Comparison of the White-Nose Syndrome Agent Pseudogymnoascus destructans to Cave-Dwelling Relatives Suggests Reduced Saprotrophic Enzyme Activity


previous item | next item

Citation
Comparison of the White-Nose Syndrome Agent Pseudogymnoascus destructans to Cave-Dwelling Relatives Suggests Reduced Saprotrophic Enzyme Activity

Material Information

Title:
Comparison of the White-Nose Syndrome Agent Pseudogymnoascus destructans to Cave-Dwelling Relatives Suggests Reduced Saprotrophic Enzyme Activity
Series Title:
PLOS One
Creator:
Reynolds, Hannah T.
Barton, Hazel A.
Publication Date:
Language:
English

Subjects

Subjects / Keywords:
Wns ( local )
Pseudogymnoascus Destructans ( local )
Geomyces Destructans ( local )
Co-Evolution ( local )
Genre:
serial ( sobekcm )

Notes

Abstract:
White-nose Syndrome (WNS) is an emerging infectious mycosis that has impacted multiple species of North American bats since its initial discovery in 2006, yet the physiology of the causal agent, the psychrophilic fungus Pseudogymnoascus destructans ( = Geomyces destructans), is not well understood. We investigated the ability of P. destructans to secrete enzymes that could permit environmental growth or affect pathogenesis and compared enzyme activity across several Pseudogymnoascus species isolated from both hibernating bats and cave sediments. We found that P. destructans produced enzymes that could be beneficial in either a pathogenic or saprotrophic context, such as lipases, hemolysins, and urease, as well as chitinase and cellulases, which could aid in saprotrophic growth. The WNS pathogen showed significantly lower activity for urease and endoglucanase compared to con-generic species (Pseudogymnoascus), which may indicate a shift in selective pressure to the detriment of P. destructans’ saprotrophic ability. Based on the positive function of multiple saprotrophic enzymes, the causal agent of White-nose Syndrome shows potential for environmental growth on a variety of substrates found in caves, albeit at a reduced level compared to environmental strains. Our data suggest that if P. destructans emerged as an opportunistic infection from an environmental source, co-evolution with its host may have led to a reduced capacity for saprotrophic growth.

Record Information

Source Institution:
University of South Florida Library
Holding Location:
University of South Florida
Rights Management:
This item is licensed with the Creative Commons Attribution License. This license lets others distribute, remix, tweak, and build upon this work, even commercially, as long as they credit the author for the original creation.
Resource Identifier:
K26-00041 ( USFLDC: LOCAL DOI )
k26.41 ( USFLDC: LOCAL Handle )

USFLDC Membership

Aggregations:
University of South Florida
Karst Information Portal

Postcard Information

Format:
serial

Downloads

This item is only available as the following downloads:


Full Text

PAGE 1

ComparisonoftheWhite-NoseSyndromeAgent Pseudogymnoascus toCave-Dwelling RelativesSuggestsReducedSaprotrophicEnzyme Activity HannahT.Reynolds * ,HazelA.Barton DepartmentofBiology,UniversityofAkron,Akron,Ohio,UnitedStatesofAmerica Abstract White-noseSyndrome(WNS)isanemerginginfectiousmycosisthathasimpactedmultiplespeciesofNorthAmericanbats sinceitsinitialdiscoveryin2006,yetthephysiologyofthecausalagent,thepsychrophilicfungus Pseudogymnoascus destructans (= Geomycesdestructans ),isnotwellunderstood.Weinvestigatedtheabilityof P.destructans tosecrete enzymesthatcouldpermitenvironmentalgrowthoraffectpathogenesisandcomparedenzymeactivityacrossseveral Pseudogymnoascus speciesisolatedfrombothhibernatingbatsandcavesediments.Wefoundthat P.destructans produced enzymesthatcouldbebeneficialineitherapathogenicorsaprotrophiccontext,suchaslipases,hemolysins,andurease,as wellaschitinaseandcellulases,whichcouldaidinsaprotrophicgrowth.TheWNSpathogenshowedsignificantlylower activityforureaseandendoglucanasecomparedtocon-genericspecies( Pseudogymnoascus) ,whichmayindicateashiftin selectivepressuretothedetrimentof P.destructans’ saprotrophicability.Basedonthepositivefunctionofmultiple saprotrophicenzymes,thecausalagentofWhite-noseSyndromeshowspotentialforenvironmentalgrowthonavarietyof substratesfoundincaves,albeitatareducedlevelcomparedtoenvironmentalstrains.Ourdatasuggestthatif P. destructans emergedasanopportunisticinfectionfromanenvironmentalsource,co-evolutionwithitshostmayhaveledto areducedcapacityforsaprotrophicgrowth. Citation: ReynoldsHT,BartonHA(2014)ComparisonoftheWhite-NoseSyndromeAgent Pseudogymnoascus toCave-DwellingRelativesSuggests ReducedSaprotrophicEnzymeActivity.PLoSONE9(1):e86437.doi:10.1371/journal.pone.0086437 Editor: JasonE.Stajich,UniversityofCalifornia-Riverside,UnitedStatesofAmerica Received September24,2013; Accepted December10,2013; Published January22,2014 Copyright: 2014Reynolds,Barton.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: USFishandWildlifeServices(www.fws.gov).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparati onof themanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:hannah.t.reynolds@gmail.com Introduction White-noseSyndromeisafungaldiseasethathaskilledmillions ofhibernatingbatsinNorthAmericasinceitsdiscoveryin2006 [1,2].ThecausalagentofWNSwasidentifiedasanovelfungal pathogen, Geomycesdestructans [1,3,4],whichwasrecentlyreclassifiedintothegenus Pseudogymnoascus (Pseudeurotiaceae, incertaesedis , Leotiomycetes)[5].Whileimportantdetailsofphysiologyinthis organismhavebeenestablishedsuchasgrowthtemperature[6] anditsabilitytoinvadewingtissue[7,8],manyimportantaspects ofitsdiseaseecologyremainunknown. Invasivefungalpathogensaredevastatingplantandanimal populationsacrosstheglobe,andunderstandingtheirecologyis crucialinpredictingdiseaseoutcomes[9].Theabilitytocarryout saprotrophicgrowthoutsideahosthasimportantimplicationsfor diseasemanagementinemergentmycoses;modelsofhostpathogendynamicsindicatethathighlevelsofsaprotrophic growthcanleadtohostextinction[9].Forexample,theemergent amphibianmycosis Batrachochytryiumdendrobatidis ( Bd )maybe capableofgrowthoutsideitshost,asitcanbeculturedon multiplecarbonandnitrogensources[10],includingfeathers[11]. Althoughdefinitiveevidenceof Bd environmentalgrowthhasnot beenobtained,modelsofthe Bd infectioncycleindicatethatin additiontosporepersistence,environmentalgrowthcouldincrease theriskoflocalhostextinction[12].Thesaprotrophicgrowthof P. destructans couldpermityear-roundgrowthincaves,increasing boththechanceofinfectionofnaõ ¬ vebatsfromanenvironmental reservoir,forwhichanecdotalevidencealreadyexists(A.Hicks, pers.comm.2011),andtheriskofspreadofthispathogentonew cavesthroughcontaminatedsediment.Lorchetal.2013[13] found Pd DNAandviable Pd sporesincavesedimentsafterthe departureoftheWNS-infectedbathosts;however,itisunknown whetherthepresenceof Pd inthesesedimentsisdueto environmentalpropagationortothepersistenceofsporesshed frominfectedhosts.Ifthelatterwerethecase,anobligatebat pathogenwouldbelimitedtogrowthinthehibernationseason whenbatbodytemperaturesduringtorporpermitfungalgrowth, reducingthelikelihoodofenvironmentalspread. Currentevidencesuggeststhat P.destructans maybeafacultative pathogen;itcangrowonavarietyoflaboratorymedia,while obligatepathogensoftencannotbeculturedoutsideoftheirhosts onsuchmedia[4,14].Whilethecurrentevidenceisconsistent withanintroductionfromEurope,thecavesinwhichNorth Americanbatshibernatecontaindiverse,presumablynative Pseudogymnoascus and Geomycesspp. [5,16Ð19].However,theirrole inthecaveenvironmentremainsunknown[17,19].Thepartially PLOSONE|www.plosone.org1January2014|Volume9|Issue1|e86437 destructans destructans

