Rapamycin Exerts Antifungal Activity In Vitro and In Vivo - Ec Asm

12 Pages·2012·1.6 MB·English

Rapamycin Exerts Antifungal Activity In Vitro and In Vivo against
Mucor circinelloides via FKBP12-Dependent Inhibition of Tor
RobertJ.Bastidas,aCeceliaA.Shertz,aSooChanLee,aJosephHeitman,a,b,candMariaE.Cardenasa
DepartmentsofMolecularGeneticsandMicrobiology,aMedicine,bandPharmacologyandCancerBiology,cDukeUniversityMedicalCenter,Durham,NorthCarolina,USA
ThezygomyceteMucorcircinelloidesisanopportunisticfungalpathogenthatcommonlyinfectspatientswithmalignancies,
diabetesmellitus,andsolidorgantransplants.Despitethewidespreaduseofantifungaltherapyinthemanagementofzygomy-
cosis,theincidenceofinfectionscontinuestoriseamongimmunocompromisedindividuals.Inthisstudy,weestablishedthat
thetargetandmechanismofantifungalactionoftheimmunosuppressantrapamycininM.circinelloidesaremediatedviacon-
servedcomplexeswithFKBP12andaTorhomolog.Wefoundthatspontaneousmutationsthatdisruptedconservedresiduesin
FKBP12conferredrapamycinandFK506resistance.DisruptionoftheFKBP12-encodinggene,fkbA,alsoconferredrapamycin D
o
andFK506resistance.ExpressionofM.circinelloidesFKBP12(McFKBP12)complementedaSaccharomycescerevisiaemutant w
strainlackingFKBP12torestorerapamycinsensitivity.ExpressionoftheMcTorFKBP12-rapamycinbinding(FRB)domain n
conferredrapamycinresistanceinS.cerevisiae,andMcFKBP12interactedinarapamycin-dependentfashionwiththeMcTor lo
a
FRBdomaininayeasttwo-hybridassay,validatingMcFKBP12andMcTorasconservedtargetsofrapamycin.Weshowedthatin d
e
vitro,rapamycinexhibitedpotentgrowthinhibitoryactivityagainstM.circinelloides.InaGalleriamellonellamodelofsystemic d

mucormycosis,rapamycinimprovedsurvivalby50%,suggestingthatrapamycinandnonimmunosuppressiveanalogshavethe f
r
o
potentialtobedevelopedasnovelantifungaltherapiesfortreatmentofpatientswithmucormycosis. m

h
t
Comparativegenomicstostudycloselyrelatedorganismshas manfungalpathogens,suchasCryptococcusneoformans,Aspergil- tp
:
/
emergedasanimportanttoolforunderstandingphenotypic lusfumigatus,Fusariumoxysporum,andseveralpathogenicPeni- /e
differences,suchaspathogenicity,andallowsforidentificationof cillium species (13, 58), and it was later found to have potent c
.
a
conservedmolecularpathwaysthatcanbetargetedinthedevel- immunosuppressiveactivity(35).Inyeastandmammaliancells, s
opmentofbroad-spectrumantimicrobialdrugs.Conservedpro- rapamycin inhibits Tor through its association with the prolyl m
teinkinasescontrollinggrowthandproliferationoffungalpatho- isomeraseFKBP12,formingabinarycomplexthatbindstothe .o
r
gensrepresentattractivedrugtargets,bothbecausetheyarelikely highly conserved FRB (FKBP12-rapamycin binding) domain of g
/
tobeessentialforfungalpropagationanddevelopmentandbe- Tor.Thismechanismofactionissupportedbytheidentification o
causetheseenzymesarelikelytobeconservedamongfungalspe- n
ofmutationsintheFRBdomainthatconferrapamycinresistance
cies, allowing the use of inhibitors of these kinases as broad- A
byblockingFKBP12-rapamycinbindingtoTorandbystructural p
spectrumantimicrobialtherapeuticagents. r
Among the repertoire of conserved protein kinases essential sbtiutidoinesodfeTfionrin(5g,t1h1e,m23o,le3c4u,l5a2r,d5e9ta).ilsofFKBP12-rapamycininhi- il 1
forcellularphysiology,theTorproteinkinaseisthefocusofin- 4
FKBP12 catalyzes cis-trans peptidyl-prolyl isomerization, a ,
tenseinvestigation,givenitsroleasacentralelementofnutrient- 2
rate-limiting step in protein folding (reviewed in reference 27). 0
regulatedcellgrowthpathways.InSaccharomycescerevisiae,Tor 1
Remarkably,FKBP12alsoservesasthereceptorfortheantimicro-
inhibitionbythenaturalproductrapamycinelicitsmanyofthe 9
bial and immunosuppressive drug FK506. Both rapamycin and b
cellularresponsesthataretriggeredbynutrientstarvation,suchas
y
inhibition of protein synthesis, downregulation of amino acid FK506 bind to the active site of FKBP12 and inhibit its prolyl g
permeases,proteindegradation,autophagy,andcellcyclearrest isomeraseactivity.ThetargetofFKBP12-FK506iscalcineurin,a u
Ca2(cid:1)-calmodulin-regulated serine-threonine-specific protein e
(reviewedinreference45).Theseeffectsaremediatedlargelyby s
phosphataseconsistingofacatalyticA(CnA)andaregulatoryB t
theactivityofTorinpromotingtheexpressionofgenesencoding
tRNAs, ribosomal proteins, rRNAs, and amino acid permeases (CnB)subunit(32).Inhumans,calcineurinregulatesnuclearlo-
andsuppressingnitrogencataboliterepressionandtheaminoacid calizationofthetranscriptionfactorNFATduringtheresponseto
generalcontrolresponse(4,6,10,22,29,41).InS.cerevisiae,two antigen presentation (32). In S. cerevisiae, calcineurin regulates
Torproteins,Tor1andTor2,formtwodistinctmultiproteincom- cationhomeostasisandcellintegrityviathetranscriptionfactor
plexes, named TORC1 and TORC2 (33, 57). Collectively, these Crz1(46).InC.neoformans,mutantslackingcalcineurinarevia-
complexescontrolproteinsynthesis,mRNAsynthesisanddegra-
dation, ribosome biogenesis, nutrient transport, and autophagy
(TORC1), as well as actin polarization and cell wall integrity Received2November2011 Accepted22December2011
(TORC2)(reviewedinreference60). Publishedaheadofprint30December2011
TheTorkinasehasalsoreceivedwideattentionasanantifun- AddresscorrespondencetoMariaE.Cardenas,[email protected].
galtargetduetoitsinhibitionbythenaturalproductrapamycin. Supplementalmaterialforthisarticlemaybefoundathttp://ec.asm.org/.
Rapamycin was first identified as an antimicrobial with potent Copyright©2012,AmericanSocietyforMicrobiology.AllRightsReserved.
activityagainstCandidaalbicans(1,56).Subsequently,rapamycin doi:10.1128/EC.05284-11
wasshowntohaverobustantifungalactivityagainstseveralhu-
270 ec.asm.org 1535-9778/12/$12.00 EukaryoticCell p.270–281
RapamycinActivityagainstMucorcircinelloides
TABLE1Strainsusedinthisstudy
Strain Genotype Referenceorsourcea
Phycomycesblakesleeanusstrain
NRRL1555((cid:3)) Wildtype SantiagoTorres-Martinez
Rhizopusoryzaestrain
FGSC9543 Wildtype SantiagoTorres-Martinez
Mucorcircinelloidesf.lusitanicusstrainsb
R7B((cid:3)) leuA(cid:3)((cid:3)) SantiagoTorres-Martinez
NRRL3631((cid:1)) Wildtype((cid:1)) SantiagoTorres-Martinez
SM2 NRRL3631((cid:1))fkbA-1(A316G) Thisstudy
SM4 R7B((cid:3))fkbA-2(L91P) Thisstudy
MU402 R7B((cid:3))pyrG(cid:3) 36
RBM1 MU402fkbA::pyrG Thisstudy
RBM2 MU402fkbA::pyrG Thisstudy D
o
Saccharomycescerevisiaestrains w
JK9-3da MATahis4HMLaleu2-3,112rme1trp1ura3-52 23 n
JHY3-3B JK9-3dafpr1::URA3 23 lo
a
CHY251 MATahis3(cid:4)200leu2-3,112trp1(cid:4)101ura3-52vph6(cid:4)::TRP1 25 d
CHY516 MATahis3(cid:4)200leu2-3,112trp1(cid:4)101ura3-52vph6(cid:4)::TRP1fpr1::ADE2 25 e
d
SMY4-1 MATatrp1-901his3leu2-3,112ura3-52ade2gal4gal80URA3::GAL-lacZ 34
f
LYS2::GAL-HIS3TOR1-3fpr1::ADE2 ro
m
aTheindicatedstrainswerekindlyprovidedbySantiagoTorres-Martinez,DepartamentodeGenéticayMicrobiología,FacultaddeBiología,UniversidaddeMurcia,Murcia,Spain.
bMatingtypeforMucorstrainsisindicatedby“(cid:3)”or“(cid:1)”. h
t
t
p
:
/
/
bleat24°Cbutinviableat37°C,andtheyareavirulentinanimal circinelloides,acommonetiologicalagentofhumanzygomycosis. e
c
modelsofcryptococcosis(18,39). We found that rapamycin’s antifungal action is mediated via .
a
Whilethemechanismofrapamycinactionhasbeenconserved FKBP12-rapamycininhibitionofTor,basedontheisolationand s
m
inbothascomyceteandbasidiomycetefungalspecies(3,13,15), characterization of spontaneous FKBP12 mutations that confer
.
littleisknownaboutitsactivityinbasalfungallineages,suchasthe resistance to rapamycin. We also demonstrate that M. circinel- o
r
zygomycetes.OnegroupwithinthezygomycetesistheorderMu- loidesTor(McTor)andFKBP12interactinthepresenceofrapa- g
/

