhyphalgrowth wasobservedondefinedMNB,requiring~6weekstoformamycelialmatacrossthesurface. Hyphal-growthoccurredmorequicklyoncomplexmedia(~4weeksforconsiderablehyphal mass),butthemycelialmassformedthicklocalizedclumpsratherthanthethinmycelialmat observedonMNBsurfaces.GrowthonMNBresultedinconsistentsecretedproteinprofiles (observedbySDS-PAGE)regardlessofproteinsupplementused(G,E,C,orK),whilegrowth oncomplexmediaproducedvariableproteinprofiles.Proteasezymogramscreeningofculture mediashowedtwodifferentactivityprofilesrepresentedbyMNB+GandTSB,whichindicated differencesinthedominantproteaseactivitiespresent.Thelocationofproteaseactivityatthe topofthegelMNBmediaindicatedtheproteinmigratedintothegelpoorly,whereasthe Fig1. Pseudogymnoascusdestructans growthinstaticliquidcultures. Leftpaneldepictstypicalculture morphologyinminimalnutrientbrothwithgelatin(A)comparedtoanutritionallycomplextrypticsoybroth(B). TherightpaneldepictsaSDS-PAGE(Coomassie-BrilliantBlueG-250stain)ofextracellularproteins recoveredfromculturemediaandanativegelzymogram(casein)illustratesproteaseactivityfromtotal proteinextract. doi:10.1371/journal.pone.0120508.g001 PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 6/18

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dominantactivityintheTSBmigratedmidwaydownthegel,indicatingdistinctdifferencesin sizeandchargefortheassociatedprotein. Qualitativedifferenceswereobservedintotalproteaseactivity(determinedwithFITCcaseinsubstrateassay)secretedintoculturemedia( Fig.2 ). P destructans grownonMNBwith supplementedproteinsaccumulated~3Xmoretotalproteaseactivitycomparedtothecomplex mediatreatments.MNBsamplestreatedwithrepresentativeproteaseinhibitors(EDTA, PMSF,andE-64)showedadramaticreductioninactivityoccurredwithPMSF,indicating dominantserineproteaseactivity.E-64treatmentresultedinnoproteaseactivityinhibition,indicatingmeasurablecysteineproteaseswerenotsecretedinmediatested.EDTAtreatmentresultedinpronouncedinhibitioninMNBmediaonly.WhileEDTAgenerallyinhibitsmetalloproteases,itcanalsode-stabilizeotherbroadclassactivities,includingcertainserineproteases. Thoughmetallo-proteasesmaybesecretedinMNBmedia,theylikelyrepresentaminorclass comparedtoserineproteasesduetothesingularactivitybandobservedbyzymogramanalysis ( Fig.1A ).MNB+Gprovidedthehighesttotalproteaseactivityyield,withnearlycompleteinhibitionofserineproteaseactivitywithPMSF.Furtherexperimentationtoisolateserineproteasesfocusedonculturing P destructans withMNB+G.IsolationoftheserineproteaseactivityWhiletheextracellularproteinprofileinMNB+Gculturemediaissimple,theconsiderablesolutionviscosity(possiblyduetosecretedpolysaccharides)ofconcentratedsampleswasproblematicforisolatingtheserineproteasepresent.Toaddressthisissue,twoseparationmethods wereevaluated — preparativeIEFandaffinitychromatography.Forthefirst,theconcentrated Fig2.Totalproteaseactivity(unitsx103/ml)secretedby P destructans growninstaticliquid cultures. Variousprotein-supplementedminimalnutrientbrothmediaandcomplexculturemediawereused. Controlsaretotalactivityaccumulatedinculturemedium,andresidualactivitywasdeterminedfollowing treatmentwiththeproteaseinhibitorsEDTA,PMSF,andE-64.MinimalnutrientbrothwithK,keratin,G, gelatin,E,elastin,C,casein.Complexmedia:TSB,trypticsoybroth,TP,tryptonepeptone,BHI,brain-heart infusion,PP,proteosepeptone.(Meanandstandarderrorfortriplicatesamples.) doi:10.1371/journal.pone.0120508.g002 PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 7/18

