Rapamycin Antifungal Action Is Mediated via Conserved Complexes with FKBP12 and TOR Kinase ...

12 Pages·1999·0.47 MB·English

MOLECULARANDCELLULARBIOLOGY,June1999,p.4101–4112 Vol.19,No.6
0270-7306/99/$04.0010
Copyright©1999,AmericanSocietyforMicrobiology.AllRightsReserved.
Rapamycin Antifungal Action Is Mediated via Conserved
Complexes with FKBP12 and TOR Kinase Homologs in
Cryptococcus neoformans
M.CRISTINACRUZ,1LORAM.CAVALLO,1,2JENIFERM.GO¨RLACH,1GARYCOX,3
JOHNR.PERFECT,3,4MARIAE.CARDENAS,1ANDJOSEPHHEITMAN1,2,3,4,5*
DepartmentsofGenetics,1PharmacologyandCancerBiology,5Microbiology,4andMedicine,3and
HowardHughesMedicalInstitute,2DukeUniversityMedicalCenter,Durham,NorthCarolina27710
Received6November1998/Returnedformodification9December1998/Accepted1March1999 D
o
w
Cryptococcus neoformans is a fungal pathogen that causes meningitis in patients immunocompromised by n
AIDS, chemotherapy, organ transplantation, or high-dose steroids. Current antifungal drug therapies are lo
limitedandsufferfromtoxicsideeffectsanddrugresistance.Here,wedefinedthetargetsandmechanismsof a
d
antifungalactionoftheimmunosuppressantrapamycininC.neoformans.IntheyeastSaccharomycescerevisiae e
andinTcells,rapamycinformscomplexeswiththeFKBP12prolylisomerasethatblockcellcycleprogression d

byinhibitingtheTORkinases.WeidentifiedthegeneencodingaC.neoformansTOR1homolog.Usinganovel fr
o
two-hybrid screen for rapamycin-dependent TOR-binding proteins, we identified the C. neoformans FKBP12 m
homolog, encoded by the FRR1 gene. Disruption of the FKBP12 gene conferred rapamycin and FK506
h
resistance but had no effect on growth, differentiation, or virulence of C. neoformans. Two spontaneous t
t
mutationsthatconferrapamycinresistancealterconservedresiduesonTOR1orFKBP12thatarerequired p
:
forFKBP12-rapamycin-TOR1interactionsorFKBP12stability.Twootherspontaneousmutationsresultfrom //
m
insertionofnovelDNAsequencesintotheFKBP12gene.Ourobservationsrevealthattheantifungalactivities
c
of rapamycin and FK506 are mediated via FKBP12 and TOR homologs and that a high proportion of b
spontaneous mutants in C. neoformans result from insertion of novel DNA sequences, and they suggest that .a
nonimmunosuppressiverapamycinanalogshavepotentialasantifungalagents. s
m
.
o
r
Cryptococcus neoformans is an opportunistic fungal patho- protein-folding step (for reviews, see references 24, 32, and g
/
genthatcausessystemicmycosisinimmunocompromisedpa- 56).
o
tients (20, 45). Cryptococcosis in patients with AIDS is char- Rapamycin and FK506 bind to the FKBP12 active site and n

acterizedbyapoorresponsetotherapyandariskofrecurrent inhibitprolylisomeraseactivity,butthisisnotthemechanism D
disease requiring lifelong suppressive antifungal regimens. of toxic action. Instead, FKBP12-rapamycin and FKBP12- e
c
Current treatments include amphotericin B, flucytosine, and FK506complexestargetproteinsrequiredforsignaltransduc- e
fluconazole.However,treatmentfailuresandtoxicityarecom- tionandcellgrowth.ThetargetofFKBP12-FK506andcyclo- m
mon,andnewantifungalagentsareneeded. philin A-CsA is calcineurin, a Ca21-regulated protein b
e
C.neoformansissensitivetotheimmunosuppressantsrapa- phosphatase (8, 26, 41). In humans, calcineurin regulates nu- r

mycin, FK506, and cyclosporine (CsA) (48, 49). These com- clearlocalizationofthetranscriptionfactorNFATduringthe 2
1
pounds are natural products with antifungal activity and are response to antigen presentation (for a review, see reference ,

also potent immunosuppressants used to treat graft rejection 53). In S. cerevisiae, calcineurin regulates cation homeostasis 2
0
intransplantrecipients(7,38).Rapamycinisamacrolideorig- andcellintegrityviathetranscriptionfactorCrz1/Tcn1,which 1
inally discovered in a screen for antimicrobial activity against regulates transcription of genes encoding ion pumps or cell 8

Candidaalbicansandlaterfoundtohavepotentimmunosup- wall biosynthetic enzymes (FKS2, PMR2, PMC1, and PMR1) by
pressiveactivity(62). (44, 65). In C. neoformans, calcineurin is also the target of g
Rapamycin, FK506, and CsA suppress the immune system FK506andCsAandisessentialforgrowthat37°C,in5%CO , u
byinhibitingsignaltransductionstepsrequiredforT-cellacti- 2 e
or at alkaline pH, conditions found in the host (48). As a s
vation(forreviews,seereferences19and59).Themechanisms consequence, calcineurin is required for virulence of C. neo- t
of action of these compounds have been studied in lympho-
formans(48).