corales, which arose early during fungal radiation and has mycin and that disruption of the FKBP12-encoding gene, fkbA, o
n
emerged as an increasingly important cause of infection in hu- confers rapamycin and FK506 resistance. Finally, we show that
A
mans.ThemajorityofzygomyceteinfectionsarecausedbyRhi- rapamycintreatmentimprovessurvivaloftheinvertebrateGalle- p
zopus,Mucor,Rhizomucor,Cunninghamella,andAbsidiaspecies riamellonellainamodelofsystemicmucormycosis,suggestinga ril
andareprevalentamongpatientswithdiabetesmellitusandneu- therapeuticeffectofrapamycin.Insummary,thisstudydemon- 1
4
tropeniaandalsoamongorganandhematopoieticstemcelltrans- stratesthehighconservationofrapamycinactionamongfungal ,

plantrecipients(43,50).Mortalityratesofzygomycosis(alsore- pathogens, providing insights for novel therapeutic vantage 2
0
ferredtoasmucormycosis)canexceed65%and90%,especially points. 1
9
amongtransplantpatients,withanestimatedannualincidenceof
MATERIALSANDMETHODS b
1.7infectionspermillionindividualsintheUnitedStatesalone y
(42,51).Infectionsareacquiredbyinhalationofinfectiousspores Strains,media,drugs,andgrowthconditions.Strainsusedinthisstudy g
arelistedinTable1.Strainsweregrownat30°Corroomtemperatureon u
fromsoilordustand,lessfrequently,throughbreachesorinjuries e
totheskin.Clinicalmanifestationsofzygomycosisincludesevere YPD (1% yeast extract, 2% Bacto peptone, and 2% dextrose), potato s
dextroseagar(PDA),andRPMI(RPMI1640liquidmediumwithglu- t
necrosisofnasal,facial,andsubcutaneoustissues,aswellasrhi-
tamineandwithoutsodiumbicarbonate[Gibco],supplementedwith2%
nocerebral,pulmonary,anddisseminateddisease(44).
dextroseandbufferedwith0.165MMOPS[morpholinepropanesulfonic
Treatment of zygomycosis relies on early detection of infec- acid][pH7])media.Strainsweregrowneitheronsolidmediacontaining
tion,surgicalremoval(debridement)ofnecrotictissue,andag- 2%agarorinliquidcultures.Rapamycin(LCLaboratories)wasdissolved
gressiveantifungaltherapy.Primaryantifungaltherapyhasrelied in90%ethanol-10%Tween20.FK506(Prograph)wasobtainedfromthe
mostlyonmonotherapywithpolyenesandlipidformulationsof DukeUniversityMedicalCenterpharmacy.RapamycinandFK506stock
amphotericin B. Whereas most azoles, including fluconazole, solutionswereaddedtomediaattheindicatedconcentrations.
voriconazole,anditraconazole,exhibitunreliableactivityagainst Antifungaldrugtesting.Druginteractionswereassessedbyacheck-
erboardtitrationfollowingtheguidelinesestablishedbytheClinicaland
zygomycosis,posaconazolehasemergedasanoptionforsalvage
LaboratoryStandardsInstitute(CLSI)(11a)forantifungalsusceptibility
therapyinpatientswhoarerefractorytopolyenetreatment(44,
testingofmolds.InvitrotestingwasperformedinRPMI1640medium.
51, 53). Despite aggressive surgical and antifungal therapies for Aliquotsof50(cid:1)lofeachdrugata4(cid:2)concentrationweredispensedto
treatmentofzygomycosis,themortalityrateassociatedwithin-
wellsofa96-wellmicrotiterplatetoprovide77drugcombinations.Ad-
fectionsbythesepathogenshasremainedhigh,warrantingnovel ditionalrowswereusedtodeterminetheMICofeachagentaloneandfor
strategiesforthetreatmentofzygomycosis. thegrowthcontrolwell(drug-free).Finaldrugconcentrationstestedwere
HereweidentifytheFKBP12andTorhomologsfromMucor asfollows:rapamycin,200(cid:1)g/mlto3.12(cid:1)g/ml(6dilutions);andFK506,
March2012 Volume11 Number3 ec.asm.org 271
Bastidasetal.
25.0(cid:1)g/mlto0.1(cid:1)g/ml(9dilutions).Toeachwell,aninoculumof5(cid:2) homokaryoticmycelialgrowthwasobserved.Sporesuspensionswerecol-
104sporangiospores/mlwasadded,andmicrotiterplateswereincubated lectedandtestedforrapamycinresistanceonYPDsolidmediumcontain-
at35°Cwithoutshakingfor24h.Growthinhibitiondeterminationswere ing100(cid:1)g/mlrapamycin.TotalDNAwasisolatedfromM.circinelloides
performedwiththeaidofaconcavemirror.TheMICandMIC ofdrugs myceliagerminatedinliquidYPDmediumat30°Cbyharvestingmycelia
80
weredefinedasthelowestdrugconcentrationsinthewellsproducinga overMiracloth(Calbiochem,LaJolla,CA)andvacuumdryingmyceliaby
completeabsenceofvisualgrowthandan80%reductionofgrowth,re- lyophilization.Driedmyceliaweretrituratedwithglassbeadsbymanual
spectively. agitationandresuspendedin1mlCTABextractionbuffer(100mMTris-
Identification of M. circinelloides FKBP and Tor homologs. M. HCl[pH8.4],1.4MNaCl,25mMEDTA,2%cetyltrimethylammonium
circinelloidesFK506bindingproteinswereidentifiedbysearchingtheMu- bromide[CTAB]).Astandardphenol-chloroformextractionprocedure
corcircinelloidesCBS277.49genomedatabase(http://genome.jgi-psf.org wasused,followedbyDNAprecipitationusinga1/10volumeof3M
/Mucci2/Mucci2.home.html) for genes encoding proteins containing sodium acetate and 2 volumes of 100% ethanol. The DNA pellet was
predicted FK506 binding domains with high homology to the FK506 resuspendedin1mlofTris-EDTAbuffer(10mMTris-HCl,1mMEDTA,
bindingdomainofS.cerevisiaeFKBP12(ScFKBP12;encodedbyFPR1) pH7.0).GenomicDNAwasusedtoamplify700-bpfragmentsbyPCR,
(seeFig.2).PercentidentityandsimilarityweredeterminedbyBLAST including1-kbsequencesupstreamanddownstreamofthefkbAcoding
protein sequence alignments utilizing bl2seq from NCBI. Protein do- sequence,andeachfragmentwassequenced.DNAanalysiswascompared
mainswereidentifiedusingthePrositedatabaseofproteindomains,fam- bygenomicDNABLASTsearchesagainsttheM.circinelloidesgenomic
D
ilies,andfunctionalsites(http://prosite.expasy.org/).TheTorhomolog databasesequences.
o
wasidentifiedbysearchingtheMucorcircinelloidesCBS277.49genome Geneexpressionanalyses.M.circinelloidesculturesweregrownin w
databaseforproteinscontainingaTorFRBdomainwithhighhomology liquidYPDmediumat30°C,andmyceliumpowderextractswerepre- n
totheS.cerevisiaeTorFRBdomain.Phylogeneticreconstructionswere paredasdescribedabove.TotalRNAwasisolatedusingTRIzolreagent lo
a
performedwiththeGeneious(Biomatters)platformforsequencealign- (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions, d
ment,utilizingtheBlosum62costmatrixandtheJukes-Cantormethod and30(cid:1)goftotalRNAwasloadedontoa1%formaldehydeagarosegel. e
d
forgeneticdistancemodeling. Followingtransfer,membraneswerehybridizedtoradioactivefkbAand
f
Plasmidconstruction.ThefkbAopenreadingframewasPCRampli- actADNA-basedprobes.Hybridizedprobesignalsweredetectedusinga ro
fiedfromanR7BcDNAlibrarybyuseofprimerscontainingahemagglu- phosphorimager.cDNAwassynthesizedfrom1.5(cid:1)goftotalRNAbyuse m
tinin(HA)epitopesequence(directlyupstreamofthefkbAcodingse- ofanAffinityScriptmultiple-temperaturereversetranscriptasekit(Strat- h
quence)andPstIandSalIrestrictionsites.Theampliconwasdigestedwith agene). tt
p
PstI and SalI and cloned into PstI/SalI-digested pCu415CUP1 (30) to Westernblotanalyses.M.circinelloidesproteinextractsweregener- :
/
generateplasmidpCu415-HA-FKBP12.TheFRBdomain-encodingre- atedfromculturesgrowninYPDmediumat30°Cbyharvestingmycelia /e
gionoftorAwasamplifiedfromR7BgenomicDNAbyuseofprimers overMiracloth(Calbiochem,LaJolla,CA)andvacuumdryingmyceliaby c
.
containingaMycepitopesequence(directlyupstreamoftheFRBdomain lyophilization.Driedmyceliaweretrituratedwithglassbeadsbymanual a
s
sequence)andEcoRIandPstIrestrictionsites.Theampliconwasfirst agitation,and20mgwasresuspendedin200(cid:1)lofsamplebuffer(1% m
digested with EcoRI, blunted with T4 DNA polymerase, and digested SDS, 9 M urea, 25 mM Tris-HCl, pH 6.8, 1 mM EDTA, 0.7 M beta- .o
with PstI. The digested amplicon was cloned into SmaI/PstI-digested mercaptoethanol) supplemented with phenylmethylsulfonyl fluoride r
g
pCu414CUP1(30)togenerateplasmidpCu414-Myc-FRB.Theyeasttwo- (PMSF)andtheindicatedconcentrationofRocheCompleteEDTA-free /