PAGE 2

annotated P.destructans genomeindicatesthepresenceofnumerous genescodingforsaprotrophicenzymes,includingcellulasesand chitinases[15].Iftheseenzymesconferafunctionalphenotype, theycouldallow P.destructans togrowsaprotrophicallyincave microhabitatsandprovideanenvironmentalreservoirfor infectionofsusceptiblehosts.Questionsregardingsaprotrophic Pseudogymnoascus enzymeactivityandhowitcomparestothe activityof P.destructans maynotonlyindicatethepotentialofan environmentalreservoirforWhite-noseSyndrome,butmayhelp usunderstandthenaturalhistoryofthispathogen. Inthisresearch,weexaminedenzymesthatcouldplayarolein fungalgrowthonvariouscavemicrohabitats,someofwhichcould alsobeexploitedinpathogenesis.Wecomparedenzymeactivityof P.destructans withthatofasuiteof Pseudogymnoascusspp. isolated fromcaveenvironmentsandthecloserelative P.pannorum var. pannorum .Asadditionalcontrols,weincludedtheestablished saprotroph Penicilliumpinophilum (Trichocomaceae,Eurotiales, Eurotiomycetes),and Oidiodendronmaius (Myxotrichaceae, incertae sedis ,Leotiomycetes),whichcanformmutualisticassociationswith ericoidplantsandhasalsobeenfoundasasaprobeinmultiple locales[20],includingcavesediments[16].Ourresultsindicate that P.destructans producesseveralenzymesthatcouldpermit environmentalgrowthandsupportpathogenesis,butthat P. destructans showslowersaprotrophicpotentialthancloselyrelated environmentalisolates.MaterialsandMethods CulturesandGrowthConditionsUnlessotherwisenoted,allreagentswerepurchasedfromSigma Aldrich(St.Louis,MO). Pseudogymnoascus specieswereisolated fromCudjo'sCave,CumberlandGapNationalHistoricPark (NPScollectionpermit # CUGA-2009-SCI-0007)byswabbing hibernatinglittlebrownbats( Myotislucifugus )andcavesurfaces withpre-moistened(dH2O)sterilecottonswabs.Thiswasfollowed byswabbingonEmmon'smodificationofSabourauddextrose agar(SDA)augmentedwithcycloheximide(100mg/L)[21]. Cultureswereincubatedat5 u Ctoselectforpsycrophilicand psychrotolerant Pseudogymnoascus species,andalibraryof668 isolateswasgenerated.Preliminaryidentificationwasperformed usingcolonymorphologyandmicroscopy,andPCRamplification andsequencingoftheinternaltranscribedsequence( ITS ),whichis consideredthemostrobustgeneticsequenceforphylogenetic inferenceatthespecieslevelinthefungi[22],wasusedtoconfirm theidentityofputative Pseudogymnoascusspp. andtoassessthe phylogeneticdiversityofstrainsusedinphysiologicalassays.The ZRSoilMicrobeDNAMiniprepKit(ZymoResearch,Irvine,CA) wasusedtoextractDNAfromcultures.Sampleswereprepared forPCRusing20Ð50ngoftemplategenomicDNA,2XTaq MasterMix(NewEnglandBiolabs,Ipswich,MA),and500nM eachoftheprimersITS5andITS4inatotalvolumeof20 m L [23].PCRconditionswere:initialdenaturationat94 u C(2min), 36cyclesofdenaturationat94 u C(45s),annealingat52 u C(45s), elongationat72 u C(1min30s),andafinalextensionat72 u C (10min).Toassessthediversityofstrainsusedinenzymeanalysis, twoprotein-codinggeneswereamplified:thetranscription elongationfactorEF1a ( TEF1 )usingtheprimers983F/2218R [24]andDNAreplicationlicensingfactorMCM7( MCM7 )using theprimers Mcm7709for/Mcm7-1348[25].PCRconditionswere thesameasforthe ITS ,with200nMofeachprimerandtheTaq MasterMixwasusedat1.2Xconcentration.CollectioninformationandGenbankaccessionnumbersforthestrainsusedin physiologicaltestsareprovidedinTable1,andfullprimer sequencesareavailableinTableS2. Labstrainsusedinanalysiswerecomparedtospeciesisolated frombathibernaculafromLorch etal [13]andsequencedin Minnis etal [5](TableS1).Sequenceswerevisuallyinspectedand assembledinGeneiousversion6.0[26]andalignedusing MUSCLEversion3.7[27]ontheCIPRESserver[28].The alignmentwasvisuallyexaminedinMesquiteversion2.75[29]to excludeambiguouslyalignedregions.Treesweregeneratedfor individualgenesandaconcatenateddataset,whichincludedall taxa,eventhosewithmissingdata.Evolutionmodelswereselected usingtheAICcriterioninjModelTestversion2.1.4[30,31],and weredeterminedtobeGTR + I + gammaforeachofthethree genes.MaximumlikelihoodtreesweregeneratedusingRaxML [32]usingthedefaultsettingsplusGTRGAMMA + IforRAxMLHPCBlackBoxversion7.27ontheCIPRESserver;thebestML treeand500bootstrapreplicateswereproducedinasinglerun. Forlong-termstorageat 2 80 u C,sporeswerecollectedby washingsporulatingcultureswith5mLof0.1%deoctylsulfosuccinate(DSS)inSabouraudDextroseBroth(containing25% glycerolv/v)tosuspendthehydrophobicspores.Fivegenetically distinct[5],unnamed Pseudogymnoascus speciesoriginallyisolatedby Lorch etal .[13]wereobtainedfromtheCenterforForest MycologyResearch(CFMR;Madison,WI).Culturesof P. destructans ATCCMYA-4855and P.pannorum var. pannorum ATCC 16222wereobtainedfromtheAmericanTypeCultureCollection (ATCC,Manassas,VA),wheretheyarestoredundertheprior name Geomyces .Controlspecies,alsoobtainedfromtheATCC, weretheascomycetes Penicilliumpinophilum ATCCMYA-9644and Oidiodendronmaius ATCCMYA-4765,anericoidmycorrhizoid fungusintheMyxotrichaceae[33]. Pseudogymnoascus ,thoughlong thoughttobeamemberoftheMyxotrichaceaewith Oidiodendron [34Ð36],hasrecentlyshowntobeinadifferentLeotiomycete family[37Ð39],while Penicillium isadistantlyrelatedfilamentous ascomyceteintheclassEurotiomycetes[40].Fungiwerecultured atroomtemperature(20 u C),exceptfor P.destructans ,whichwas culturedat10 u Conpotatodextroseagar(PDA;20g/Ldextrose, 4g/Lpotatoextract,15g/Lagar,pH6.8)fromfrozenspore suspensionspriortouseinenzymeassays.Forplate-basedassays, pointinoculationwasusedtotransferfungalsporesfrom sporulatingculturestothecenterofpreparedmedia.Toinoculate filterpapercultures,sporessuspensions(dilutedto107spores/mL) werepreparedbywashingculturesonPDAwith5mLofDSS, whichhaspreviouslybeenshowntobenon-toxictoboth Geomyces and Pseudogymnoascus species[41].Plate-basedAssaysEnzymetestsinsolidmediawereperformedatpH6.8inagarbasedmedia(15g/L).Toassessthetemperatureoptimafor Pseudogymnoascus growth,10replicatesofeachspecieswere cultivatedonPDAat5,10,15,20,25,and30 u C,withthe diameterofeachcolonycheckedweeklyforsixweeks.Theweekly growthrateforeachsinglereplicatewascalculatedinusingthe lmListfunctioninRversion2.15.1[42],andthemeanand standarddeviationforeachspeciesatagiventemperaturewas calculated.Toassessenzymeactivity,boththecolonyand clearancezonediameterswereusedtocalculatetheRelative EnzymeActivity(REA),whichistheratiooftheclearancezoneto colonysize.Todeterminethebestmediumforhemolysisanalysis, apilotstudywasconductedonfourmedia,eachwith5%sheep's blood:brainheartinfusionagarandtrypticsoyagar(TSA)from HardyDiagnostics(SantaMaria,CA),andlab-preparedTSAand PDA.ThePDAwaspreparedusing10mLofPDAand5mLofa PDA/bloodoverlay.Basedontheresultsofthispilotstudy, hemolysiswasassessedat10 u Cforthesix Pseudogymnoascus species usingTSAamendedwith5%defibrillatedsheep'sblood(Cleve-White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org2January2014|Volume9|Issue1|e86437