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enzymesolutionwasmixedwithbroadrangeampholytes(pH3 – 10)andfractionatedina Rotoforcell.Proteaseactivitywasresolvedasapeakoverfractions10 – 13(pH7 – 9; Fig.3 ). PMSFcompletelyinactivatedproteaseactivityinthepooledfraction,confirmingpresenceof theserineprotease(datanotshown).TheSDS-PAGEprofileforproteinsinpooledpeakfractionsshowedenrichmentoftwomajorbandsat27.9kDaand55.0kDainthepooledfraction (theirco-isolationindicatesasimilarpIforbothproteins). Furthertreatmentofconcentratedculturemediumevaluatedaffinitybindingtobenzamidine-SepharoseandConAchromatographymedia.Noproteinoractivitywasadsorbedtobenzamidine-Sepharose,suggestingtheserineproteaseisnotafamilyS1-typeserinepeptidase [ 38 ].Incontrast,serineproteaseactivityinextracellularpreparationsboundtotheConAlectin affinitycolumn,indicatingitcontainsan N -linkedglycan.SDS-PAGEofproteinselutedfrom ConA( Fig.4 )showedthe27.9kDabandwasgreatlyenrichedbythisseparation,andthe55.0 kDabandbeingeliminated.ThespecificactivityoftheConA-boundfractionwasincreased33fold,implicatingthe27.9kDaproteinasthe P destructans serineprotease(PdSP). ElectrophoreticandbiochemicalpropertiesdeterminedfortheConA-treatedPdSPare summarizedin Table1 .TheenzymeshowedabroadpHoptimumcoveringpH6to8.Itstemperatureoptimumwas~60C,suggestingmodestthermalstability.AdditionorDTTorSDSto substrate(casein)solutionshowedincreasedenzymeactivity,whiledivalentcationaddition didnotfurtherincreaseactivity.MALDI-TOFMSscreeningtoidentify P destructans proteinsTherewere7majorbandsobservedconsistentlybySDS-PAGEforproteinsisolatedfrom P destructans MNB+Gculturemedium( Fig.1 ).All7proteinswereexcisedfromSDS-PAGE gels,trypsin-digested,andthenanalyzedbyMALDI-TOFMStoestablishidentitybasedonsequencebymatchingtrypticpeptidesetsusingtheMASCOTsearchenginewiththeBroadInstitute ’ s P destructans genomicsequencedatabase[ Geomycesdestructans SequencingProject, Fig3.PreparativeIEFseparationandanalysisof P destructans extracellularproteins. SecretedproteinswererecoveredfromMBN-Gculturemedium withbroad-rangeampholytes.Activityprofileseparatedwithbroad-rangeampholytesintheRotoforcell(A).pHgradientindicatedbydashedline. Protease activityassayedwithFITC-casein:activityindicatesfluorescenceunitsperml.Pooledactivitypeakfractions(10 – 13)resolvedbySDS-PAGEandstained withCoomassie-brilliantblue. doi:10.1371/journal.pone.0120508.g003 PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 8/18