o
hybridconstructpGBKT7-FRBwasgeneratedbyfirstamplifyingtheFRB proteaseinhibitorcocktail.Sampleswereboiledfor2min,vortexedfor1
n
domain sequence of torA from R7B genomic DNA by use of primers min,andboiledagainfor1min.Anadditionalcentrifugationstepwas A
containing NcoI and BamHI restriction sites. The amplicon was then carriedoutfor15minat16,000rpm,andsupernatantswerecollected.For p
digestedwithNcoIandBamHIandligatedinframewiththeGAL4DNA Westernblotting,0.3mgoftotalproteinextractwasloadedona4to20% ril
binding domain (BD) into NcoI/BamHI-digested pGBKT7 (Clontech SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) 1
4
LaboratoriesInc.).ThepGADT7-FKBP12plasmidwasgeneratedbyam- membrane(AmershamBiosciences,Piscataway,NJ).Blotswereblocked
,

plifyingthefkbAcodingsequencefrompCu415-HA-FKBP12byuseof with5%nonfatmilk-0.05%Tween20inTris-bufferedsaline,andmem- 2
0
primerscontaining30bpofpGADT7ADsequence(ClontechLaborato- braneswereincubatedovernightat4°Cwitha1:1,000dilutionofantise- 1
riesInc.)directlyupstreamoftheEcoRIsite(includingtheEcoRIsite)and rum against ScFKBP12, raised in rabbits (7). Horseradish peroxidase 9