cytes and Sacharomyces cerevisiae (for reviews, see references
RapamycinpreventsproliferationofS.cerevisiaecellsandT
11,18,32,and59).Thesehydrophobiccompoundsdiffuseinto
lymphocytes by inhibiting cell cycle progression from G to S
thecellandbindintracellularreceptorsknownasimmunophi- 1
phase. An FKBP12-rapamycin complex is the active intracel-
lins. The immunophilins are ubiquitous and conserved from
lular agent, but this complex does not inhibit calcineurin. In-
yeasttohumans.FK506andrapamycinbindtheimmunophilin
stead,FKBP12-rapamycininhibitstheTORkinases(15,33,35,
FKBP12, whereas CsA binds cyclophilin A. Although the cy-
40).TheyeastS.cerevisiaeexpressestwoTORproteins,TOR1
clophilinandFKBPproteinshavenosequencehomology,both
andTOR2.AmammalianTORhomologhasalsobeeniden-
catalyze cis-trans peptidyl-prolyl isomerization, a rate-limiting
tified(9,21,54,55).TheTORproteinshavehomologytolipid
andproteinkinasesandbelongtoafamilyofphosphatidylino-
sitol kinase-related kinases that regulate the cell cycle. The
*Corresponding author. Mailing address: 322 Carl Building, Re-
TORkinasedomainisconservedandisessentialforcellcycle
search Drive, Duke University Medical Center, Durham, NC 27710.
Phone: [phone]. Fax: [phone]. E-mail: heitm001@mc progression (1, 17, 74). The TOR signaling pathway also reg-
.duke.edu. ulates translation in yeast and mammalian cells (4, 5, 23). In
4101
4102 CRUZ ET AL. MOL.CELL.BIOL.
mammalian cells, mTOR regulates translation initiation via rapamycinwasfromtheNationalCancerInstitute.Drugstocksolutionswere
the p70s6k kinase and 4E-BP1/PHAS-I proteins (reviewed in preparedin90%ethanol–10%Tween20.
Isolation and characterization of the C. neoformans gene encoding TOR1.
reference 11). The S. cerevisiae TOR2 protein has an addi-
AmplificationbyPCRofC.neoformanscDNAwastheinitialapproachusedto
tionaluniquefunctioninvolvingactincytoskeletalpolarization characterize the C. neoformans TOR homolog. Degenerate oligonucleotide
(57,58). primersweredesignedbasedonconservedregionsinmammalianmTORand
Recentmodelsofmitogen-activatedTORsignalinginmam- yeast TOR1 and TOR2. The forward primer was 59-CA(G/A)GCITGGGA
maliancellsinvokeaninitialactivationofphosphatidylinositol (T/C)ITITA(T/C)TA(775),andthereverseprimerwas59-TC(G/A)AA(G/A)C
A(G/A)TCICC(A/G)TCIATGATG(781)(Iisinosine).PCRamplificationwas
3-kinase, which signals to p70s6k and 4E-BP1/PHAS-I via
performedunderconditionsofmoderateannealingstringencywithapooledC.