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BroadInstituteofHarvardandMIT( www.broadinstitute.org )]andNCBI Â’ sReferenceSequencedatabase[ 39 ].Tentativeidentificationswereinitiallymadefor5proteins( Table2 ).The largestband,115kD,matchedtoaputativecatalase/peroxidaseHPI.Thedominant106kD protein,whichco-separatedbyConAbinding,iswithoutannotatedfunction,butstructuralelementsandsequencehomologyimplicateittobeaFAD-bindingoxidoreductase.Aminor 84kDproteinisalsoputativelyidentifiedasaC-Nligase.The55kDaprotein,enrichedinthe IEFpeakfractionsbutnotConA,isputativelyidentifiedasaglycosidefamily18chitinase. Thesmallestproteinidentified,12.5kDa,isputativelyidentifiedasaphosphatidylglycerol/ phosphatidylinositoltransferprotein.The70kDaproteinremainsunidentified,butis Fig4.SDS-PAGEof P destructans extracellularproteinsseparatedbyConAlectinaffinity chromatography. Lane1,BioradPrecisionPlusmassstandards;lane2,Crudeenzymesubstrate;lane3, ConAelution(fromnon-adjacentgellane);lane4,westernblotanalysisdetectioninenrichedprotein concentrateusingaPdSP1antiserum. doi:10.1371/journal.pone.0120508.g004 Table1.Propertiesobservedforthe Pseudogymnoascusdestructans serineproteaseisolatedby ConAlectinaf nitychromatography. Molecularmass(SDS-PAGE)27,900Da EstimatedpIpH7to8 Concanavalin-AbindingYes( N -linkedglycoprotein) N-terminalpeptideALETRSGAT pHoptimumpH6to8 Temperatureoptimum60C PMSF(1mM) > 90%inhibition 0.1%DTT155%activityincrease 0.5%SDS178%activityincrease CaCl2(5mM)Nochange MgCl2(5mM)Nochange ZnCl2(5mM)Nochange doi:10.1371/journal.pone.0120508.t001 PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 9/18

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eliminatedastheserineproteasesinceitwasseparatedfromtheisolatedenzymeactivityby bothIEFandaffinitychromatographytreatments. Thetrypticpeptidemassspectrum(byMALDI-TOFMS)forthe P destructans 27.9kDa proteinisshownin Fig.5A .Whiletheoverallsamplequalityappearedgood,thetrypticpeptide iondatawasnotabletoprovidestatisticallysignificantmatchingtoanyexistingprotein.Severalexogenouspeptidesareidentifiedinthemassspectrum — theautolytictrypsinpeptide, m/z 842.50andthreepeptidesderivedfromConA, m/z 1318.63, m/z 2104.06,and m/z 2832.34. Furtheranalysisoftrypticpeptidesfromthe27.9kDaproteinwasperformedwithanOrbitrap (ThermoScientificLTQVelos)massspectrometertoobtainsequencedatafrompeptideionsto establishidentityasthePdSP. Denovo sequencingwasobtainedforfivepeptideions: m/z 904.46, m/z 892.49(observed m/z 908.49forMOx+16), m/z 1435.70, m/z 2011.01,and m/z 3802.74,andthedataareincludedin Table3 .ArepresentativeMS/MSspectrumisshownfor peptideion m/z 908.49(SVISMOxSLR)( Fig.5B ).ThecombinedMSandMS/MSdatamatched unequivocallytoaspecificopenreadingframe(UniProtGMDG_08491)fromthe P destructans 20631 – 21genome,confirmingstructurewithfunctionalserineproteaseactivityfor PdSP1.Thecalculatedtrypticpeptideionsfromthetranslatedproteinsequence(GenBank ELR07576.1)aresummarizedin Table3 .SequenceanalysisforthetranslatedPdSP1geneThecompletetranslatedaminoacidsequenceforthe P destructans geneproductELR07576.1, representedbyPdSP1,isshownin Fig.6 .BLASTpdatabasesearching(NCBInrDB)indicated twoconservedstructuraldomainsforPdSP1.Thefirstincludesa99aminoacidpropeptidesequencerepresentingaMEROPSfamilyI9inhibitordomain,followedbythe256aminoacidsequencerepresentingaMEROPSproteinaseK-likepeptidaseS8family(SBclan)domain[ 20 ]. Twoserineproteasehomologuesarepresentinthe P destructans genome.Thefirst,PdSP2, shows90%sequenceidentitywithPdSP1,andasecondhomologue,PdSP3,showedamuch