30bpofpGADT7ADsequencedirectlydownstreamoftheBamHIsite (HRP)-conjugatedanti-rabbitIgGantibodywasusedasthesecondary b
y
(including the BamHI site). The amplicon was cotransformed with antibody,atadilutionof1:2,000.Equalloadingofsampleswasmonitored
g
EcoRI-digestedpGADT7ADintoS.cerevisiaestrainSMY4-1(Table1)by bystrippingandincubatingmembranesfor1hwitha1:2,000dilutionof u
astandardlithiumacetatetransformationmethod.Strainsinwhichthe aratanti-alpha-tubulinantibody(SantaCruzBiotechnology,SantaCruz, e
s
fkbAcodingsequencewasclonedinframewiththeGAL4activationdo- CA).HRP-conjugatedanti-ratIgGantibodywasusedasthesecondary t
main(AD)inpGADT7ADbyhomologousrecombinationwereselected antibody, at a 1:2,000 dilution. Proteins were visualized by using ECL
oncompletesyntheticmediumlackingleucine(CSM(cid:3)Leu).Thepres- Westernblottingsubstrate(PierceBiotechnology,Rockford,IL)follow-
enceoftheclonedfkbAcodingsequencewasconfirmedbycolonyPCR. ingthemanufacturer’sprotocol.
TotalgenomicDNAwasextractedfrompositivetransformants,and2(cid:1)l S.cerevisiaetotalproteinextractswerepreparedbydisruptingcells
ofthisDNAwastransformedintoEscherichiacoliDH5(cid:2)cells(Invitrogen withglassbeadsinlysisbuffer(20mMTris-HCl,pH7.5,100mMKCl,0.1
Corporation)torecoverpGADT7-FKBP12plasmids. mMEDTA,100(cid:1)MNa VO,25mMbeta-glycerophosphate,25mMNaF)
3
Yeasttwo-hybridassay.Thetwo-hybridstrainSMY4-1(Table1)was supplemented with PMSF and the indicated concentration of Roche
cotransformedwiththetwo-hybridfusionplasmidspGBKT7-FRBand CompleteEDTA-freeproteaseinhibitorcocktail.Fiftymicrogramsofto-
pGADT7-FKBP12,cellsweregrownonCSM(cid:3)Leu(cid:3)Trp,and(cid:3)-galacto- talproteinextractwasusedforWesternblottingasdescribedabove.
sidaseactivitywasassayedusingthechlorophenol-(cid:3)-D-galactopyranoside DisruptionoftheM.circinelloidesfkbAgene.AnR7BfkbAdisrup-
(CPRG)substrateaspreviouslydescribed(8). tioncassettewasgeneratedbyoverlapPCRamplificationofanamplicon
Molecular analysis of spontaneously drug-resistant M. circinel- containing1,000bpoffkbAgenomicDNAsequencedirectlyupstreamof
loides isolates. Spontaneously FK506 and rapamycin cross-resistant theATGcodon,withanaddedXbaIrestrictionsite;a2,000-bppyrGgene
strainswereisolatedbyspreading500M.circinelloidessporangiosporeson amplicon;andanampliconwith1,000bpoffkbAgenomicDNAsequence
YPD solid medium containing 1 (cid:1)g/ml FK506 and passaging FK506- directlydownstreamofthefkbAcodingsequencestopcodon,withan
resistantmycelialoutgrowthsonFK506-containingsolidmediumuntil addedXmaIrestrictionsite.TheresultingfkbA::pyrGdisruptioncassette
272 ec.asm.org EukaryoticCell
RapamycinActivityagainstMucorcircinelloides
TABLE2Radialcolonydiametersofzygomycetespeciesgrowninthe
presenceofYPD,rapamycin,andFK506a
Mean(SE)colonydiam(cm)b
YPD(cid:1)
Strain YPD rapamycin YPD(cid:1)FK506
MucorcircinelloidesR7B 6.8(0.065) 5.0(0.061) 0.4(0.053)
Mucorcircinelloides 7.2(0.077) 4.9(0.080) 0.6(0.042)
NRRL3631
Rhizopusoryzae 8(0) 3.7(0.086) 0.5(0.030)
FGSC9543
Phycomycesblakesleeanus 8(0) 4.0(0.061) 0(0)
NRRL1555
aSpores(500sporesperplate)fromrepresentativezygomycetespeciesweregerminated
onYPDagarcontainingeither100nMrapamycinor1(cid:1)g/mlFK506.Sporeswere
germinatedfor72hatroomtemperature,andcolonydiametersweremeasured.
bStandarderrorswerecalculatedfor8replicates. D
o
w
n
mus) and FK506 (tacrolimus) have been established for several lo
a
ascomycetous and basidiomycetous fungal pathogens, little is d
known about the activities of these antifungal agents and their e
d
FIG1Growthofrepresentativezygomycetespeciesiscompromisedwhen mechanismsofactioninbasalfungallineages,whichincludesev- f
organismsareexposedtorapamycinandFK506.Spores(500sporesperplate) r
eralfungalpathogensofclinicalandenvironmentalimportance. o
fromrepresentativezygomycetespeciesweregerminatedonYPDagarcon- m
tainingeither100nMrapamycinor1(cid:1)g/mlFK506.Sporesweregerminated Tofurtherassesstheactivitiesofthesenaturalproductsamong
for72hatroomtemperature. basalfungalspecies,growthassaysofthezygomycetespeciesM. h
t
t
circinelloides,Rhizopusoryzae,andPhycomycesblakesleeanuswere p
:
performedinthepresenceofbothrapamycinandFK506.Growth //
wasdigestedwithXbaIandXmaIandligatedintopJAF12,yieldingplas- ofM.circinelloidesstrainsR7B((cid:3)matingtype)andNRRL3631 ec
mid pJAF12/fkbA::pyrG. The disruption plasmid was linearized with ((cid:1)matingtype)andofR.oryzaeandP.blakesleeanusstrainswas .a
XhoIanddephosphorylatedwithcalfintestinalphosphatase(CIP;New s
EnglandBioLabs),and50(cid:1)gofdigestedplasmidwastransformedinto evaluated on rich agar media containing rapamycin or FK506. m
twoindependentMU402(R7BpyrG(cid:3)strain)(Table1)cultures.Trans- Whileallfourstrainsexhibitedreducedradialmycelialgrowthin .o
formationwasperformedasdescribedinreference55,withamodified thepresenceofrapamycin,P.blakesleeanusgrowthexhibitedthe rg
protoplastpreparationstepasdescribedinreference37,inwhich2.5(cid:2) highest sensitivity to rapamycin, with a clear reduction in both /
o
108germinatedsporeswereincubatedwith5mg/mlchitosanase(molec- radialmycelialgrowthandapicalsporangiophoreformation(Fig. n
ulargrade;U.S.BiologicalsRD)and0.5mg/mllysingenzymes(Sigma)for 1andTable2).Asobservedwithrapamycin,FK506stronglyin- A
90minat30°C.fkbAdisruptionwasverifiedbyPCR(seeFig.6B)utilizing hibited the growth of all zygomycete species tested (Fig. 1 and p
r
primersP1(JOHE20967;5=-GAAAATAAGAAAAGAATAAATTAGATT Table2). il
AC-3=),P2(JOHE20968;5=-AAGCACCTATTATATGGAGATAGAAC- 1
In addition, we also determined the rapamycin and FK506 4
3=),P3(JOHE20965;5=-AAAGTATATTAAAGGCAATAAAGTACAAT- ,
MICs for inhibition of growth of all three zygomycete species,
3=),andP4(JOHE20966;5=-AATAGACAATAATTCTTTCTTTGTAGT 2
followingCLSI’sguidelinesforantifungalsusceptibilitytestingof 0
AAC-3=)andbySouthernblotanalysis(datanotshown). 1
Galleriamellonellamodelofsystemiczygomycosis.M.circinelloides molds by broth microdilution (11a). We found that rapamycin 9
inoculumswerepreparedinphosphate-bufferedsaline(PBS)bysuspend- inhibitedupto80%ofgrowthatconcentrationsabove6.26(cid:1)g/ b
ingsporangiosporesharvestedfrommyceliallawnsgrownonPDAfor5 ml,12.5(cid:1)g/ml,and100(cid:1)g/mlforP.blakesleeanus,R.oryzae,and y
g
daysatroomtemperature,andsporetitersweredeterminedbyhemocy- M.circinelloides,respectively(Table3).Asexpected,P.blakesleea- u
tometercounting.Drugstockswerepreparedbydilutionin90%ethanol- nusgrowthinhibitionrequiredlowerconcentrationsofrapamy- e
s
10%Tween20.Measurementsofthehemolymphvolumeof20larvaeof cin, while surprisingly, M. circinelloides growth inhibition re- t
variousweightsanddeterminationofthemeanhemolymphvolume/kgof quired high concentrations of rapamycin during liquid culture
bodyweightbylinearregressionanalysiswereusedtodeterminethedrug
growth(Table3).Interestingly,growthofR.oryzaeexhibitedthe
dosesadministered.LarvaeinthefinalinstarwereobtainedfromVander-
leastsensitivitytoFK506onagarmediumcontainingFK506(Fig.
host,Inc.Tenlarvae(300mg(cid:5)25mg)wereusedpergroup.Eachlarva
wasinjectedwith5(cid:1)lofPBScontaining500sporangiospores,PBSonly,
drugs,orvehicleintothehemocoelviaaproleg.At4hpostinfection,5(cid:1)l
ofPBScontainingdrugsorvehiclecontrolwasinjectedintoeachlarvavia TABLE3RapamycinandFK506MIC80sduringgrowthofzygomycete
aseparateprolegfromtheoneusedduringinfection.Larvaewereincu- species
batedatroomtemperature,andthenumberofdeadlarvaewasscored MIC ((cid:1)g/ml)a
80
daily.Killcurveswereplotted,anddifferencesinsurvivalrates(logrank
Strain Rapamycin FK506
test) were determined by the Kaplan-Meier method, using GraphPad
Prismsoftware. MucorcircinelloidesR7B((cid:3)) 100–200 0.8–1.6
MucorcircinelloidesNRRL3631((cid:1)) 100–200 0.4–0.8
RESULTS RhizopusoryzaeFGSC9543 12.5–25 0.2–0.4
Growth of representative zygomycete species is sensitive to PhycomycesblakesleeanusNRRL1555 6.3–12.5 0.2–0.4
rapamycin.Whilethemechanismsofactionofrapamycin(siroli- aDrugdilutionstestedrangedfrom200to0.09(cid:1)g/ml.
March2012 Volume11 Number3 ec.asm.org 273
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FIG2M.circinelloidesfamilyofFK506bindingproteins.FamilymemberswereidentifiedbasedontheirproteinsequencehomologytotheFK506binding c
domainofS.cerevisiaeFKBP12.IdentityandsimilarityscorescorrespondtosimilaritiesbetweentheproteinsequenceoftheFK506bindingdomainofeachM. .a
circinelloidesFKBPfamilymemberandthecorrespondingdomainofS.cerevisiaeFKBP12.ThenomenclatureforeachFKBP(FK506bindingprotein)family s
m
memberisbasedonitspredictedmolecularmass(denotedinaparenthesisbeloweachhomolog).ProteinIDsarefromtheMucorcircinelloidesCBS277.49v2.0
genomedatabase,hostedattheJointGenomeInstitute(JGI)website.TPR,tetratricopeptiderepeat. .o
r
g
/