PAGE 3

landScientific,Bath,OH).LipaseassayswereperformedonSpirit BlueAgar(BDDifco,FranklinLakes,NJ);theagarmixwas sterilizedbyautoclaving20minandcooledto55 u Cbeforeadding 30mLofsterilizedlipasereagent(100:1oliveoil:Tween80in 400mLdeionizedwater).Colloidalchitinagarwaspreparedfrom shrimpshellchitinfollowingtheprotocolinHsuandLockwood [43],withthefollowingchanges:thechitinwasdissolvedovernight ratherthanfor30minutesinconcentratedHCl,andwasused immediatelyinagarpreparationasawetcakeinsufficient quantitytoyield4g/Ldryweight.Theproductionofureasewas testedusingChristensen'sureaagar[44].Totestcellulose degradation,mediacontainingoneofthreedifferenttypesof celluloseÐcarboxymethyl-cellulose(CMC),crystallinecellulose (Avicel),andD-cellobioseÐwerepreparedfollowingYoon etal 2007[45]withthefollowingalterations:yeastextractwasused ratherthanyeastnitrogenbase,anddyeswereusedaspost-stains basedonSaczi etal [46]ratherthanbeingincludedinthemedia. Cellulasetestswererepeatedfor Pseudogymnoascusspp. usingyeast nitrogenbaseratherthanyeastextract,withthecelluloseasthe solecarbonsource.Whilethechitin,Avicel,SpiritBlue,andblood platesshowdistinctclearancezones,theCMC-agarandDcellobioseagarrequirefurtherprocessing.Basedon Saczietal [46], theCMC-agarwaspost-stainedwithCongoredfor1hourand destainedwith0.7MNaClfor15minutes.Bromcresolpurple (pH6.8)wasselectedasapost-stainforD-cellobioseagarduetoits abilitytodetectpHchangesundermildlyacidictonear-neutral conditions. Humicandfulvicacidswereextractedfrompottingsoilas describedinBhullar etal [47]andusedtoprepareagarmediawith , 20mg/Lofeitherhumicorfulvicacidasthesolesourceof nutrients.Fungalcultureswereinoculatedusingpointinoculation andkeptforoneweekateither20 u Cor10 u C,with10replicates foreachtemperature/mediumcombination.Growthwasrecordedastheincreaseincolonydiameterovertime.LiquidAssaysFurthertestsofcellulaseabilitywereperformedin50mLliquid mediawith9cmWhatman # 4filterpapercutinto1cm2pieces. Aninoculatingdoseof106sporeswasaddedtothisliquidassay, whichalsocontained0.1MNaNO3,11.48 m MK2HPO4, 7.35 m MKH2PO4,6.10 m MMgSO4,30 m MFeCl3,1.24 m M ZnSO4,and1.22 m MMnSO4.Twoflaskswereinoculatedwith P. destructans whileonewasusedforeachoftheBL308,BL549, BL578,BL606, P.pannorum and Penicilliumpinophilum cultures.After onemonthofshakingincubation(120rpm)at10 u C,thecultures werecentrifugedat6000 6 g .Thesupernatantwasfilteredto removecellularmaterialandstoredat4 u Cpriortouseinliquid cellulaseassays.Thepresenceofreducingandnon-reducing sugarswasassessedwithamodificationofthedinitrosalicylicacid (DNSA)reagentmethodfromMiller[48].Briefly,2mLof cellulosesolution(1%CMCorAvicelin0.05Msodiumcitrate) and2mLofculturefiltratewereincubatedina50 u Cwaterbath foronehour.Sampleswerethensubdivided;thefirsthalfofthe samplewasuseddirectlytodeterminethepresenceofreducing sugarsbyadding1mLDNSAreagent,incubatingat95 u Cfor 15min,andaddingof1mLof40%potassiumsodiumtartrate. Theotherhalfofthesamplewasusedtoassessthepresenceof non-reducingsugars:beforeproceedingwiththeDNSAreagent, thesamplewasfirsthydrolyzedat95 u Cfor5minutesusingHCl (finalconcentration:0.24M)andneutralizedwithKOH(final concentration:0.25M).Absorbanceat575nmwasmeasured usingaDR2800Spectrophotometer(HachCo.,Loveland,CO). Triplicatetestswereperformedforeachculturefiltrateandfor controltestsusingeithertheenzymesolutionorthecellulose Table1. Originofisolatesusedinthisstudy*.IsolateSp.CollectionOriginLocationTestITSMCM7TEF1 BL308 1Barton Mlf Tennessee,USA10xKF686750KF686779KF686764 BL549 1Barton Mlf Tennessee,USA10xKF686751KF686771KF686767 BL578 1Barton Mlf Tennessee,USA10xKF686752KF686772NA BL606 1Barton Mlf Tennessee,USA10xKF686753KF686780NA MYA4855 3ATCC Mlf NewYork,USA10xKF686759KF686773KF686768 16222 2ATCCwfsGermany10xNAKF686777KF686766 MYA4765 4ATCC Vmr Poland10xNANANA 9644 5ATCCradioPapuaNewGuinea10xNANANA FI204 1BartoncsTennessee,USA3xKF686754NAKF686763 FI590 1BartoncsTennessee,USA3xKF686755NANA FI606 1BartoncsTennessee,USA3xKF686756NANA FI609 1BartoncsTennessee,USA3xNAKF686778KF686761 FI687 1BartoncsTennessee,USA3xKF686757KF686775KF686769 FI698 1BartoncsTennessee,USA3xKF686758KF686776KF686765 13PA1 1FCMRcsPennsylvania,USA3xNAKF686774KF686770 4NY16 1FCMRcsNewYork,USA3xJX270377KF017653KF017762 4NY17A 1FCMRcsNewYork,USA3xJX270378KF017654KF017763 KF017763 5NY8 1FCMRcsNewYork,USA3xJX270387KF017656KF017765 KF017765 * Speciescodes: 1Pseudogymnoascussp.;2Pseudogymnoascuspannorum,;3Pseudogymnoascusdestructans;4Oidiodendronmaius;5Penicilliumpinophilum. Origincodes: cs,cavesediment;MlfMyotislucifugusfur;rsradioset;wfswheatfieldsoil;VmrVacciniummyrtilisroots. doi:10.1371/journal.pone.0086437.t001 White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org3January2014|Volume9|Issue1|e86437

PAGE 4

solutioninisolation,withstandardizedcurvesgeneratedfor glucoseandsucroseusing0,2.5,5,7,9,and10mMsolutions. LinearregressionequationswereusedtocalculatethemMof reducingandnon-reducingsugarsproducedfromeachcellulase/ enzymeassay. Theactivityof b -glucosidasewasassessedwiththestandardpnitrophenylb -glucopyranoside(pNP b G)method.Briefly,0.1mL cellulosefiltratewasaddedto0.9mLofa0.02%solutionof pNP b Gin0.1Msodiumacetatebuffer(pH4.8)andincubatedat 50 u Cfor30minbeforeadding2mLofClarkandLubsbuffer (pH9.8)[49]andmeasuringabsorbanceat430nm.Astandard curvewaspreparedusing102 1,102 2,102 3,102 4,102 5,and 102 6m Mp-nitrophenolsolutions,andtheexponentialregression equationwasusedtocalculatetheamountofp-nitrophenol releasedfrompNP b G,anindicationof b -glucosidaseactivity.StatisticalAnalysisAllstatisticaltestsofenzymeactivitiywereperformedinR[42] usinganANOVAtotesttemperatureandspecieseffectsandthe Tukey'sHighlySignfiicantDifferences(Tukey'sHSD)testto assesssignificantdifferencesbetweeneachREA.ResultsBeforebeginningourcomparativeenzymaticanalyses,itwas importanttodeterminetherelatednessofthebatandenvironmental Pseudogymnoascus isolatestoeachotherand P.destructans. In ordertodothis,weusedRAxML[32]togenerateaphylogenetic treeoftheseisolates,including P.destructans, usingaconcatenated sequenceofthreegeneticloci(ITSregion:876, MCM7 :486, TEF1 :806).Thistreeincluded72taxa(Tables1andS1),which allowedustoreliablyplaceouridentifiedisolatesineachofthe Pseudogymonascus clades[5].Thephylogeny(Figure1)confirmsthe resultsofotherinvestigators,inthat P.destructans remainsina distinctcladefromotherNorthAmerican Pseudogymnoascus isolates [4,5].Theresultsalsosuggestthatourenvironmentalisolate BL578iscloselyrelatedto P.pannorum ATCC16222,possiblyasa subtypeorstrain. Basedonthephylogenyforour Pseudogymnoascus strains,two geneticallydiversesubsetsof Pseudogymnoascusspp wereselectedfor physiologicaltests:(1)asetofsix Pseudogymnoascusspp. representing fiveclades,includingfourofourisolates(BL308,BL549,BL578 andBL606), P.destructans, thecontrolspecies P.pannorum , Penicilliumpinophilum ,and O.maius (usedin10xreplicatetests); and(2)asetofeightisolatesfrombatfurandcavesediments representingeightadditional Pseudogymnoascus lineages(13PA1to FI698)usedintriplicatetests.The10xsetwasusedtopermit statisticalanalysiscomparingREAamongspecies,whilethe3xset wasusedtogainabroaderassessmentoftherangeofREAin Pseudogymnoascus . Theoptimaltemperatureof P.destructans growthrangesfrom 12.5Ð15.8 u C,whereitgrowsbothradiallyandproducesasexual spores,althoughgrowthon Myotislucifugus batsintheenvironmentalhasbeenobservedaslowas2 u C[6].Toexaminetherole temperaturemightplayinenzymeactivity,weexaminedthe growthofourstrainsatvarioustemperaturesandcalculatedthe lineargrowthratesbasedontheweeklygrowth(Figure2).Allof thespeciesexaminedgrewbetween5and25 u C,withoptimal growthvaryingbetween10Ð25 u C.BatisolateBL308wasmore restrictedinitsgrowth,showinganoptimumgrowthof10 u Cwith nogrowthat25 u C,similarto P.destructans .Strainsthatwere closelyrelatedtoeachother,suchasBL578and P.pannorum ATCC16222,showedsimilarresponsestotemperature,butthere wasotherwisenoimmediatelyevidentcorrelationbetween physiologyandstrainrelationships.Thebat-isolatedstrainsin ourstudythusdemonstratedbothpsychrophilicandpsychrotolerantgrowth,inlinewithothermembersof Pseudogymnoascus and Geomyces .Basedonthesegrowthtemperaturesandourunderstandingofthetemperaturerangeofhibernacula( , 12 u C),we examinedenzymeactivityforthesaprotrophic Pseudogymnoascus spp. attwotemperatures:10 u C,whichisclosetotheaverage temperatureofbathibernaculaandtheoptimalgrowthtemperaturefor P.destructans andstrainBL308;and20 u C,theoptimal growthtemperatureofsomeofourbat-isolated Pseudogymnoascus species.Usingthesetemperaturesallowedustoexaminewhether fungishowahigherREAattheiroptimalgrowthtemperatureor thetemperatureoftheenvironmentfromwhichtheywere isolated. Fulvicandhumicacidsarecomplexaromaticandpolyaromatic organicmoleculesrichincarboxylandphenolategroupsproduced bycellulosedecompositionintheenvironment[50].Wetestedthe abilityof Pseudogymnoascusspp. togrowontheseacidswhich,asthe dominantformofdissolvedorganiccarbon(DOC)inbothsoils andcaves[51,52],maysupportfungalgrowthinhibernacula.All fungitestedshowedgrowthoveroneweekonbothfulvicand humicacidmedia; P.destructans and O.maius hadtheslowest growthrates,whilethesaprotroph Penicilliumpinophilum hadthe highestgrowthrate(Figure3).Boththetemperatureandspecies testedsignificantlyaffectedgrowthrateonbothfulvicandhumic acids(ANOVAresultsinTableS3).ATukey'sHSDtestofthe temperatureandspecieseffectsfoundthat P.destructans had significantlylowergrowththanmostother Pseudogymnoascus species, butwasnotsignificantlydifferentfromenvironmentalisolates BL308(10 u Cand20 u C),BL549(10 u C),orfrom O.maius (10 u C) (significancevaluesareshowninTableS4).Acomparisonof psychrotolerant Pseudogymnoascus speciesatambient(20 u C)and cooltemperatures(10 u C)indicatedthatformostspecies, temperaturedidnotaffectgrowthrateonthismedium,while strainBL578andBL549grewsignificantlybetterat20 u Cthanat 10 u Confulvicacid,andBL578and P.pannorum grewsignificantly betterat20 u Cthanat10 u Conhumicacid(significancevaluesare showninTableS6). Thepresenceofcelluloseandchitinincavesmaybedependent onenvironmentaleffectsthatvaryamongsites,suchasfloodingor thepresenceofbatand/orinsectpopulations[53,54];cavesthat experienceperiodicalfloodingcanaccumulatelargequantitiesof plantdebris[55]andtheguanoofinsectivorousbatscontainsa highamountofchitinousinsectcuticle[56].Wetherefore investigatedtheabilityof P.destructans toproducecellulasesand chitinases(Figure4),whichcouldprovideanenvironmental carbonandenergysourceforthegrowthof P.destructans .While mostofthespeciesexaminedcouldbeassessedforchitinaseatone week,theREAof O.maius and P.destructans wereassessedattwo andfourweeks,respectively.Allexaminedspeciesproduced chitinases,with P.destructans showingsimilaractivitytoasuiteof relatedfungalspecies.Fourofthefiveenvironmental Pseudogymnoascus isolatesshowedlowerchitinaseREAat10 u Cthanat20 u C, butthisdifferencewassignificantonlyforBL308(Tukey'sadjusted p-value , 0.001;seeTableS6forothervalues).Thelowest chitinaseactivityobservedwasforthe Penicilliumpinophilum at 20 u C.At10 u C, P.destructans chitinaseactivitydidnotdiffer significantlyfromthatoftheotherspecies(TableS5),butchitinase activityfor P.destructans at10 u Cwassignificantlylowerthanthatof BL308andBL549andhigherthanthatof P.pinophilum at20 u C. While P.destructans and P.pinophilum showedsimilarREAtothe otherfungi,theclearancezoneforthesetwospeciesremained slightlycloudy,suggestinganincompletehydrolysis.Growth remainedscantforthesespecies,withlittleaerialmycelia.White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org4January2014|Volume9|Issue1|e86437