mTOR (28). The 4E-BP1/PHAS-I protein inhibits the eIF4E neoformanscDNAlibraryfromtheserotypeDstrainB3501(Stratagene).PCR
translationinitiationfactor,whichrecognizesthe59N7-meth- conditionswereasfollows:3minat95°C;35cyclesof30sat95°C,1minat45°C,
ylguaninecaptoinitiatetranslation.4E-BP1/PHAS-Ibindsto and2minat72°C;andafinal10-min72°Cstep.A795-bpproductwascloned
andsequenced.TheremainingportionsoftheC.neoformansTOR1genewere
and inhibits translational initiation by eIF4E, and the TOR
isolatedbyscreeningthreesize-selectedsubgenomiclibrariesoftheserotypeA
kinasephosphorylatesPHAS-ItoreleaseeIF4Eandpromote H99strainandbyanalysisofa59RACE(rapidamplificationofcDNAends)
translation (12, 13, 30, 60). Rapamycin blocks translation by product.TotalRNAforthecDNARACEwaspreparedfromC.neoformans
pTrOevRenatlsinogrePgHulAatSe-sIphpohsopshpohroyrlyaltaiotinonofapn7d0s6rke(le1a4s)e.RoafpaemIFy4cEin. HsGyG9st9eGmgGro()wv(en2r1sin2io1Yn),P25D.90-)CaatTn3Td0T°tCTh.CeRCTAAOCAREG1-wAspaCesCcpiTfiecrTfGoprrGmimAeedGrswA5iTt9h-GCt)hTe(A2GG12iAb2c)G,oTaBnCRdAL5C95-GC9ATRGAacGAe Dow
blocks translation in yeast by destabilizing the eIF4E-associ- n
ated translation factor eIF4G (4, 6, 23). Direct substrates of TFRGRG1AgAenTeAdCeTsCcrCibTeGdGbeGlo)w(2w12a3s)p.eSrefqourmenecdewanitahlyasisPeorfktihne-ETlOmRer1AgepnpeliaenddBtihoe- loa
yeastTORremaintobeidentified(forreviews,seereferences systemsDNAsequencermodel377(Amplitaqdyeterminatorchemistry). d
29and66). Two-hybrid screen to clone the C. neoformans FKBP12 homolog. Plasmids e
expressingtheGAL4DNAbindingdomain[GAL4(DB)]-C.neoformansTOR1 d
Biochemical,genetic,andstructuralstudieshavedefinedthe
FRBdomainproteinfusionwereusedtoscreenatwo-hybridcDNAlibraryfrom f
molecular details of FKBP12-rapamycin inhibition of TOR. C.neoformansH99.Thetwo-hybridlibraryconstructionwasasfollows.H99was ro
An X-ray crystal structure of an FKBP12-rapamycin-mTOR grownovernightat30°CinYPDmediumtosaturation.Aliquotsweretrans- m
complex revealed that FKBP12-rapamycin binds to a small ferredtofreshmedium(1:50dilution)andgrownforanother6hunderthe h
regiononTOR,theFKBP12-rapamycin-bindingdomain(FRB fsotallrovwatiinogncSoLnAdiDtiomnes:dYiuPmDamt3e0d°iCu.mToatta3l0R°CN,AYaPnDdmpoeldyi(uAm)1atR3N7°ACs,wanerdeniistorloagteend ttp
domain) located adjacent to the carboxy-terminal kinase do- andpooledsuchthatthetotalpopulationconsistedof50%mRNAfromYPDat :/
main (22). Mutations in the FRB domain confer rapamycin 30°C,37%mRNAfromYPDat37°C,and13%mRNAfromSLADmediumat /m
resistance by blocking FKBP12-rapamycin binding to TOR 30°C. The two-hybrid cDNA library was constructed by using the Clontech c
(15,17,35,43,63,74). MATCHMAKERlibrarykit;thecDNApopulationwassizefractionatedona b
Here we describe the identification of the FKBP12 and columntobelargerthat300bp,andH99cDNAswerethenfusedtotheGAL4 .a
activationdomain[GAL4(AD)]inplasmidpGAD10. s
TOR1 homologs from C. neoformans. We demonstrate that To fuse the TOR1 FRB domain to the GAL4(DB), the FRB domain was m
rapamycinantifungalactionismediatedviaFKBP12-rapamy- amplifiedwithprimers59-GACGTTCAGGAGCTTGAGCTG(849)and59-GC .