o
n
1),whileitexhibitedahighersensitivitytoFK506thanthoseofthe theM.circinelloidesgenomeidentifiedafamilyof5genesencod-
A
MucorandP.blakesleeanusstrainsinabrothmicrodilutionassay ing 5 putative proteins with closely related FK506 binding do- p
(Table 3). While the source of these discrepancies remains un- mains. We named these genes fkbA, fkbB, fkbC, fkbD, and fkbE ril
known,theyarelikelyaresultofdifferencesinR.oryzaegrowth (FK506binding),andbasedonthepredictedmolecularweightsof 1
4
properties on semisolid surfaces and liquid medium. Neverthe- the proteins they encode, we named their products FKBP12, ,

less,bothassaysclearlyindicatethatR.oryzaeishighlysusceptible FKBP19, FKBP22, FKBP30, and FKBP42, respectively (Fig. 2). 2
0
to FK506. In contrast to the fungicidal activity of rapamycin FK506bindingproteinswithvariousmolecularweightsareubiq- 1
9
against the pathogens C. albicans and C. neoformans (MICs of uitous in nature, and members of the FKBP family have been
(cid:6)0.09 (cid:1)g/ml and (cid:7)0.19 (cid:1)g/ml, respectively [15]), zygomycete identifiedinmultipleorganisms,includinghumans,plants,and by

growthwasnotentirelyinhibitedbyrapamycin,andinhibition fungi (40). FKBP12 family members contain only one FK506 g
u
requiredsignificantlyhigherconcentrationsofdrug,whichcould bindingdomain,whileFKBPswithhighmolecularweightspos- e
beduepartlytodecreasedpermeabilityofzygomycetecellwalls sessextradomains,suchastetratricopeptiderepeatdomainsand s
t
and/ormembranestorapamycinorhigher-affinitydrugpumps calmodulin binding and transmembrane motifs (20). Based on
ortoocclusionoftheFRBdomaininzygomyceteTorhomologs. the12-kDapredictedmolecularmassoftheM.circinelloidespu-
Nevertheless,theeffectofrapamycinonthegrowthofthetested tativehomologandonphylogeneticanalysesoftheMcFKBPfam-
zygomycetespeciesprovidesevidencethatintheseorganisms,Tor ilyofproteinsandseveralfungalFKBP12homologs(Fig.3),we
alsofunctionstocontrolgrowth.TheMIC sofFK506weresig- identifiedfkbAasthegeneencodingtheMcFKBP12homolog.
80
nificantlylowerthanthoseobservedforrapamycin(Table3),in TotestwhethertheproductoffkbAfunctionsastherapamy-
agreement with the phenotypes observed during growth on cin/FK506receptorinM.circinelloides,weperformedfunctional
FK506-containing agar medium. These results illustrate that complementation studies using S. cerevisiae as a heterologous
FK506exhibitspotentantifungalactivityagainstthezygomycete host.FromanM.circinelloides(R7B)cDNAlibrary,weamplified
speciestested. anHA-taggedfkbAcodingsequenceampliconandcloneditintoa
TheM.circinelloidesFKBP12homologandtheFRBdomain yeastlow-copy-numberplasmidunderthecontrolofaninducible
ofTorarerequiredforrapamycinaction.Wefoundthatgrowth copper promoter (pCu415CUP1) (30). The resulting plasmid
of M. circinelloides strains was compromised during rapamycin (pCu415-HA-FKBP12) was introduced into a rapamycin-resis-
treatment.Thepresumedtargetsofrapamycininthisfungalspe- tantS.cerevisiaestrainlackingtheFKBP12homolog(fpr1(cid:4))(23)
ciesaretheFKBP12andTorhomologs.Comparativeanalysisof and tested on rapamycin-containing YPD agar plates with or
274 ec.asm.org EukaryoticCell
RapamycinActivityagainstMucorcircinelloides
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FIG3PhylogeneticreconstructionofM.circinelloidesFKBPfamilyofproteinsandfungalFKBP12homologs.TheMcFKBP12homologwasidentifiedbyprotein m
sequencecomparisonswithPhycomycesblakesleeanus(P.b.),Candidaglabrata(C.g.),S.cerevisiae(S.c.),Schizosaccharomycespombe(S.p.),Aspergillusfumigatus h
(A.f.),Neurosporacrassa(N.c.),Ustilagomaydis(U.m.),Cryptococcusneoformans(C.n.),Rhizopusoryzae(R.o.),andBatrachochytriumdendrobatidis(B.d.) t
t
FKBP12homologs. p
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c
.
a
without exogenous copper. Heterologous expression of McfkbA straintransformedwithvectoraloneremainedsensitivetorapa- s
restoredrapamycinsensitivityinthefpr1(cid:4)strain,indicatingthat mycin (Fig. 4B). These results indicate that the M. circinelloides m
.
McHA-FKBP12 can functionally complement an S. cerevisiae FRB domain can effectively compete for rapamycin binding o
r
strainlackingendogenousFKBP12(Fig.4A). (whenoverexpressedinthepresenceofcopper)andistherefore g
/

We also tested whether McHA-FKBP12 can mediate FK506- thelikelytargetoftheFKBP12-rapamycincomplexinM.circinel- o
n
dependentinhibitionofS.cerevisiaecalcineurinbytransforming loides.
theMcfkbA-containingconstructintoanS.cerevisiaevph6(cid:4)fpr1(cid:4) Theyeasttwo-hybridreportersystemwasusedtodetermine Ap
dpoounbenletmofuthtaenvtascturaoilnar(H25(cid:1))-.ALTosPsaosefVasPseHm6b(lwyhcoicmhpelnecxo)dreessualtcsoimna- wthheetphreerseMncceFKoBfPra1p2aimntyecrianc.tsTwoitthhitsheenFdR,BadroampaaminycoifnM-recTsiostranint ril 1
4
synthetic lethal phenotype during inhibition of calcineurin by yeasttwo-hybridhoststraincontainingaTOR1-3mutationand ,
FK506,whereasavph6(cid:4)fpr1(cid:4)mutantstrainalsolackingFKBP12 lacking endogenous FKBP12 was employed (SMY4-1) (8). This 2
0
isFK506resistant(seeFig.S1Ainthesupplementalmaterial)(24). strain was transformed with plasmids expressing McFKBP12 1
9
Surprisingly,heterologousexpressionofMcHA-FKBP12didnot fusedtotheGAL4AD(pGADT7-FKBP12)andtheMcTorFRB
render the vph6(cid:4) fpr1(cid:4) strain sensitive to FK506 (see Fig. S1), domainfusedtotheGAL4BD(pGBKT7-FRB),andinteractions by