PAGE 5

Duetothepotentialroleofplantdetritusinpromotingfungal growthincaves,weassessedtheactivityofthreedifferent functionalcategoriesofcellulases:endoglucanases,whichcleave internalcellulosebonds,exoglucanases,whichcleavethenonreducingendofthecellulosepolymer,and b -glucosidases,which breakdownthedisaccharidecelluloseby-product.Duetovarying growthrates, P.destructans wasassessedforendoglucanaseatthree weeks, b -glucosidaseattwoweeks,andcellobiohydrolaseatfour weeks.Theother Pseudogymnoascusspp. wereassessedatoneweek forendoglucanaseatbothtemperatures,whiletheREAof b glucosidaseandcellobiohydrolasewasmeasuredatoneweekfor the20 u Cassayandtwoweeksforthe10 u Cassay. P.pinophilum and O.maius wereassessedatoneweekandtwoweeks,respectively,for allcellulasecategories.Theendoglucanaseassay(usingCMC)was negativefor P.destructans ,butpositiveforallotherspecies examined(Figure4).Thisassaydidnotyieldtheclassicred-toblackcolorchangethatindicatesmediumacidification,but showedapaleredzoneindicatingwherecellulosehadbeen degradedsurroundingcolonies.Whenthetestswererepeated usingCMCasthesolecarbonsource,theREAincreasedforeach species,butremainednegativefor P.destructans. Despiteits apparentlackofendoglucanaseactivity, P.destructans wasableto growandsporulateonCMCmedia.The b -glucosidaseassay indicatedthat P.destructans hadsignificantlyhigherREAthanall otherfungiexaminedexceptforBL308andtheplantmutualist O. maius ,whichwerenotsignificantlydifferent(TableS5).CellobiohydrolaseREAwaspositiveforeachfungus,and,thoughmuch slowertodevelopin P.destructans ,notsignificantlydifferentfrom Figure1.Bestmaximum-likelihoodtreeforthePseudogymnoascusspp.usedinthisstudy(lnL= 2 10622.006982). Aconcatenated alignmentoftheTEF-1,MCM7,andpartialITSgeneswasusedtogenerateaMaximum-likelihoodtreeinRAxMLusingstrainsidentifiedinMinnisand Lindner[5].Valuesshowbootstrapsupportfrom500bootstrapreplicates;closedcirclesindicate $ 95%supportateachbranchpoint,whileopen circlesindicate . 70%support.Thetaxashowninredwereusedin10Xtests,whilethetaxainbluewereusedintriplicatetests. Geomycesauratus andmembersofthe Leuconeurospora wereusedastheoutgroups.AccessionnumbersforeachsequencecanbefoundinTables1andS1. doi:10.1371/journal.pone.0086437.g001 White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org5January2014|Volume9|Issue1|e86437