o
cininhibitionofTOR1.First,spontaneousFKBP12andTOR1 GAGAGCACTCTCCCAAGATCGTTGC(889)andclonedinthetwo-hybrid r
vectorpGBT9,yieldingplasmidpGBT9-CnTORFRB.SMY87-4,anisogenic g
mutations that confer rapamycin resistance destabilize /
fpr1::hisG derivative of strain PJ69-4A, was used as the two-hybrid strain to
FKBP12 or prevent FKBP12-rapamycin binding to C. neofor- screentheC.neoformanscDNAlibrary.SMY87-4wascotransformedwiththe on
mansTOR1.Second,disruptionoftheFKBP12-encodinggene GAL4(DB)-C.neoformansTOR1FRBdomainandthetwo-hybridcDNAlibrary
FRR1 confers rapamycin and FK506 resistance. Mutant cells described above. In addition, a URA31 plasmid (pTR17) was introduced to De
expressadominant,rapamycin-resistantalleleoftheTOR2gene(TOR2-1).Cells
lackingFKBP12exhibitedwild-typegrowthunderavarietyof c
weregrownonsyntheticmediumlackingleucine,tryptophan,uracil,andadenine e
differentconditionsandhadnodefectsinmating,sporulation, (SD-Leu-Trp-Ura-Ade)andsupplementedwith1mgofrapamycinperml.The m
or virulence. In summary, our studies reveal that a conserved cells were replica plated to synthetic medium lacking histidine (SD-Leu-Trp- b
TOR homolog is the target of FKBP12-rapamycin in C. neo- Ura-His)andsupplementedwith3mM3-aminotriazoleand1mgofrapamycin e
formans and that the mechanism of rapamycin action is con- ppyerramnol.sibd-eG(aClaPcRtoGsi)daassedaecstcirviibtyedwapsreavsisoauyseldyb(1y6u).singchlorophenol-b-D-galacto- r 2
servedfrompathogenicfungitoyeastsandhumans,andthey 1
Site-directed mutagenesis of C. neoformans TOR1. The C. neoformans ,
suggest that nonimmunosuppressive rapamycin analogs have GAL4(DB)-TOR1(Ser1846Leu)mutantwascreatedbyPCRoverlapmutagen- 2
potentialasnovelantifungalagents.Finally,ahighproportion esis(36)withplasmidGAL4(DB)-TOR1wild-typeFRBdomainasatemplateby 0
ofspontaneousmutationsresultedfromtheinsertionofnovel usingprimers59-GGAAGAAGCTTTAAAGCACTAC(1113)and59-GTAGT 1
GCTTTAAAGCTTCTTCC (1114) (mutations are in boldface) and flanking 8
sequencesintotheFKBP12locus,whichhasimportantimpli- primers 59-CTTACCGGAATTCATCCAAACGCCACGCCAGTCTATAC b
cations for the mutation, evolution, and virulence of this hu- (1115) and 59-CTCGCAACTGCAGCTATGGAACAGCAATATCCAAGTC y

manpathogen. TCG(1116).First-roundPCRoverlapproductsweregelpurifiedastemplates g
forsecond-roundPCRwithflankingprimers.ThePCRprotocolwas3minat u
e
95°C;35cyclesof30sat95°C,30sat55°C,and30sat72°C;andafinal5-min s
MATERIALSANDMETHODS 72°Cstep.TheresultingPCRproductwascleavedwithEcoRIandPstI,cloned t
Strains,media,antisera,andcompounds.TheS.cerevisiaetwo-hybridstrains intothesesitesinplasmidpGBT9,andconfirmedbysequencing.