lower53%sequenceidentity.TherewereBLASTphitsfor43putativeS8serineproteasesin the P pannorum genome,withsequencesimilarityfallingbetweenthetwo P destructans homologues.TheprimarysequencesforthethreePdSPhomologuesfrom P destructans andthe highestmatchinghomologuefrom P pannorum areincludedin Fig.6 FeaturesofPdSPprimarysequencesin Fig.6 includea20aminoacidsecretorysignalpeptideandthelocationforthe N -terminalaminoacidinthematureprotein(determineddirectly Table2.Trypticpeptide-mass ngerprintanalysisbyMALDI-TOFMStoidentifymajor P.destructans1extracellularproteinsresolvedbySDSPAGE. ProteinMass (kDa) GeneIdenti cation(andProteinAccesson) Mascot Score 115UniProtKATG(L8FYA6_PSED2;GenBankELR04656.1);Catalase/peroxidaseHPI87 106UniProtGMDG_07140(L8FX91_PSED2;GenBankELR05098.1);SimilartofungalFAD-bindingoxidoreductases162 84UniProtGMDG_08145(L8G218_PSED2;GeneBankELR06854.1);Similartofungalglutamyl-tRNA(Gln) amidotransferasesubunitA 96 70Notidenti ed — 55UniProtGMDG_06569(L8FSW0_PSED2;GenBankELR04060);SimilartofungalGHfamily18chitinase85 27.9UniProtGMDG_08491(L8G6I7_PSED2;GenBankELR07576);S8serinepeptidase — 12.5UniProtGMDG_02579(L8G2X2;GenBankELR07487.1);Similartofungalphospholipidtransferprotein1371Geneidenti cationsindicateORFnamesfrom Pseudogymnoascusdestructans ATCCMYA-4855(strain20631 – 21)genomicsequence.Identi cationof the27.9kDaproteinrequiredbothMSandMS/MSdatatoestablishunequivocalidentity. doi:10.1371/journal.pone.0120508.t002 PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 10/18

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fromPdSP1byEdmansequencingchemistry; Table1 ).ThespecificorderofAsp160-His192Ser345catalytictriad(forfamilyS8serineendopeptidases),andtheconservedmotifsaround them,isindicatedbyshading.Therearetwopredictedcalcium-bindingsitesinPdSP1(C1: K294,A296,D319;andC2:G132,S135,H136).ThematurePdSP1iscalculatedtohaveapI7.21 andmolecularweight(averagemass)27,882.70.Theseareconsistentwiththeexperimentalresults( Table1 ).Thereisasingle N -glycosylationsequon(N187-Y188-S189),whichisoccupied basedonConAbinding,buttheglycanappearstocontributelittletotheapparentmassby Fig5.PeptidemassfingerprintandMS/MSspectraofPdSP1. MALDI-TOFMSspectrum( m/z 700 – 3000)fromPdSP1trypticpeptides(A).MS/MS spectrumwithb/y-seriesionsfrompeptideion m/z 908.49(Ox+16)(SVISMSLR)(B). doi:10.1371/journal.pone.0120508.g005 PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 11/18

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SDS-PAGE( Fig.4 ).Asinglecysteine(C196)presentinthematureproteinsequenceindicates nointernaldisulfidebondsinPdSP1structure. Aphylogenetictreewasconstructedwiththethree P destructans PdSPsequencesandselecteddermatophyticandplantpathogenicsubtilisin-likeserineproteases(S8A)withhighsequence relationship(identifiedfromBLASTpsearch), Endgyodontiumalbum proteinaseK, Bacillus licheniformis subtilisin(Carlsberg),andwith Aspergillusclavatus serineproteasechosenarbitrarilyasanoutgroup( Fig.7 ). P pannorum S8Aserineproteasesequenceswerenotincluded duetotheverylargenumberofgenes(43)andtheirclosesequenceconservation(asindicated byBLASTp)comparedtothe P destructans