PAGE 6

theotherfungi(TableS5),exceptBL606,whichhadsignificantly lowerREAat10 u C(Tukey'sadjustedp-value=0.001).Temperaturewasasignificantfactoroverallfor b -glucosidase(ANOVAFvalue=16.005,p-value , 0.001)andcellobiohydrolaseactivity (ANOVAF-value=35.871,p , 0.001),butnotforendoglucanase (ANOVAF-value=0.004,p=0.948).Nevertheless,BL308 showedsignificantlyhigherendoglucanaseactivityat20 u Cthan at10 u C(Tukey'sadjustedp-value=0.001),asitdidfor b glucosidaseactivity(Tukey'sadjustedp-value , 0.001).BL606was theonlystrainshowingsignificantdifferencesforcellobiohydrolase activityatthesetwotemperatures,withhigheractivityat20 u C (Tukey'sadjustedp-value=0.004)(TableS6). Todeterminewhichcleavageproductswerereleasedfrom celluloseforpotentialfungalgrowth,weexaminedtheproduction ofreducingandnon-reducingsugarsinliquidassays.UsingCMC, theseassayswerenegativefortheproductionofnon-reducing sugarsbyallthefungitested(datanotshown),whilereducing sugarswereproduced(Figure5).The P.destructans culturefiltrate releasedasmallamountofreducingsugarsfromCMC(mean 6 SD=0.333 6 0.008 m M),whichwasdoublethatofthenegative control(0.182 6 0.014 m M).Theotherfungiassayedreleased muchhigherlevelsofreducingsugarsthan P.destructans ,with BL308astheleastactive(mean=1.03 6 0.247 m M)andBL578as themostactive(mean=2.70 6 0.045 m M).Asubsequentassay usingtheinsolublecelluloseAviceltestednegativeforreducing sugarsusing P.destructans filtrate(0.265 6 0.017 m M)comparedto thenegativecontrol(0.295 6 0.019 m M).Theother Pseudogymnoascusspp. testedpositiveforreducingsugarsonAvicel,rangingfrom 0.398 6 0.103 m M(BL308)to0.882 6 0.054 m M(BL578),while Penicilliumpinophilum testednegative(0.274 6 0.038 m M).The cellobiohydrolaseassayusingpNP b Gwasnegativefor P.destructans andBL308,butpositivefortheother Pseudogymnoascusspp. and Penicilliumpinophilum (Figure5). Inadditiontoexaminingpotentialsaptrotrophicenzyme activityinour Pseudogymnoascus isolates,wecomparedthemfor enzymaticactivitythatcouldaidinpathogenesis.Todetermine whether P.destructans producesenzymesthatcouldplayarolein breakingdownhosttissues,wetestedwhethermembersof Pseudogymnoascus couldproducelipases(Figure6A&B)and hemolases(Figure6C).Weinitiallytestedhemolysisonfour complexmediacontaining0.5%sheep'sblood,eachofwhich containdifferentlevelsofnutrients.Hemolysiswasnotedonlyin TSA/bloodplates,andnotintheBHI/bloodorPDA/blood preparations(datanotshown).LipaseREAwasnotsignificantly affectedbytemperature(ANOVAF-value=0.002,p=0.961), althoughREAforBL578wassignificantlyhigherat10 u Cthanat 20 u C(Tukey'sadjustedp-value=0.025).Whilelipaseactivitywas rapid(visibleinoneweekforallspeciessave P.destructans and O. maius ,whichwereassessedattwoweeks),hemolysiswasslow. Alpha-hemolysis,whichindicatesthepartiallysisofredblood cells,wasobservedinourfungalculturesonlyafterseveralweeks: 11weeksfor P.destructans and8weeksfortheother Pseudogymnoascusspp., whichgrewmorerapidly.TheREAof P.destructans a hemolysiswasnotsignificantlydifferentfromthatoftheother Pseudogymnoascusspp. (TableS4).Thecompletelysisofredblood cellsandbreakdownofhemoglobin( b -hemolysis)wasnot observedforanyofthetestedfungi. Ifdegradedbyurease,theureainbaturineandguanocould serveasasourceofnitrogenforfungalgrowth.Ureasecouldalso playanimportantroleinpathogenesis,astheenzymehasbeen implicatedinvirulencepathwaysinotherpathogenicmicroorganisms[57].WetestedureaseactivityonChristensen'sureaagar, andfoundthatallthefungitestedexcept O.maius rapidly producedtheenzyme(Figure6A&B).The20 u Cassayswere assessedatoneweek,butthe10 u Cassaysforthe Pseudogymnoascus spp. and P.destructans weremeasuredattwoweeks,saveforBL606, whichwasassessedatoneweek.Theplantmutualist O.maius developedweaklypositiveREAforureaseonlyaftersevenweeks. The Pseudogymnoascusspp. showedwidevariationintheirurease ability,rangingfromaverageREAsof1.54for P.destructans to6.03 forBL308(20 u C).The Pseudogymnoascusspp. tendedtoshowhigh ureaseactivity,withsomespeciesproducingureasezonesoversixfoldgreaterthanthecolonydiameter.While P.destructans showed significantlylowerureaseactivitythandidallotherexamined Pseudogymnoascusspp. ,itproducedureasewithanactivityzone doubleitscolonysize,similartoBL606at10 u Cand P.pannorum at 10 u Cand20 u C.Temperaturewasnotasignificantfactorinurease REAoverall(ANOVAF-value=2.839,p=0.0947),butBL549 producedsignificantlyhigherureaseREAat10 u Cthanat20 u C Figure2.Temperature-dependentgrowthofenvironmental isolatesofPseudogymnoascus. Theplotshowstheaveragegrowth rateofeachtaxonover6weeks.Errorbarsindicatethesignificant differenceofeachassay(n=10). P.pan = P.pannorum. doi:10.1371/journal.pone.0086437.g002 Figure3.GrowthofPseudogymnoascusspp.onfulvicandhumic acidextracts. Boxplotboundariesshowthefirstandthirdquartiles, withthemeanasthecenterlineandwhiskersas1.5timestheinterquartiledistance.Outliersareplottedaspoints. P.des = P.destructans;P. pan = P.pannorum;Pen.p=Penicilliumpinophilum; and Om=Oidiodendronmaius. doi:10.1371/journal.pone.0086437.g003 White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org6January2014|Volume9|Issue1|e86437

PAGE 7

(Tukey'sadjustedp-value , 0.001),whileBL606produced significantlyhigherureaseREAat20 u C(Tukey'sadjustedpvalue , 0.001).DiscussionAstoppredatorsintheirecosystems,batsplayacriticalrolein controllinginsectpopulations.Thus,thehighmortalityinmultiple batspeciesfromWhite-noseSyndromerenders P.destructans a majorthreatnotonlytothesewildlifepopulations,butalsoto agricultureandpublichealth[1,58,59].Therecentfindingsofthe persistenceof P.destructans incavesedimentssuggestthatthe funguscansurviveinbathibernaculaevenintheabsenceofits host,whichhasaprofoundimpactonbothdiseasemanagement andtheepidemiologyofthedisease[13].Wethereforecompared thefunctionofseveralenzymesthatcouldserveinasaprotrophic nicheforthepathogenic P.destructans aswellasresident Pseudogymnoascus species.Theenzymesselectedforthisstudywere notintendedtobeanexhaustivesurveyof Pseudogymnoascus metabolism;rather,theywerechosentorepresentenzymesthat mightberequiredforgrowthonmajorcomponentsofthecave ecosystem.Wealsoexaminedasubsetofenzymesthatcouldhave dualfunctionforthesefungi,supportingsaprotrophicgrowthand potentiallycontributingtoapathogeniclifestyle. Caveenvironmentsaregenerallycarbonandenergy-limited duetogeologicisolationandthelackoflightdrivenphotosynthesis [60].Asaresult,thesubstratesavailabletofungiincavesare primarilyintheformofallochthonousinput,suchassoil-derived dissolvedorganiccarbon(DOC)ordetritus,includingleaflitter fromenteringsurfacestreams.Anothersourceofenergyisthe organicwasteoftrogloxenes,suchascricketsandotherarthropods andbatsandtheirchitin-andurea-richbatguano[53Ð54].We thereforeassessedtheactivityofchitinaseandcellulaseactivityin Pseudogymnoascus ,aswellastheabilitytogrowonDOCintheform offulvicandhumicacids.Ourresultssuggestthatwhile P. destructans demonstratesenzymaticactivityineachofthesegroups, testingpositiveforgrowthonfulvicandhumicacids(Figure3)and forchitinaseandcellulaseactivity(Figure4),itshowsslower growthandlowercellulaseactivityrelativetoother Pseudogymnoascusspp. Thechitinaserelativeenzymeactivityin P.destructans was similartoothertested Pseudogymnoasci, whileactualgrowthofthe pathogenonchitinwaslimited.Thechitinaseproductionbyother Pseudogymnoasci indicatesthatthesespeciescanusechitinasa nutritionsource,whichmayallowgrowthoninsectsand/orthe insectremnantsfoundinbatguano,althoughsuchgrowthby P. destructans wouldlikelybecomparativelyslow. Assaysofcellulasesthatwouldallowgrowthonplantdebris werecontradictory.Inplateassays, P.destructans showedthe highest b -glucosidaseREAofallexaminedspecies,butwasslowto developcellobiohydrolaseandwasnegativeforendoglucanase (Figure4).Liquidassaysfoundthat P.destructans usingfilterpaper asasourceofcarbonproducedendoglucanases,butnot b glucosidasesorcellobiohydrolases(Figure5).Thenegativeresult onsolidmediaforendoglucanasemayhavebeenduetotheassay's relianceonvisiblecolordifferencesinthesurroundingmedia, Figure4.RelativeenzymeactivityforsaprotrophicenzymesinP.destructansandotherfungi. Boxplotboundariesshowthefirstandthird quartiles,withthemeanasthecenterlineandwhiskersas1.5timestheinter-quartiledistance.Outliersareplottedaspoints. P.des = P.destructans;P. pan = P.pannorum;Pen.p=Penicilliumpinophilum; and Om=Oidiodendronmaius. doi:10.1371/journal.pone.0086437.g004 White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org7January2014|Volume9|Issue1|e86437

PAGE 8

whichmaythushaveahigherminimumdetectionlimitthanthe liquidassay.StrainBL308and Penicilliumpinophilum ,like P. destructans ,testednegativeinliquidtestsforAvicel,asynthetic microcrystallinecellulose,butpositiveforgrowthonthesubstrate onsolidmedia.Theseresultssuggestthat P.destructans does produceeachclassofcellulase,butonlytoalimiteddegreeand underrestrictedconditions.Comparedtothebat-isolatedand environmental Pseudogymnoascusspp. , P.destructans appearscapable ofdegradingcellulose,albeitatareducedcapacity. Althoughoccasional,superficialmycosesinhumansand animalsbymembersof Pseudogymnoascus havebeenobserved, membersofthisgenusareconsideredtobepredominately saprotrophic[61,62].Toassesstheexpressionofenzymesthat mightfunctionineitherasaprotrophicorpathogeniccontext,we examinedlipase,hemolaseandureaseactivity.Ourdata demonstratedthat P.destructans waspositiveforallthreeenzymes, withlipaseactivitysimilartotheothermembersofthegenus (Figure6).Lipolyticfungiareknowntoplayanimportantrolein thebeginningstagesofleaflitterdecomposition[63],andlipases canalsoaidinfectiousmycosesinhosttissueadhesionandinvasion [64].All Pseudogymnoascusspp. examinedwerepositivefor a hemolysisafterseveralweeksofincubation,although P.destructans hemolysisoccurredmoreslowly.Comparedtotherapidhemolysis seenintruepathogens,suchasthebacterialpathogens Staphylococcusaureus and Streptococcuspyogenes, whichgeneratevisiblezones ofhemolysisonbloodagarwithinhours[65,66],thehemolytic Figure5.Liquidassaysofendoglucanase, b -glucosidase,andcellobiohydrolase. ReducingsugarsreleasedfromCMC(endoglucanase activity)andAvicel( b -glucosidaseactivity)whentreatedwithfungalfilterpaperculturesupernatantandp-nitrophenolreleasefrompNP b G (cellobiohydrolaseactivity).Testswereperformedintriplicate,andtheresultswereplottedindividually.Thehorizontallineindicatesresul tsfor negativecontrolsusingculturefiltratesandH2O. doi:10.1371/journal.pone.0086437.g005 White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org8January2014|Volume9|Issue1|e86437