used here were PJ69-4A (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4D DisruptionoftheC.neoformansFRR1gene.TheFRR1genewasdisruptedby
gal80DLYS2::GAL-HIS3GAL2-ADE2met2::GAL7-lacZ)(37)andSMY4(Y190 insertinga3,000-bpKpnI/SmaIfragmentspanningtheC.neoformansADE2gene
TOR1-3fpr1::ADE2)(16).SMY87-4isanisogenicfpr1::hisGderivativeofstrain (bluntedwithT4DNApolymeraseanddeoxynucleosidetriphosphates)intoan
PJ69-4A.ThepathogenicC.neoformansserotypeAstrainH99anditsisogenic RsrIIsitewithintheFRR1geneina5-kbEcoRIgenomicfragmentclonedin
Dade2derivativeM049havebeendescribedpreviously(67).StrainsJEC20and pBluescript(Stratagene),yieldingplasmidpMCC1bearingthefrr1::ADE2dis-
JEC21areisogenicMATaandMATaserotypeDstrains(46).MutantsC20F1, ruptionallele.Approximately4mgofplasmidDNAwaspurifiedonaQiagen
C20F2, C21F2, and C21F3 were as reported previously (48). C. neoformans column,absorbedontothesurfaceofgoldmicroprojectiles,andintroducedby
strainsweregrownonrich(yeastextract-peptone-dextrose[YPD])andsynthetic biolistictransformationintostrainM049,anisogenicDade2derivativeofstrain
mediausedforS.cerevisiae(61),andimmunosuppressantandSLADmediawere H99.Adenine-prototrophictransformantswereselectedonsyntheticmedium(1
asdescribedpreviously(27,31).Regenerationmediumforbiolistictransforma- Msorbitol)lackingadenineat30°Candcolonypurified.ADE1transformants
tionswaspreparedasdescribedpreviously(67).Capsuleproductionwasassessed weretestedfortheabilitytogrowonYPDmediumlackingorcontaining1mg
inlow-ironmediumplusethylenediaminedi(o-hydroxy)phenylaceticacidasde- ofrapamycinpermlor1mgofFK506perml(at37°C).
scribedpreviously(71).MelaninproductionwasassessedonNigerseedmedium Southern blot analysis. C. neoformans genomic DNA was prepared as de-
asdescribedpreviously(3).TheS.cerevisiaeFKBP12polyclonalantiserumwas scribedpreviously(52).DNA(;10mg)wasdigestedtocompletionwithrestric-
generatedandcharacterizedasdescribedpreviously(17).TheC.neoformans tionenzymes(NewEnglandBiolabs),fractionatedbyelectrophoresisina0.8%
cyclophilinApolyclonalantiserumwasproducedbyinjectingarabbitwithC. agarosegel,transferredtoanylonmembrane,andfixedbyUVcross-linking
neoformanscyclophilinArecombinantprotein.FK506wasfromFujisawa,and (Stratalinker; Stratagene). The membrane was hybridized overnight with gel-
VOL.19,1999 RAPAMYCIN ACTION IN C. NEOFORMANS 4103
purifiedDNAprobeslabelledwith32Pbyrandompriming(PharmaciaBiotech), neoformans TOR1 (see Materials and Methods). In addition,
andthemembraneswerethenwashed,dried,andexposedtofilm. the59endofthegeneandtheapproximatetranscriptionstart
Westernblotanalysis.C.neoformansculturesweregrowninYPDmediumat site were established by 59 RACE (see Materials and Meth-
30°Cfor24h,andcellswerethenpelletedandlysedbyglassbeadagitationin
abeadbeaterin20mMTris-Cl(pH7.4)–100mMKCl.Proteinconcentrations ods).ThecompletesequenceoftheC.neoformansTOR1ho-
weredeterminedbytheBradfordassay(Bio-Rad)withbovineserumalbuminas molog was determined from genomic and 59 RACE cDNA
astandard.Equivalentamountsofprotein(;100mg/ml)werefractionatedby clones.