PdSPs.PdSP2andPdSP3were90%and53%homologous(bysequenceidentity)toPdSP1,respectively. P destructans S8serineproteases groupedclosertoplantpathogenicfungalproteases[ e g ., Botryotiniafuckeliana (graymolddisease), Sclerotiniasclerotiorum (whitemold), Marssoninabrunnea (Marssoninaleafspot)]than tohumanpathogenicdermatophytes( i e ., Arthroderma Trichophyton ,and Microsporum ).WesternBlottingThepeptideGSVDSTDTRASSSN(correspondingtoaminoacidpositions400 – 413in Fig.6 ) wassynthesizedandusedtogenerateaPdSP1-specificantiserumforsubsequentuseinwestern blottingdetectionoftheprotein.TheresultingPdSP1antiserashowedsuitabletiterandsensitivityfordetectingPdSP1(27.9kDaband)inblotsfromtotalproteinisolatedfrom P destructans culturemedium.Bovinetrypsin,proteinaseK,orother P destructans proteinsdidnot cross-react(datanotshown).DiscussionWNSisanemergentfungaldiseasewithdevastatingconsequencesforNorthAmericancavedwellingbatpopulations,andlittleisknownabout P destructans ’ ecologyorrelativepathogenicity[ 1 2 26 ].Batsplayintegralrolesinourecosystemaskeystonespecies,pollinators,and pestcontrol[ 40 41 ].Theunprecedentedmassmortalityofcavebatsmayleadtoincreasesin insectpests,pesticideusage,andinsectvectordiseases[ 42 ].Thelossofbatsandtheirfunction Table3.Trypticpeptideionmasses,positioncorrespondingtothematureprotein,andthecorrespondingaminoacidsequencestranslated (GenBankELR07576.1)fromthe P destructans genematchedtoPdSP1(27.9kDaprotein). Mass(M+1)SequencePositionPdSP1PeptideSequence 904.461,26 – 14SGATWGLGR 3802.74219 – 54ATGSNSYIYDGSAGSGSTVYVLDTGIYIEHSEFEGR 3465.54157 – 91WGANYISGSPDTDENGHGTHCAGTIAGATYGVASK 2025.00103 – 123DGFGATSATIAGINFVGQNGK 892.492127 – 134SVIS M SLR 3952.84135 – 175GHYSAAVNSAVESTVSNGVTIVVAAGNDGDDASNYSPASAK 1435.702176 – 189NAITVGSVDSTDTR 2011.011,2190 – 209ASSSNYGSVVDIFAPGVNVK 2969.582223 – 254SGTS M ATPHVAGLAAYLIGLGGLSSPAAIASK 1249.621269 – 281GSVNLIAYNGNGA1FourpeptidesarecommonwithahomologuePdSP2(GenBankELR03877.1).2Peptideionsobservedexperimentallyonly. Onlypeptideionswithamassgreaterthan m/z 800areincluded.(Theoreticaltrypticpeptideionsfromthepropeptideandtheirsequencesarenot included.) doi:10.1371/journal.pone.0120508.t003 PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 12/18

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innaturalpestcontrolisestimatedtocostatleast$3.7billion/yearindamagestoagriculture [ 43 ].Aclearunderstandingofthebiochemicalecologybetween P destructans andbatsisparamountindeterminingpropercontrolstrategiestoalleviatediseaseeffects. Extracellularproteasesplayrolesinfungalpathogenesisandarehypothesizedtofunctionin WNS[ 23 ].EpidermalwingnecrosisobservedinWNSmaybepartiallyattributedtoprotease activitiessecretedby P destructans .Secretedserineproteasesarecommoninsaprophyticfungi andaredocumentedinspeciespathogenictoplants,insects,mammals,andotherfungi [ 44 – 46 ].Inthisstudyweisolateda27.9kDaproteinconsistentlysecretedby P destructans intoliquidculturemediaandidentifieditasafamilyS8Asubtilisin-likeserineproteinase (PdSP1).Weconfirmedstructuralidentificationbymatchingpeptideionmassesand denovo peptidesequencesobtainedbyMS( Table3 and Fig.5 )tothehypotheticalprotein(GenBank