PAGE 9

activityin P.destructans appearstobeginwhennutrientsbecome limiting.Thus,hemolyticactivityby P.destructans maynotbe optimizedforsystemicgrowthwithinitshost,butrathermayaid insurvivalonthenutrient-limitedlandscapeofabatwing membrane.Thishighlipaseactivitybutlowhemolysinactivity correspondswiththehistologicalevidenceofWNSinfection, wherethefungusdegradesthewingepitheliumandbasal membranetoinvadeconnectivetissue[8,67],butdoessowithout directlyinvadingthebloodvessels[67].Thus,hemolysismaynot playanimportantroleindiseasepathology,butmaybenecessary toobtaintheironneededtosupportfungalgrowth. Allofthefungiexaminedinthisstudysave O.maius showed highureaseactivity.Ureasewouldbeadvantageousforgrowthin batguano,butasthisenzymeisbroadlydistributedamongsoil microbes[68,69],ureaseproficiencyisprobablynotaspecific adaptiontocavelife.Nonetheless,ureasehasbeenimplicatedin tissueinvasionandvirulencesignalingpathways[57,70,71], includingin Coccidioidesposadasii, whereammoniaproducedby ureasedamageshosttissues[72,73]and Cryptococcusneoformans, whereureaseplaysanimportantroleincrossingtheblood-brain barrier[74].UreasemaythusplaytworolesinWNS:permitting propagationof P.destructans onguanoandpotentiallyaidingin pathology.Furtherresearchisneededtounderstandthepossible involvementoftheseenzymesinWNSdiseaseecology,aswellas othersaprotrophicpathwaysthatmayhavebeencooptedfor pathogenesis. Wehadexaminedtheeffectoftemperatureonenzymeactivity totestwhetherspecieswouldshowhigherREAattheiroptimal growthtemperatureoratthetemperatureoftheenvironment fromwhichtheyhadbeenisolated.Infact,wefoundthat temperaturerarelycausedasignificantdifferenceinrelative enzymeactivity,andinthemajorityofcasesthatdidshowa significantdifference,theREAwashigherat20 u C,regardlessof thetemperaturepreferencesoftheorganismorsiteoforigin.For instance,BL308,whichhasanoptimalgrowthat10 u Candwas isolatedfromacoldcave,showedsignificantlyhigherchitinase, endoglucanase,and b -glucosidaseactivityat20 u C.Theonlytwo instancesofsignificantlyhigherREAat10 u CwereforBL549 ureaseandBL578lipase;thesetwospecieshaveoptimalgrowthat 20 u Cand25 u Crespectively.Thus,whilesomeofthecave-isolated fungigrowmorequicklyatthecooltemperaturesfoundin northernbathibernacula,theirenzymefunctionindicatesthat theyarenotlikelyrestrictedtotheseenvironments. Theresultsofthisstudyindicatethatthe Pseudogymnoascusspp. foundincavescandegradeavarietyoforganicmoleculesfor growthinmultiplemicrohabitats,includinginsectdetritus, decayingplantmaterial,andbatguano.Suchresultssuggestthat ratherthanhavingaspecificniche,membersofthegenus Pseudogymnoascus canfunctionasgeneralistdecomposers,afinding supportedbythepresenceof Pseudogymnoascus andrelated Geomyces speciesworldwide[75Ð82],includingAntarcticaandtheArctic. Ourfindingthat P.destructans showssimilarsaprotrophicpotential toothermembersofthisgenusmayindicateapotential environmentaloriginforthispathogen;however,thereduced growthrateandsaprotrophicenzymeactivitysuggestthat P. destructans mayhavegainedfunctionalityinpathogenesisatthe expenseofanenvironmentallifestyle.Suchadiminished saprotrophiccapacitycouldbeexplainedif P.destructans had enteredintoanevolutionaryhost-pathogenrelationshipat sometimeinthepast;asignificantlengthofcoevolutionbetween hostandpathogencouldhaveresultedinadecreaseinthe selectivepressureon P.destructans tomaintainenzymesneededfor decomposition.Todetermineifthisisindeedthecase,a phylogeneticcomparisonofthegenesinvolvedinbothsaprotrophicandpathogenicpathwaysmaybeabletodetectchangesin eitherexpressionpathwaysand/orenzymaticactivityandcould potentiallyprovideamolecularclockforthetimingofthe movementof P.destructans outofthesoil/cavesediment environmentandintoitshost. Figure6.Relativeenzymeactivityofenzymesthatmayhave pathogenicandsaprotrophicfunctioninenvironmentalPseudogymnoascusspp.andP.destructans. A)10xreplicatestesting lipaseandureaseactivityathibernaculumandambienttemperature.B) 3xreplicatestestinglipaseandureaseactivityatambienttemperature. C)10xreplicatestesting a -hemolysisactivityonontrypticsoyagar/5% sheep’sbloodafter8weeks(environmentalisolates)and11weeks( P. destructans ).Boxplotboundariesshowthefirstandthirdquartiles,with themeanasthecenterlineandwhiskersas1.5timestheinter-quartile distance.Outliersareplottedaspoints. doi:10.1371/journal.pone.0086437.g006 White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org9January2014|Volume9|Issue1|e86437