indicating that McHA-FKBP12 is unable to inhibit calcineurin were quantified by monitoring expression of a GAL4-lacZ re- g
u
function during FK506 exposure, even though the McHA- porter gene. Strong interactions between FKBP12 and the FRB e
FKBP12 protein was stably expressed in a vph6(cid:4) fpr1(cid:4) back- domainwereobservedonlyinthepresenceofrapamycin,indicat- s
t
groundinaWesternblot(datanotshown).Thismayreflectdif- ingthattheFRBdomainofTorisatargetforrapamycinwhenTor
ferences in the composite FKBP12-FK506-calcineurin binding iscomplexedwithFKBP12(Fig.4C).Insummary,theMcFKBP12
surface that arose during divergence of S. cerevisiae and M. cir- homologcanfunctionasthereceptorforrapamycinandcanme-
cinelloidesfromtheirlastcommonancestor. diateinhibitionofTorviatheFRBdomaininaheterologoushost.
Genomeanalysisalsorevealedthepresenceofonegeneencod- MutationsinfkbAconferrapamycinandFK506resistance.
ing a Tor homolog (Mucor circinelloides CBS277.49 protein ID TheobservationthatMcFKBP12canmediaterapamycininhibi-
152074),whichwedesignatedtorA.TheFRBdomainofTorwas tionofScToryetisunabletopromoteinhibitionofS.cerevisiae
PCR amplified from M. circinelloides (R7B) genomic DNA and calcineurininthepresenceofrapamycinpromptedustoemploya
cloned into a yeast low-copy-number plasmid containing a genetic approach to assess whether McFKBP12 functions as the
copper-induciblepromoter(pCu414CUP1)(30).Awild-typeS. rapamycin/FK506receptorinM.circinelloides.Exploitingthero-
cerevisiaestrain(JK9-3da)wastransformedwiththisplasmidand bustantifungalactivityofFK506towardM.circinelloides(Fig.1
tested on rapamycin-containing YPD agar in the presence and andTables2and3),weisolatedaseriesofstrainsintwoindepen-
absenceofexogenouscopper.Inthepresenceofcopper,expres- dentstrainbackgrounds,R7B((cid:3)matingtype)andNRRL3631((cid:1)
sion of the M. circinelloides FRB domain rescued the wild-type matingtype),thatexhibitedspontaneousFK506resistance,and
strainfromthefungicidalactivityofrapamycin,whileawild-type we examined the fkbA locus for the presence of genetic lesions.
March2012 Volume11 Number3 ec.asm.org 275
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FIG4FKBP12andtheTorFRBdomainofM.circinelloidesinteractandcanfunctionallycomplementS.cerevisiaewild-typeandfpr1(cid:4)strains.(A)McFKBP12 s
mediates inhibition of ScTor by rapamycin in an S. cerevisiae fpr1(cid:4) strain lacking FKBP12. Wild-type (JK9-3da) cells transformed with vector alone m
(pCu415CUP1[LEU2])aresensitivetorapamycin,whilefpr1(cid:4)cells(JHY3-3B)transformedwithvectoralone(pCu415Cup1)areresistant.Expressionof .o
McHA-FKBP12(pCu415-HA-FKBP12)rendersanfpr1(cid:4)strain(JHY3-3B)sensitivetorapamycin.(B)OverexpressionoftheFRBdomainofMcTor(pCu414- r
g
cMycFRB)inawild-typestrain(JK9-3da)competeswithendogenousTor1/Tor2forrapamycinbinding,suppressingthefungicidalactivityofrapamycin /

observedinawild-type(JK9-3da)straintransformedwithvectoralone(pCu414CUP1[TRP1]).(C)TheFRBdomainofMcTorandMcFKBP12interactinthe o
presenceofrapamycinintherapamycin-resistantyeasttwo-hybridhostSMY4-1(TOR1-3fpr1(cid:4)GAL4-lacZ).SMY4-1strainstransformedwithvectoralone n

(pGADT7ADorpGBKT7)orwithvectorsexpressingtheFRBdomainfusedtotheGAL4DB(pGBKT7-FRB)orFKBP12fusedtotheGAL4AD(pGADT7AD- A
fkbA)failtoinducestrongexpressionoftheGAL4-lacZreporterinthepresenceorabsenceofrapamycin.Resultsrepresentthreeindependentreplicates. p
r
il
1
4
,

Twoisolateswereidentified:one,SM2(NRRL3631background), prolinesubstitution(L91P)inthepredictedproteinsequenceof 2
0
wascrossresistanttorapamycinandFK506,andasecondisolate, FKBP12 that does not affect FKBP12 protein production (Fig. 1
9
SM4(R7Bbackground),wassensitivetorapamycinandresistant 5C).Leucine91isahighlyconservedresidueamongFKBP12ho-
b
toFK506(Fig.5A).Bothisolatesweresensitivetothecalcineurin mologs(Fig.5D)andhasbeenidentifiedasacriticalresiduere- y

inhibitor CsA, indicating that these are not multidrug-resistant quiredforbindingofhumanFKBP12tocalcineurin(21).InSM4, g
u
isolates(Fig.5A).SM2containsanfkbAallele(fkbA-1)harboring the L91P substitution leads to a shorter cyclic side chain that e
anA-to-Gsubstitution(A316G)intheacceptorsplicesiteofin- wouldpredictablydiminishcalcineurinbindinginthepresenceof s
t
tron2.AmplificationofthefkbAcodingdomainsequencefroma FK506 (see Discussion). Taken together, our genetic analysis
cDNAlibrarysynthesizedfromSM2totalRNArevealedthepres- stronglysuggeststhatFKBP12functionsastherapamycin/FK506
enceofseveralalternativefkbAcDNAspeciesofvarioussizes(Fig. receptorinM.circinelloides.
5B). Sequence analysis of the smallest species revealed a 28-bp DisruptionoffkbAbyhomologousrecombinationrenders
deletionofexon3sequenceduetotheuseofacrypticsplicesite M.circinelloidesresistanttorapamycinandFK506.Tofurther
located downstream from the acceptor splice site in wild-type establishthatFKBP12isthecognatereceptorforrapamycinand
fkbA (see Fig. S2 in the supplemental material). In two of the FK506,wedisruptedthefkbAlocusbyreplacingthecodingdo-
fkbA-1 mRNA species, intron 2 was retained due to the A316G main sequence with a pyrG disruption cassette. A gene replace-
substitution,leadingtolongerfkbAcDNAspecies(seeFig.S2). mentallelecomprisedofthepyrGgeneand1-kbsequencesfrom
ForallthreecDNA/mRNAspecies,thepredictedproteinscontain thefkbA5=-and3=-untranslatedregions(seeMaterialsandMeth-
prematurestopcodonsupstreamoftheFK506bindingdomain ods)waslinearizedandusedtotransformtheuridineauxotrophic
(seeFig.S2),andnoFKBP12proteinproductsweredetectableby strainMU402(leuA(cid:3)pyrG(cid:3))(36).Twoindependenttransforma-
Westernblotting(Fig.5C). tionswereperformedtoensurethatdisruptionofthefkbAgene
The FK506-resistant isolate SM4 contains an fkbA allele resultedfromtwoindependentevents.PCRanalysisoftwoinde-
(fkbA-2) in which a mutation in exon 3 results in a leucine-to- pendent homokaryotic uridine prototrophic transformants,
276 ec.asm.org EukaryoticCell
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FIG 5Spontaneous mutations in fkbA confer rapamycin and FK506 resistance. (A) Spores from strains R7B (WT (cid:3)), NRRL3631 (WT (cid:1)), SM4 [R7B :/
/
fkbA-1(L91P)],andSM2[NRRL3631fkbA-2(A316G)]wereplatedonYPDplateswithorwithout100nMrapamycin,1(cid:1)g/mlFK506,or100(cid:1)g/mlcyclosporine e
c
(cyclosporinA).SM2iscrossresistanttorapamycinandFK506,whileSM4israpamycinsensitiveandFK506resistant.(B)AnA316Gsubstitutionintheacceptor .
a
splicesiteofintron2inthefkbAlocusinSM2leadstotheproductionofalternativefkbAcDNAspecies.ThreefkbAalternativetranscriptswereamplifiedfrom s
anSM2cDNAlibrary(lane3).ThealternativecDNAspecieswerenotdetectedinno-reverse-transcriptasecontrols((cid:3)RT).fkbA-1speciesaandbretainintron m
2.fkbA-1speciescharborsa28-bpdeletioninexon3duetothealternativeuseofacrypticacceptorsplicesiteupstreamofthewild-typeacceptorsplicesite(see .
o
Fig.S2inthesupplementalmaterial).(C)SM2doesnotexpressFKBP12.FKBP12proteinexpressionwasdeterminedbyWesternblotanalysisofwild-type r
(R7B),fkbA(cid:4)1(fkbAdeletionstrain[seeFig.6]),SM2,andSM4totallysates.FKBP12wasdetectedwithantiserumraisedagainstScFKBP12(seeMaterialsand g
/
Methods).AnfkbA(cid:4)deletionstrainwasusedasacontrolforFKBP12antiserumspecificity.Alpha-tubulinlevelsservedasloadingcontrols.(D)Protein o
alignmentsofthe80sloopinMucorcircinelloides(R7B)(Mc),SM4(L91P),human(h),bovine(b),Batrachochytriumdendrobatidis(Bd),Rhizopusoryzae(Ro), n