sodium dodecyl sulfate–17% polyacrylamide gel electrophoresis (SDS–17%
The open reading frame of the C. neoformans TOR1 gene
PAGE),transferredtonitrocellulosemembranes,andprobedwithrabbitpoly-
clonalantiserumdirectedagainstyeastFKBP12ortheC.neoformanscyclophilin spans8,439bpandencodesa2,360-amino-acidprotein.Twen-
Aproteinasaloadingcontrol.DetectionwasperformedwiththeECLsystem ty-sixintronswereidentifiedbycomparingTOR1genomicand
(AmershamCorp.). cDNAsequencesandbyalignmentwiththehomologousTOR
Analysisoffrr1mutants.Mutantstrainswereassayedforcapsuleandmelanin
proteins from S. cerevisiae, Schizosaccharomyces pombe, and
production.C.neoformanscellswereincubatedonliquidlow-ironmediumplus
ethylenediaminedi(o-hydroxy)phenylaceticacidfor48h,andthepolysaccharide humans. The introns have an average size of 52 bp and have
capsulewasstainedwithastandardIndiainkpreparation.Toassessmelanin consensussplicedonor(GTNNGY)andacceptor(YAG)sites,
production,C.neoformansfrr1strainsweregrownat30°Cfor72honNigerseed with the exception of intron 25 (with GCAAGT as splice do-
agar,onwhichstrainsthatproducemelaninarebrown,whilestrainsthatdonot D
produce melanin are white. Sequence analysis of the mutant strains C20F1, nor) and intron 20 (with AAG as splice acceptor). CTRAY o
C20F2,andC21F3wasdoneasfollows.First,genomicDNAwasisolatedac- intronicbranchsiteswerepresentin25ofthe26introns. w
cording to a protocol described previously (51) and used as a template for The overall size and order of established protein domains n
duplicatePCRamplificationswitholigonucleotides59-GCATGGGATCCCAT aresimilarinthe C.neoformans TOR1proteinandtheTOR lo
GGGTGTTACTGTTGAG(1769)and59-GTACGAGAATTCTTAGTTGACC a
TATGGAAAGTGTAAAGA(C17C7T0G)f(o2r7s8t5r)aiannCd2117F730faonrdstorlaiginosnCuc2l0eFot1idaensd5C9-2C0GF2T.TTGhCeAPCARC hproomteoilnosgoisfS;.2c0e0reavmisiianeoaancdidhsusmhoarntse.rTthheanCt.hneeomfoamrmmaanlsiaTnOaRnd1 ded
conditionswere35cyclesof94°Cfor1min,47°Cfor1min,and72°Cfor3min. S.cerevisiaecounterpartsbecauseitlacksanN-terminalregion
f
The resulting DNA fragments spanning the frr1 gene were cloned into the andoneshortinternalsegment.TheC-terminalregionofthe ro
pCR2.1TAcloningvector(Invitrogen)andsubsequentlysequencedinduplicate C. neoformans TOR1 homolog contains the conserved FRB m
withaPerkin-ElmerAppliedBiosystemsDNAsequencermodel377(Amplitaq
dyeterminatorchemistry). andkinasedomains.TheC.neoformansTOR1kinasedomain h
Rabbitmodelofcryptococcalmeningitis.NewZealandWhiterabbitsweighing has78,77,and73%identitytotheTOR1,TOR2,andmTOR tt
p
2to3kgwerehousedinseparatecagesandprovidedwithwateradlibitumand proteins,respectively(Fig.1).WithintheC.neoformansTOR1 :
Pfrur1rimnautraanbtbCit.cnheoowfo.rImsoagnesnsiecroFtRypRe1Awisldtr-atyinpsew(eMre04p9reApDarEe2dbreycgornoswtitthutfeodr)7a2nhd FRB domain, Ser1862 is homologous to the serine residue //m
onYPDmediumandresuspendedin0.015Mphosphate-bufferedsaline.Eight implicatedinrapamycinbindingtotheS.cerevisiaeTOR1and c
rabbitswereadministeredcortisoneacetateat2.5mg/kgintramuscularly1day TOR2 proteins and mammalian mTOR (Ser1972 in TOR1, b
.