PAGE 10

SupportingInformationTableS1CollectioninformationandGenbankaccession numbersforstrainsusedinassessingphylogeneticdiversity. (XLSX)TableS2Primersusedforsequencing Pseudogymnoascus isolates. (XLSX)TableS3Resultsoftwo-wayANOVAassessingtemperature andspecieseffectsinrelativeenzymeactivity. (DOCX)TableS4Tukey'sHighlySignificantDifferencesTestforgrowth onorganicacidscomparing P.destructans tootherspecies. (DOCX)TableS5Tukey'sHighlySignificantDifferencesTestfor relativeenzymeactivitycomparingmultiplefungalspeciesto P. destructans. (DOCX)TableS6Tukeys'HighlySignificantDifferencesTestcomparingintraspecificgrowthorrelativeenzymeactivityat20 u Cand 10 u C. (DOCX)AcknowledgmentsWethanktheUSFishandWildlifeServiceforfundingthisstudyandthe CumberlandGapNationalHistoricParkforprovidingcollectionpermits. WewouldalsoliketothankDanielLindnerandAndrewMinnisatthe CenterforForestMycologyResearch(CFMR)forprovidingculturesused inthisstudy,TonishaHenryandJosephineLandenbergerforassistanceon temperature-dependentgrowthandlipasestudies,andMatõ `asCafarofor adviceregardingcellulaseassays.AuthorContributionsConceivedanddesignedtheexperiments:HTRHAB.Performedthe experiments:HTR.Analyzedthedata:HTR.Contributedreagents/ materials/analysistools:HAB.Wrotethepaper:HTRHAB.References1.BlehertDS,HicksAC,BehrM,MeteyerCU,Berlowski-ZierBM,etal.(2009) Batwhite-nosesyndrome:anemergingfungalpathogen?Science323:227. 2.ReynoldsHT,BartonHA(2013)White-noseSyndrome:Humanactivityinthe emergenceofanextirpatingmycosis.MicrobiologySpectruminpress. 3.LorchJM,MeteyerCU,BehrMJ,BoylesJG,CryanPM,etal.(2011) Experimentalinfectionofbatswith Geomycesdestructans causeswhite-nose syndrome.Nature480:376Ð378. 4.GargasA,TrestMT,ChristensenM,VolkTJ,BlehertDS(2009) Geomyces destructans sp.nov.associatedwithbatwhite-nosesyndrome.Mycotaxon108: 147Ð154. 5.MinnisAM,LindnerDL(2013)Phylogeneticevaluationof Geomyces andallies revealsnocloserelativesof Pseudogymnoascusdestructans ,comb.nov.,inbat hibernaculaofeasternNorthAmerica.MycologicalResearch117:638Ð649. 6.VerantML,BoylesJG,WaldrepW,Jr.,WibbeltG,BlehertDS(2012) Temperature-dependentgrowthof Geomycesdestructans ,thefungusthatcausesbat White-noseSyndrome.PLoSOne7:e46280. 7.PikulaJ,BandouchovaH,NovotnyL,MeteyerCU,ZukalJ,etal.(2012) Histopathologyconfirmswhite-nosesyndromeinbatsinEurope.Journalof WildlifeDiseases48:207Ð211. 8.MeteyerCU,BucklesEL,BlehertDS,HicksAC,GreenDE,etal.(2009) Histopathologiccriteriatoconfirmwhite-nosesyndromeinbats.Journalof VeterinaryDiagnosticInvestigation21:411Ð414. 9.FisherMC,HenkDA,BriggsCJ,BrownsteinJS,MadoffLC,etal.(2012) Emergingfungalthreatstoanimal,plantandecosystemhealth.Nature484: 186Ð194. 10.PiotrowskiJS,AnnisSL,LongcoreJE(2004)Physiologyof Batrachochytrium dendrobatidis ,achytridpathogenofamphibians.Mycologia96:9Ð15. 11.JohnsonML,SpeareR(2005)Possiblemodesofdisseminationoftheamphibian chytrid Batrachochytriumdendrobatidis intheenvironment.DiseaseofAquatic Organisms65:181Ð186. 12.MitchellKM,ChurcherTS,GarnerTWJ,FisherMC(2008)Persistenceofthe emergingpathogen Batrachochytriumdendrobatidis outsidetheamphibianhost greatlyincreasestheprobabilityofhostextinction.ProceedingsoftheRoyal SocietyB275:329Ð334. 13.LorchJM,MullerLK,RussellRE,O'ConnorM,LindnerDL,etal.(2013) DistributionandenvironmentalpersistenceofthecausativeagentofWhite-nose Syndrome, Geomycesdestructans ,inbathibernaculaoftheeasternUnitedStates. AppliedandEnvironmentalMicrobiology79:1293Ð1301. 14.ChaturvediV,SpringerD,BehrM,RamaniR,LiX,etal.(2010) Morphologicalandmolecularcharacterizationsofpsychrophilicfungus Geomyces destructans fromNewYorkbatswithwhite-nosesyndrome(WNS).PLoSOne5: e10783. 15. Geomycesdestructrans SequencingProject.http://www.broadinstitute.org:Broad InstituteofHarvardandMIT. 16.LorchJM,LindnerDL,GargasA,MullerLK,MinnisAM,etal.(2013)A culture-basedsurveyoffungiinsoilfrombathibernaculaintheeasternUnited Statesanditsimplicationsfordetectionof Geomycesdestructans ,thecausalagentof batwhite-nosesyndrome.Mycologia. 17.JohnsonLJ,MillerAN,McCleeryRA,McClanahanR,KathJA,etal.(2013) Psychrophilicandpsychrotolerantfungionbats: Geomyces acommonfunguson batwingspriortothearrivalofWhiteNoseSyndrome.Appliedand EnvironmentalMicrobiologyinpress. 18.WarneckeL,TurnerJM,BollingerTK,LorchJM,MisraV,etal.(2012) InoculationofbatswithEuropean Geomycesdestructans supportsthenovel pathogenhypothesisfortheoriginofwhite-nosesyndrome.ProcNatlAcad SciUSA109:6999Ð7003. 19.VanderwolfKJ,McAlpineDF,MallochD,ForbesGJ(2013)Ectomycota associatedwithhibernatingbatsineasternCanadiancavespriortothe emergenceofWhite-noseSyndrome.NortheasternNaturalist20:115Ð130. 20.RiceAV,CurrahRS(2006) Oidiodendronmaius :saprobeinsphagnumpeat, mutualistinericaceousroots?In:SchulzB,SieberTN,editors.MicrobialRoot Endophytes.BerlinHeidelberg:Springer-Verlag.227Ð246. 21.EmmonsCW,BinfordCH,UtzJP,Kwon-ChungKJ(1977) Medicalmycology . Philadelphia,Pennsylvania,USA:Lea&Febiger. 22.SchochCL,SeifertKA,HuhndorfS,RobertV,SpougeJL,etal.(2012)Nuclear ribosomalinternaltranscribedspacer(ITS)regionasauniversalDNAbarcode markerfor Fungi .ProceedingsoftheNationalAcademyofSciences109:6241Ð 6246. 23.WhiteTJ,BrunsT,LeeS,TaylorJW,editors(1990)Amplificationanddirect sequencingoffungalribosomalRNAgenesforphylogenetics.NewYork: AcademicPress,Inc. 24.RehnerSA,BuckleyE(2005)A Beauveria phylogenyinferredfromnuclearITS andEF1a sequences:evidenceforcrypticdiversificationandlinksto Cordyceps teleomorphs.Mycologia97:84Ð98. 25.SchmittI,CrespoA,DivakarPK,FankhauserJD,Herman-SackettE,etal. (2009)Newprimersforpromisingsingle-copygenesinfungalphylogeneticsand systematics.Persoonia23:35Ð40. 26.Geneiousversion6.0createdbyBiomatters.Availablefromhttp://www. geneious.com. 27.EdgarRC(2004)MUSCLE:multiplesequencealignmentwithhighaccuracy andhighthroughput.NucleicAcidsResearch32:1792Ð1797. 28.MillerMA,HolderMT,VosR,MidfordPE,LiebowitzT,etal.(2010)Creating theCIPRESScienceGatewayforinferenceoflargephylogenetictrees. ProceedingsoftheGatewayComputingEnvironmentsWorkshop(GCE).New Orleans,LA.1Ð8. 29.MaddisonWP,MaddisonDR(2011)Mesquite:amodularsystemfor evolutionaryanalysis.Version2.75.http://mesquiteproject.org. 30.DarridaD,TaboadaGL,DoalloR,PosadaD(2012)jModelTest2:more models,newheuristicsandparallelcomputing.NatureMethods9:772. 31.GuindonS,GascuelO(2003)Asimple,fastandaccuratemethodtoestimate largephylogeniesbymaximum-likelihood.SystematicBiology52:696Ð704. 32.StamatakisA(2006)RAxML-VI-HPC:maximumlikelihood-basedphylogenetic analyseswiththousandsoftaxaandmixedmodels.Bioinformatics22:2688Ð 2690. 33.PerottoS,GirlandaM,MartinoE(2002)Ericoidmycorrhizalfungi:somenew perspectivesonoldacquaintances.PlantandSoil244:41Ð45. 34.CurrahRS(1985)TaxonomyoftheOnygenales:Arthrodermataceae, Gymnoascaceae,Myxotrichaceae,andOnygenaceae.Mycotaxon24:1Ð216. 35.UdagawaS,UchiyamaS,KamiyaS(1993) Gymnostellatospora ,anewgenusofthe Myxotrichaceae.Mycotaxon48:157Ð164. 36.RiceAV,CurrahRS(2006)Twonewspeciesof Pseudogymnoascus with Geomyces anamorphsandtheirphylogeneticrelationshipwith Gymnostellatospora. Mycologia 98:307Ð318. 37.SogonovMV,SchroersH-J,GamsW,DijksterhuisJ,SummerbellRC(2005) Thehyphomycete Teberdiniahygrophila gen.nov.,sp.nov.andrelatedanamorphs of Pseudeurotium species.Mycologia97:695Ð709. 38.WangZ,BinderM,SchochCL,JohnstonPR,SpataforaJW,etal.(2006) Evolutionofhelotialeanfungi(Leotiomycetes,Pezizomycotina):Anuclear rDNAphylogeny.MolecularPhylogeneticsandEvolution41. 39.WangZ,JohnstonPR,TakamatsuS,SpataforaJW,HibbettDS(2006)Toward aphylogeneticclassificationoftheLeotiomycetesbasedonrDNAdata. Mycologia98.White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org10January2014|Volume9|Issue1|e86437