ELR07576.1),translatedfromthegeneGMDG_08491presentinthedraftgenomesequenceof P destructans isolate20631 – 21( www.broadinstitute.org ).ThepropertiesdeterminedexperimentallyforPdSP1( Table1 and Fig.5 )matchedvaluespredictedforthe281AAmature Fig6.Sequencealignmentof Pseudogymnoascusspp .FamilyS8serineproteases. SequencesidentifiedfromGenBankaccessions: P destructans PdSP3,ELR10046.1; P destructans ,PdSP1,ELR07576.1; P destructans ,PdSP2,ELR03877.1;and P pannorum KFZ06449.1.Locationofcatalytictriad, D160,H192,andS345(indicatedby belowsequence)withconservedmotifsforS8Asubfamilyindicatedingrayboxes.Residuespredictedtoparticipatein calcium-bindingarealsoindicatedbelowsequencesforsitesC1(+)andC2(#).AminoacidssequencesdeterminedexperimentallyfromPdSP1are underlined. N -glycosylationsequons(N-X-S/T)areindicatedinbolditalics.AlignmentpreparedwithClustalWOmega. doi:10.1371/journal.pone.0120508.g006 PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 13/18

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proteasehavingatheoreticalmolecularweightof27,882.7andpIof7.20.Thenearlycomplete inhibitionoftotalproteaseactivitybyPMSFindicatesserineproteaseisthepredominantproteasetypeproducedby P destructans culturedwithproteinassolenitrogensource.Subtilisinlikeserineproteasesaredominantsecretedenzymesinfungiassociatedwithsymbioticinteractions,particularlywithanimals[ 46 ].WhilePdSP1isisolatedasanextracellularproteinproduced invitro ,itmayfunctionasakeyproteasein P destructions Â’ virulence. P destructans hasthreehomologousgenesforS8subtilisin-likeserineproteases.ThetranslatedsequenceforPdSP2(NCBIaccession#ELR03877.1)exhibitshighsequenceidentity(90%) withPdSP1,whilePdSP3(NCBIaccession#ELR10046.1)retains53%sequenceidentity.ConsideringthehighsequenceconservationbetweenPdSP1andPdSP2,thereareonly4trypticpeptides( > m/z 750)incommon,withtwooftheseobservedexperimentally( m/z 892.49and 1435.70; Table3 ).FourtrypticpeptideionsuniquetoPdSP1areobservedinsamplespectra, readilyconfirmingidentitymatching.ArecentanalysisbyMuszewskaetal.(2011)suggestedindependentexpansionsofsubtilisin-likeserineproteasesinfungimayberelatedtouseofmammaliansubstrates,ratherthanpathogenicity[ 46 ].Threeisoformsin P destructans mayreflecta moreobligaterelationshipwithitshost(s),whereastheentomopathogen M anisopliae canhave asmanyas25isoformsand P pannorum has43S8Aserineproteasegenespresent[ 47 ],withthe latterreflectingageneralsaprophyticlifestyle[ 26 44 ].Thelownumberofadditionalisoforms maysupportpreviousresultsthat P destructans exhibitsreducedsaprophyticgrowth[ 26 ]. Fig7.PhylogenetictreegeneratedfromS8serineproteasesprimarysequencefrom P destructans andotherselectmicrobialorganisms. Includedarethreeserineproteasesfrom P destructans ,selectedserineproteasesequences(S8Aproteinase-Ksubfamily)fromplantpathogenicand humandermatophyticfungi,proteinaseK,Carlsbergsubtilisin,and Aspergilluscavatus serineprotease.Phylogenetictreegeneratedwithprogram Phylogeny.fr(S8Aaccessionsarelistedin S1Fig .) doi:10.1371/journal.pone.0120508.g007 PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 14/18