priortoinoculationwithC.neoformansandthendailyfor14days.Twenty-four Ser1975inTOR2,andSer2035inmTOR)(10,15,17,22,35, a
hoursfollowinginitiationofsteroidtreatment,rabbitswereanesthetizedwith s
43,63,74).Inaddition,TORresiduesthatbindrapamycinin m
intramuscularxylazineandketamineandtheninoculatedintracisternallywith0.3
ml of yeast suspension through a 25-gauge needle on a 3-ml syringe. Three the crystal structure of the mTOR-rapamycin-FKBP12 com- .o
rabbitsreceivedwild-typestrainH99,andfiverabbitsreceivedtheisogenicfrr1 plex(Trp2101,Asp2102,Tyr2105,Phe2108,Leu2038,Tyr2038, r
g
mutantstrain.Therabbitsweresedatedwithketamineandxylazineondays4,7, andGly2039)areconservedintheC.neoformansTOR1FRB /
10, and 14 after inoculation, and cerebrospinal fluid (CSF) was withdrawn. domain, with the sole exception that Thr2098 of mTOR is o
QuantitativeyeastcultureswereperformedbyplatingserialdilutionsofCSFin n
phosphate-bufferedsalineonYPDmediumandincubatingfor3daysat30°C. replaced by Gln1925 in C. neoformans TOR1 (Fig. 1). All D
Murinemodelofsystemiccryptococcosis.FRR1wild-typeandfrr1mutantC. presumptiveTORactive-siteresiduesareconserved(Arg2276 e
neoformansstrainswereusedtoinfect4-to6-week-oldfemaleBALB/cmice and Asp2294 in TOR1, Asp2279 in TOR2, and Asp2338 and c
(CharlesRiverLaboratories,Raleigh,N.C.)bylateraltailveininjections.Ten e
micewereinfectedwith107yeastcellsofeachstraininavolumeof100ml,and Asp2357 in mTOR) (10, 17, 58, 74). These findings indicate m
themiceweremonitoredbytwice-dailyinspections.Micethatappearedtobe thatabonafideTORhomologhasbeenidentifiedinC.neo- b
moribundorinpainweresacrificedbyCO inhalation. formans, with conserved structural features important for e
Nucleotidesequenceaccessionnumbers.T2heGenBankaccessionnumbersfor FKBP12-rapamycinbindingandenzymaticactivity. r 2
theC.neoformansTOR1genomicandcDNAsequencesandfortheC.neofor-
Cloning of the C. neoformans FKBP12 homolog. Because 1
mansFRR1genomicandcDNAsequencesareAF098972,AF098973,AF097888, ,
andAF097889,respectively. FKBP12istheintracellularreceptorforrapamycinandFK506 2
inotherorganisms,wesoughttoidentifytheFKBP12homolog 0
1
from C. neoformans. Previous attempts to clone the gene by
RESULTS 8
severalapproacheswereunsuccessful.Wethereforedeveloped
b
CloningoftheC.neoformansTOR1homolog.InS.cerevisiae, anoveltwo-hybridscreentoisolatetheFRR1(forFK506and y

rapamycinactionismediatedbytheFKBP12prolylisomerase, rapamycin resistance) gene, encoding the C. neoformans g
u
whichbindstoandinhibitstheTORkinasehomologstoblock FKBP12homolog. e
cellcycleprogression(15,33,39,40).Wehavetestedwhether For this two-hybrid cloning approach, the FRB domain of s
t
homologous FKBP12 and TOR proteins mediate rapamycin theC.neoformansTOR1proteinwasfusedtotheGAL4(DB).
actioninC.neoformans. The resulting GAL4(DB)-TOR1 fusion protein was used as
The C. neoformans TOR1 homolog was identified by PCR baittoidentifyproteinsthatinteractwithcryptococcalTOR1
amplification under reduced-stringency conditions with prim- inthepresenceofrapamycin.Second,aC.neoformanscDNA
erstoconservedTORsequencesandDNAfromaC.neofor- two-hybrid library was constructed by fusing cDNAs isolated
mansserotypeDstrainB3501cDNAlibraryasatemplate.A from the C. neoformans serotype A strain H99 to the
795-bp product was obtained, purified, and cloned. Sequence GAL4(AD).Third,weconstructedacustomizedtwo-hybridS.
analysis revealed a unique open reading frame encoding 265 cerevisiae strain, isogenic with strain PJ69-4A (37), which had
amino acids with marked identity to other TOR proteins. three important features: first, the FKBP12 gene was deleted
Southern blot analysis confirmed that this sequence was de- to reduce competition for rapamycin; second, the strain has
rived from C. neoformans and hybridized to a single genomic threeGAL4-dependentreportergenes,ADE2,HIS3,andlacZ;
locus,andthisfragmentwasusedasaprobetoclonetheTOR1 and third, because our screen requires rapamycin, which is
gene. Three size-selected subgenomic libraries were con- normally toxic to S. cerevisiae, a plasmid expressing a domi-
structedfromgenomicDNAoftheserotypeAstrainH99,and nant,rapamycin-resistantTOR2mutantwasintroduced.