PAGE 11

40.GeiserDM,GueidanC,MiadlikowskaJ,LutzoniF,KauffF,etal.(2006) Eurotiomycetes:EurotiomycetidaeandChaetothyriomycetidae.Mycologia98: 1053Ð1064. 41.ShelleyV,KaiserS,WilliamsT,KramerM,HamanK,etal.(2012)Evaluation ofstrategiesforthedecontaminationofequipmentfor Geomycesdestructans ,the causativeagentofWhite-noseSyndrome(WNS).JournalofCaveandKarst Studies75:1Ð10. 42.TeamRC(2012)R:Alanguageandenvironmentforstatisticalcomputing. Vienna,Austria:RFoundationforStatisticalComputing. 43.HsuSC,LockwoodJL(1975)Powderedchitinagarasaselectivemediumfor enumerationofactinomycetesinwaterandsoil.AppliedMicrobiology29:422Ð 426. 44.ChristensenWB(1946)Ureadecompositionasameansofdifferentiating Proteus andparacolonculturesfromeachotherandfrom Salmonella and Shigella types. JournalofBacteriology52:461Ð466. 45.YoonJH,ParkJE,SuhDY,HongSB,KoSJ,etal.(2007)Comparisonofdyes foreasydetectionofextracellularcellulasesinfungi.Mycobiology35:21Ð24. 46.SacziA,RadfordA,ErenlerK(1986)Detectionofcellulolyticfungibyusing Congoredasanindicator:acomparativestudywiththedinitrosalycilicacid reagentmethod.JournalofAppliedBacteriology61. 47.BhullarK,WaglechnerN,PawlowskiA,KotevaK,BanksED,etal.(2012) Antibioticresistanceisprevalentinanisolatedcavemicrobiome.PLoSOne7: e34953. 48.MillerGL(1959)Useofdinitrosalicylicacidreagentfordeterminationof reducingsugar.AnalyticalChemistry31:426Ð428. 49.BowerVE,BatesRG(1955)pHvaluesoftheClarkandLubsbuffersolutionsat 25 u C.JournalofResearchattheNationalBureauofStandards55:197Ð200. 50.ClappCE(2001)Humicsubstances:considerationsofcompositions,aspectsof structure,andenvironmentalinfluences.SoilScience166:723Ð737. 51.Saiz-JiminezC,HermosinB(1999)Thermallyassistedhydrolysisand methylationofdissolvedorganicmatterindrippingwatersfromtheAltamira Cave.JournalofAnalyticalandAppliedPyrolysis49. 52.MudarraM,AndreoB,BakerA(2011)Characterisationofdissolvedorganic matterinkarstspringwatersusingintrinsicfluorescence:Relationshipwith infiltrationprocesses.ScienceoftheTotalEnvironment409:3448Ð3462. 53.WeckerlyFW(2012)Cavecricketexitcounts:environmentalinfluencesand durationofsurveys.JournalofCaveandKarstStudies74:1Ð6. 54.PolseelaR,VittaA,NateeworanantS,ApiwathnasornC(2011)Distributionof cave-dwellingphlebotominesandfliesandtheirnocturnalanddiurnalactivityin PhitsanulokProvince,Thailand.SoutheastAsianJournalofTropicalMedicine andPublicHealth42:1395Ð1404. 55.KelleyRH,JackJD(2002)Leaflitterdecompositioninanephemeralkarstlake (ChaneyLake,Kentucky,U.S.A.).Hydrobiologia482:41Ð47. 56.Chron a «kova «A,Hora «kA,Elhottova «D,Kris tu û fekV(2009)DiverseArchaeal communityofabatguanopileinDomicaCave(SlovakKarst,Slovakia).Folia Microbiologica54:436Ð446. 57.NavarathnaDHMLP,HarrisSD,RobertsDD,NickersonKW(2010) Evolutionaryaspectsofureautilizationbyfungi.FEMSYeastResearch10: 10.1111/j.1567-1364.2009.00602.x. 58.FrickWF,PollockJF,HicksAC,LangwigKE,ReynoldsDS,etal.(2010)An emergingdiseasecausesregionalpopulationcollapseofacommonNorth Americanbatspecies.Science329:679Ð682. 59.DzalY,McGuireLP,VeselkaN,FentonMB(2011)Going,going,gone:the impactofwhite-nosesyndromeonthesummeractivityofthelittlebrownbat ( Myotislucifugus ).BiologyLetters7:392Ð394. 60.SimonKS,PipanT,CulverDC(2007)Aconceptualmodeloftheflowand distributionoforganiccarbonincaves.JournalofCaveandKarstStudies69: 279Ð284. 61.Christen-ZaechS,PatelS,ManciniAJ(2008)Recurrentcutaneous Geomyces pannorum infectioninthreebrotherswithichthyosis.JAmAcadDermatol58: S112Ð113. 62.GianniC,CarettaG,RomanoC(2003)Skininfectiondueto Geomycespannorum var. pannorum .Mycoses46:430Ð432. 63.DillyO,BartschS,RosenbrockP,BuscotF,MunchJC(2001)Shiftsin physiologicalcapabilitiesofthemicrobiotaduringthedecompositionofleaflitter inablackalder( Alnusglutinosa (Gaertn.)L.)forest.SoilBiologyandBiochemistry 333:921Ð930. 64.StehrF,FelkA,Ga «cserA,KretschmarM,Mahn b B,etal.(2004)Expression analysisofthe Candidaalbicans lipasegenefamilyduringexperimentalinfections andinpatientsamples.FEMSYeastResearch4:401Ð408. 65.BhakdiS,Tranum-JensenJ(1991)Alpha-toxinof Staphylococcusaureus. MicrobiologyandMolecularBiologyReviews55:733Ð751. 66.NizetV(2002)Streptococcalbeta-hemolysins:geneticsandroleindisease pathogenesis.TrendsinMicrobiology10:575Ð580. 67.CryanPM,MeteyerCU,BoylesJG,BlehertDS(2010)Wingpathologyof white-nosesyndromeinbatssuggestslife-threateningdisruptionofphysiology. BMCBiology8:doi:10.1186/1741-7007-1188-1135. 68.MobleyHL,HausingerRP(1989)Microbialureases:significance,regulation, andmolecularcharacterization.MicrobiologyandMolecularBiologyReviews 53:85Ð108. 69.TaylorJP,WilsonB,MillsMS,BurnsRG(2002)Comparisonofmicrobial numbersandenzymaticactivitiesinsurfacesoilsandsubsoilsusingvarious techniques.SoilBiologyandBiochemistry34:387Ð401. 70.GhoshS,NavarathnaDHMLP,RobertsDD,CooperJT,AtkinAL,etal.(2009) Arginine-inducedgermtubeformationin Candidaalbicans isessentialforescape frommurinemacrophagelineRAW264.7.InfectionandImmunity77:1596Ð 1605. 71.LeeIR,MorrowCA,FraserJA(2013)Nitrogenregulationofvirulencein clinicallyprevalentfungalpathogens.FEMSMicrobiologyLetters345:77Ð84. 72.Mirbod-DonovanF,SchallerR,HungCY,XueJ,ReichardU,etal.(2006) Ureaseproducedby Coccidioidesposadii contributestothevirulenceofthis respiratorypathogen.InfectionandImmunity74:504Ð515. 73.ColeGT(1997)Ammoniaproductionby Coccidioidesimmitis anditspossible significancetothehost-fungusinterplay.In:BosscheH,StevensDA,OddsFC, editors.Host-FungusInterplay.NewYork,NY:PlenumPress.247Ð263. 74.OlszewskiMA,NoverrMC,ChenGH,ToewsGB,CoxGM,etal.(2004) Ureaseexpressionby Cryptococcusneoformans promotesmicrovascularsequestration,therebyenhancingcentralnervoussysteminvasion.AmericanJournalof Pathology164:1761Ð1771. 75.AnbuP,HildaA,GopinathSC(2004)Keratinophilicfungiofpoultryfarmand featherdumpingsoilinTamilNadu,India.Mycopathologia158:303Ð309. 76.KochkinaGA,IvanushkinaNE,AkimovVN,GilichinskiiDA,OzerskayaSM (2007)Halo-andpsychrotolerant Geomyces fungifromarcticcryopegsandmarine deposits.Microbiology76:39Ð47. 77.MarshallWA(1998)Aerialtransportofkeratinaceoussubstrateanddistribution ofthefungus Geomycespannorum inAntarcticsoils.MicrobialEcology36:212Ð 219. 78.RiceAV,CurrahRS(2006)Twonewspeciesof Pseudogymnoascus with Geomyces anamorphsandtheirphylogeneticrelationshipwith Gymnostellatospora. Mycologia 98:307Ð318. 79.FeniceM,SelbmannL,ZucconiL,OnofriS(1997)Productionofextracellular enzymesbyAntarcticfungalstrains.PolarBiology17:275Ð280. 80.DuncanSM,MinasakiR,FarrellR,ThwaitesJM,HeldBW,etal.(2008) Screeningfungiisolatedfromhistoric Discovery HutonRossIsland,Antarctica forcellulosedegradation.AntarcticScience205. 81.RosaLH,LAVMd,SantiagoIF,RosaCA(2010)Endophyticfungicommunity associatedwiththedicotyledonousplant Colobanthusquitensis (Kunth)Bartl. (Caryophyllaceae)inAntarctica.FEMSMicrobiologyEcology73:178Ð189. 82.Vohnõ « kM,FendrychM,Albrechtova «J,Vosa «tkaM(2007)Intracellular colonizationof Rhododendron and Vaccinium rootsby Cenococcumgeophilum , Geomyces pannorum and Meliniomycesvariabilis. FoliaMicrobiologica52:407Ð414.White-NoseSyndromeEnzymeActivity PLOSONE|www.plosone.org11January2014|Volume9|Issue1|e86437


printinsert_linkshareget_appmore_horiz

Download Options

close


  • info Info

    There are only PDFs associated with this resource.

  • link PDF(s)



Cite this item close

APA

Cras ut cursus ante, a fringilla nunc. Mauris lorem nunc, cursus sit amet enim ac, vehicula vestibulum mi. Mauris viverra nisl vel enim faucibus porta. Praesent sit amet ornare diam, non finibus nulla.

MLA

Cras efficitur magna et sapien varius, luctus ullamcorper dolor convallis. Orci varius natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Fusce sit amet justo ut erat laoreet congue sed a ante.

CHICAGO

Phasellus ornare in augue eu imperdiet. Donec malesuada sapien ante, at vehicula orci tempor molestie. Proin vitae urna elit. Pellentesque vitae nisi et diam euismod malesuada aliquet non erat.

WIKIPEDIA

Nunc fringilla dolor ut dictum placerat. Proin ac neque rutrum, consectetur ligula id, laoreet ligula. Nulla lorem massa, consectetur vitae consequat in, lobortis at dolor. Nunc sed leo odio.