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TofacilitatepreparationofPdSP1-specificantiseraforfuture invivo studies,weselectedthe peptidesequenceGSVDSTDTRASSSNforsynthesizingapeptideantigen.Thispeptideshowsa suitablehydrophilicityprofile,avoidsthesingle N -glycosylationsite,andappearstorepresentan exteriorloop(determinedfroma3-DmodelgeneratedbyFirstGlanceinJmol).Weobtaineda monospecificpolycolonalantiserum(rabbits)fromthesyntheticpeptidethatdetectedthe27.9 kDaPdSP1inwesternblotswithgoodsensitivity( Fig.4 ).WhilePdSP2maycross-reactwiththis antiserum,themoredivergentsequenceforthecorrespondingpeptideinPdSP3suggestsitwill notcross-react.Pendingfurthercharacterizationoftheantiserum,itwillproveusefulserviceto detectthepresenceofPdSP1intissuewashingsorinhistologicalsectionsfrombatsdisplaying WNS.AntiseramayalsobegeneratedfortheothertwoPdSPisoformsinasimilarapproach. Proteasesfunctioningasvirulencefactorsinfungalepithelialinfectionsmayprovidetargets forvaccinesanddiseaseprophylactics[ 23 ].Whileextracellularly-secretedproteasescanbemolecularbiomarkersindiseaseprocesses,non-pathogenicfungisecretemanysimilarenzymes. Currently,thedifferentialexpressionofPdSPsin P destructans uponhosttissueinfectionand establishmentisunknown.Here,weprovidetwonewtoolsforPdSP1detection,PdSP-specific genesequencesthatcanbeusedforselectingPCRprimerprobesandanantiserumtodirectly detecttheexpressedproductfrom P destructans genes.Currentevidencesuggestspathogenesis isacomplexprocessinvolvinghost,pathogen,andenvironmentalinteractions[ 48 ],hence manyfactorsotherthansimplyenzymesecretioncontributetodiseasemanifestation.Furthermore,ifcertainenzymesinvolvedindiseaseprocessesareinhibited,fungimaycompensateby expressingalternativeenzymes. Invitro fermentationsystemsprovideastartingpointforidentifyingfungal-specificextracellularenzymes.Thetemporalpatternsofsuchenzymes,thepresenceoftheiractivevs.precursorformsatdifferenttimepoints,andtheirsecretion insitu within battissueremainsanareaavailableforfutureresearch.Theavailabilityofthe P destructans genomicsequenceandapplicationofhighlyaccuratemassspectrometrictechniqueswillfacilitate suchstudies.Inconclusion,weisolatedandidentifiedadominantsubtilisin-likeserineprotease produced invitro by P destructans ,providingnewtoolstodetermineitsputativefunctionin batwingnecrosis[ 6 ],aswellasforassessingexpressionofotherPdSPisoformsduringWNS.SupportingInformationS1Fig.AccessionnumbersforS8Asequencesusedwith P destructans proteasesforpreparingphylogenetictree( Fig.7 ). (DOCX) S1Table.Summarydatafortotalproteaseactivitymeasuredinculturemediaandresponse toproteaseinhibitortreatments( Fig.2 ). (XLSX)AcknowledgmentsTheauthorswouldliketothankJoseTovarandDavidGilmoreforusefuldiscussionsduring thecourseofthestudyandtheanonymousreviewersforcriticalinputusedforrevising themanuscript.AuthorContributionsConceivedanddesignedtheexperiments:ELPTSRBJS.Performedtheexperiments:ELPBJS. Analyzedthedata:ELPBJS.Contributedreagents/materials/analysistools:TSRBJS.Wrotethe paper:ELPTSRBJS. PseudogymnoascusDestructans SecretedSerineProtease PLOSONE|DOI:10.1371/journal.pone.0120508March18,2015 15/18

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