Phycomycesblakesleeanus(Pb),Saccharomycescerevisiae(Sc),andSchizosaccharomycespombe(Sp)FKBP12homologs.Leucine91inM.circinelloidesandthe A
L91PmutationinSM4areboxedinred. p
r
il
1
namedRBM1(fkbA(cid:4)1)andRBM2(fkbA(cid:4)2),confirmedintegra- wassensitivetorapamycinandFK506,growthofbothfkbAmu- 4,

tionofthepyrGdisruptionalleleatthefkbAlocus(Fig.6AandB). tant strains was unaffected in the presence of either drug. All 2
0
Disruption of fkbA in these two strains was also confirmed by strainsweresensitivetogrowthinhibitioninthepresenceofcy- 1
9
Southernblotanalysis(datanotshown). closporine (Fig. 6E). Taken together, these results confirm that
b
Northernanalysiswasfurtheremployedtoconfirmthelossof FKBP12,encodedbythefkbAgene,functionsastherapamycin/ y

the fkbA gene. Employing a probe that hybridizes to exon 2 of FK506receptorinM.circinelloides. g
u
fkbA,wewereabletodetectfkbAmRNAproductioninthewild- RapamycinimprovessurvivalofGalleriamellonellalarvae e
typeparentalstrain(MU402)andfailedtodetectthemessagein infectedwithalethaldoseofM.circinelloidesspores.Clinical s
t
eachofthetwoindependentfkbA(cid:4)deletionstrains(Fig.6C).An treatmentofhuman-invasivefungalinfectionscausedbyzygomy-
antiserumraisedagainstS.cerevisiaeFKBP12(7)wasemployedto cete (Rhizopus and Mucor) species is notoriously difficult to
confirmthelossoftheFKBP12proteininbothofthegenedele- achieveduetotheintrinsicresistanceofmostzygomycetespecies
tionstrains.ByWesternanalysis,wewereabletodetectabandof tothecurrentantifungaldrugarmamentarium(reviewedinref-
approximately12kDa(determinedbycomigrationwithappro- erences44and50).Inlightoftheinhibitoryeffectsofrapamycin
priatesizemarkers[datanotshown])thatwasnotdetectedinthe onM.circinelloidesgrowth,wetestedwhethertargetingtheTor
deletionstrains(Fig.6D).InthesameWesternblot,extractsfrom pathway with rapamycin could serve as a potential antifungal
anS.cerevisiaewild-typestrain(JK9-3da)andanfpr1(cid:4)strainwere therapeuticapproach.Tothisend,weusedlarvaefromthewax
included as positive controls for the anti-FKBP12 serum used mothGalleriamellonellainfectedwithM.circinelloidessporesas
(Fig.6D). an invertebrate model of disseminated infection. The use of G.
AfterbothfkbAdeletionstrainswererigorouslyverified,spores mellonellaasamodelofinfectionisbecomingincreasinglypopu-
fromeachstrainweregerminatedonYPDagarmediumsupple- larduetoitseaseofuseandhighcorrelationwithmurinemodels
mentedwithrapamycinandFK506inparallelwithsporesfrom ofdisseminatedinfection(9,12).Healthylarvaearelightcolored
the parental background strains, i.e., R7B and MU402 (MU402 andactive,whilelarvaekilledbyafungalinfectionaredarkand
wasderivedfromR7B).Whilethegrowthofeachparentalstrain immobile,facilitatingscoringofmortalityduringassays.
March2012 Volume11 Number3 ec.asm.org 277
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FIG6 TargeteddisruptionofthefkbAlocusconfersrapamycinandFK506resistance.(A)Schematicrepresentationofgenotypingprimersusedtoverify s
disruptionofthefkbAlocus.(B)PCRgenotypingoftwoindependentfkbA(cid:4)deletionstrains.(C)NorthernanalysisoffkbAexpressioninthewild-typeparental m
strain(MU402)andthetwoindependentfkbA(cid:4)strains.(D)WesternanalysisofFKBP12proteinexpressioninthewild-typeparentalstrain(MU402)andthe .
o
twoindependentfkbA(cid:4)strains.ExtractsfromeachsiblingstrainwereusedforWesternanalysis.FKBP12proteinwasdetectedwithantiserumraisedagainstS. r
g
cerevisiaeFKBP12.Alpha-tubulin(loadingcontrol)wasdetectedusingamonoclonalantibodyraisedagainstS.cerevisiaealpha-tubulin.(E)Sporesuspensions
/
fromtheparentalstrainsR7B(parentofMU402)andMU402andfromthefkbA(cid:4)strainswerespottedonYPDagarandYPDagarsupplementedwith100nM o
rapamycin,1(cid:1)g/mlFK506,or100(cid:1)g/mlcyclosporine(cyclosporinA)andwereincubatedfor48hatroomtemperature. n

A
p
r
Inthismodelofinfection,injectionofcontrolPBShadnoeffect lentinG.mellonella(Fig.7),consistentwithrecentpublishedresults il 1
onsurvivaloflarvae(Fig.7).Injectionof500sporesoftheM.circinel- (31),andwerenotusedinsubsequentexperiments. 4
,

loidesR7Bstraincaused100%deathwithin5daysafterinfection, TreatmentofG.mellonellainfectedwithM.circinelloidesR7B 2
0
whilesporesfromtheM.circinelloidesNRRL3631strainwereaviru- sporeswithadoseof33mgofrapamycin/kgresultedina50% 1
survivalrate,astatisticallysignificant(P(cid:7)0.0133;logranktest) 9

b
improvementinsurvivalcomparedtothe0%survivalrateofPBS-
y
treatedinfectedcontrols(Fig.8).Similarrapamycintreatmentof g
G.mellonellalarvaeinfectedwithsporesfromtwoindependently u
e
derivedR7BfkbAdeletionstrains(RBM1andRBM2)didnothave s
t
anyeffectonitsvirulence,indicatingthatinhibitionofMcTorby
rapamycin is protective in G. mellonella. Notably, treatment of
uninfectedlarvaewiththesamedoseofrapamycinhadanegligi-
bleeffectontheirsurvivalinrelationtoPBScontrols,indicating
that rapamycin’s known immunosuppressive effect in humans
hasanegligibleimpactonG.mellonellasurvival(Fig.8).
Surprisingly,andcontrarytoourinvitroresults,monotherapy
withFK506didnotimprovesurvivalofG.mellonellalarvaein-
fectedwithR7Bsporesatthedoseadministered(0.13mgFK506/
kg)(datanotshown).ThisdosefollowedtheMICthateliciteda
growth inhibitory effect in our in vitro assays. However, FK506
FIG7InfectionbyM.circinelloidesR7BsporesresultsinacutemortalityofG. provednottobebeneficialintheinvivomodelofinfection,pos-
mellonellalarvae.Thirtylarvaepertreatmentwereinfectedwith500M.cir-
siblyduetoareducedstabilityofFK506inG.mellonella,higher
cinelloidessporesfromtheR7BandNRRL3631wild-typestrains.InfectingG.
ratesofdrugclearance,orcompetitionfordrugbindingbyendog-
mellonellawithM.circinelloidesR7Bsporesresultedinacutemortalityrates,
whileinfectionsbyM.circinelloidesNRRL3631sporesdidnot. enousFKBP12.
278 ec.asm.org EukaryoticCell
RapamycinActivityagainstMucorcircinelloides
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FIG8PharmacologicalinhibitionofTorenhancessurvivalofGalleriamellonellainfectedwithalethaldoseofM.circinelloidesspores.InhibitionofMcTorby
n
rapamycinexhibitsatherapeuticbenefitintheG.mellonellamodelofM.circinelloidespathogenesis.TenlarvaepergroupwereinfectedwithPBSalone, lo
rapamycinalone,or500sporesofM.circinelloidesR7BorthefkbA(cid:4)strainRBM1orRBM2.LarvaeweretreatedwithPBSorrapamycin(33mgrapamycin/kg a
ofbodyweight)at4hpostinfectionandgrownatroomtemperature,andviabilitywasscoreddaily.Anasteriskdenotesastatisticallysignificantchangeinsurvival d
ofinfectedG.mellonellalarvaeaftertreatmentwithrapamycininrelationtountreatedlarvae.Rapamycinwasnotbeneficialtolarvaeinfectedwithsporesfrom e
d
thefkbA(cid:4)strainsRBM1andRBM2.Survivalcurvesarerepresentativeoftwoindependentexperiments.
f
r
o
m
Overall,thetherapeuticbenefitimpartedbytargetingtheTor betweenMcFKBP12andtheMcTorFRBdomainintheyeasttwo- h
pathwayduringinfectionofaninvertebratemodelofM.circinel- hybridassay(Fig.4C).Theselinesofevidencedemonstrateacon- tt
p
loidesdisseminationestablishestheTorpathwayasanattractive served mechanism of FKBP12-mediated inhibition of Tor by :
/
/
target for the development of antifungal therapy against life- rapamycininazygomycetefungalpathogen. e
c
threateningzygomycosis. Second,weisolatedaspontaneouslyrapamycin-FK506doubly .
a
resistantmutantharboringamutationintheacceptorsplicesite s
DISCUSSION m
ofintron2offkbAthatabrogatesFKBP12proteinexpression(Fig.
.
TheTorsignalingpathwayservesasanattractivetargetforanti- 5AtoC),presumablybynonsense-mediateddecay.Asecondin- or
fungaltherapyduetoitscentralroleinregulatingfungalgrowth g
dependentmutant,inwhichleucine91(aconservedcalcineurinA /

anditsbroadconservationamongspeciesinthefungalkingdom, o
bindingresidueinFKBP12)isreplacedbyproline,whilesensitive
n
withthenotableexceptionoftheMicrosporidia(28,47).TheTor torapamycin,displayedFK506resistance(Fig.5D).Inthecrystal A
signalingcascadeevolvedpriortothelastcommonancestortothe structureofthehumanFKBP12-FK506-calcineurinternarycom- p
metazoanandfungallineages,inaccordwithitsknownconserva- plex,isoleucine90(I90)(correspondingtoleucine91inM.cir- ril
tioninmetazoans,landplants,algae,andfungi(2,47).Inaccord 1
cinelloidesFKBP12)changesconformationintheternarycomplex 4
with the deep ancestral roots of the Tor pathway, comparative andoccupiesahydrophobicpocketinthecalcineurinAsubunit ,
genome analyses across multiple species throughout the fungal 2
(21). Futer et al. identified I90 as a constituent of the human 0
kingdomhaverevealedaremarkableconservationintheamino 1
FKBP12(hFKBP12)80sloop,acriticalcompositesurfaceessen- 9
acid sequences of Tor homologs. Of particular interest are the tialforcalcineurinbinding(19).ReplacingI90withapolaramino b
invariablyconservedresidueswithinthehydrophobicpocketof y
acidsuchaslysinedecreasestheaffinityofthehFKBP12-FK506
theFRBdomainthatarecriticalforrapamycinbindingtoTor(5, g
binarycomplexforcalcineurin26,000-fold(wild-typeK,5.5nM; u
7,34,47,52,59).Thisobservationisinagreementwithprevious i e
studies showing that rapamycin has broad-spectrum antifungal I90KmutantKi,14,300nM),withoutsignificantlyaffectingthe s
bindingaffinityforeitherFK506orrapamycin(19).Similarly,in t
activityagainstseveralhumanpathogens,includingCandidaal-
SM4,theL91Psubstitutionleadstoashortercyclicsidechainthat
bicansandCryptococcusneoformans,andthatitsactivityinthese
wouldpredictablybeoccludedfromthecalcineurinAhydropho-
species is mediated by conserved rapamycin-FKBP12 drug-
bicpocketandtherebydiminishcalcineurinbindinginthepres-
proteincomplexes(1,13,15,17,56,58).
Inthisstudy,wefurtherdemonstratethatrapamycininhibits enceofFK506.Third,ablationofthefkbAlocusbygenereplace-
growth of the zygomycetes P. blakesleeanus, R. oryzae, and M. ment conferred both rapamycin and FK506 resistance (Fig. 6),
circinelloides,demonstratingaconservedroleforTorinregulating furtherdemonstratingaroleforFKBP12inmediatingrapamycin
thegrowthofbasalfungalpathogens(Fig.1andTables2and3). andFK506antifungalaction.Takentogether,thesestudiesfurther
We also established that rapamycin’s antifungal action is medi- demonstratethatthewidespreadantifungalactivityofrapamycin
atedviaconservedFKBP12-dependentinhibitionofaTorkinase anditsmechanismofactionareconservedintheascomycetesS.
homologinM.circinelloides.First,expressionofMcFKBP12inan cerevisiaeandC.albicans,thebasidiomyceteC.neoformans,and
S.cerevisiaeFKBP12-deficientstrainrestoredrapamycinsensitiv- thezygomycetepathogenM.circinelloides(13,15,17,23).
ity(Fig.4A).Inaddition,overexpressionoftheM.circinelloides OurstudiesalsodemonstratethatinaG.mellonellamodelof
Tor FRB domain rescued rapamycin toxicity in an S. cerevisiae infection,rapamycinprotects50%oflarvaeagainstalethalinfec-
wild-typestrain(Fig.4B).Rapamycinalsopromotedinteractions tionbyM.circinelloides(Fig.8),providingakeyproofofprinciple
March2012 Volume11 Number3 ec.asm.org 279