severalscreenswereperformedtocompletethecloningofC. Thetwo-hybridscreenwasperformedbycotransformingthe
4104 CRUZ ET AL. MOL.CELL.BIOL.
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FIG. 1. C.neoformansTOR1kinaseandFRBdomainsarehighlyconserved.ThepredictedC.neoformansTOR1(CnTOR1)proteinsequencefromaminoacid s
residues1842to2200isalignedwithanalogousregionsfromS.cerevisiaeTOR1(ScTOR1)(aminoacids1952to2309)(15),S.cerevisiaeTOR2(ScTOR2)(aminoacids m
1955to2313)(40),mammalianmTOR(aminoacids2015to2372)(54),andS.pombeTOR(SpTOR)(aminoacids1814to2171).Thepositionsofkinaseconserved .
o
motifsareunderlined.AsterisksindicateconservedaminoacidresiduesoftheFRBdomainofTOR. r
g
/

o
n
C. neoformans two-hybrid cDNA library and the plasmid ex- characterizedTORmutationsthatconferrapamycinresistance
D
pressing the GAL4(DB)-TOR1 FRB domain fusion protein by preventing FKBP12-rapamycin binding (15, 17, 35, 43, 63, e
intothetwo-hybridyeaststrainlackingFKBP12andexpressing 74).WeisolatedseveralspontaneousC.neoformansmutations c
e
TOR2-1. Transformed cells were grown on synthetic medium thatconferrapamycinbutnotFK506resistance.Becausemu- m
lackingleucine,tryptophan,uracil,andadenine(SD-Leu-Trp- tationsintheTORFRBdomainconferrapamycinresistance b
Ura-Ade) and supplemented with 1 mg of rapamycin per ml. inotherorganisms,thesemutantsmightharboralterationsin e
TwelveADE1colonieswereisolated.Ofthe12cDNAclones theC.neoformansTOR1FRBdomain.Inonemutant,dupli- r 2
recovered,9activatedreportergeneexpressionwhenreintro- cate PCR amplification and sequence analysis revealed a 1
,
duced with the GAL4(DB)-TOR1 bait plasmid into the two- Ser1846LeusubstitutioninTOR1attheconservedserineres- 2
hybridstrain.Sequenceanalysisrevealedthatallninecontain 0
idueimplicatedinFKBP12-rapamycinbindingtoyeastTOR1
1
overlapping or identical portions of the same gene, which we
andTOR2. 8
namedFRR1.ThesequenceoftheC.neoformansFRR1gene
We used the yeast two-hybrid system to test if the b
predictsa108-amino-acidprotein,whichhassequenceidentity y
Ser1862Leu mutation confers rapamycin resistance by block-
with FKBP12 proteins of yeast, fungi, and mammals (Fig. 2). g
ing FKBP12-rapamycin binding toC. neoformans TOR1. The u
The predicted amino acid sequence of the C. neoformans GAL4(DB) was fused to wild-type and to mutant FRB e
FKBP12 protein is aligned in Fig. 3 with sequences of con- s
(Ser1862Leu) domains of C. neoformans TOR1. Plasmids ex- t
servedFKBP12proteinsfromotherorganisms.ThisFKBP12
pressing the GAL4(DB)-TOR1 FRB domain fusion protein,
protein has 65, 57, and 59% identity with FKBP12 homologs
the GAL4(DB)-TOR1 (Ser1862Leu) mutant FRB domain,
from S. cerevisiae, C. albicans, and humans, respectively. Im-
and the GAL4(AD)-yeast or C. neoformans FKBP12 fusion
portantly, residues of the rapamycin- and FK506-binding
proteinswerecoexpressedinatwo-hybridhost(SMY4[Y190
pocket are highly conserved or invariant between FKBP12
proteinsfromC.neoformans,S.cerevisiae,andhumans(Fig.3). TOR1-3fpr1::ADE2])inthepresenceorabsenceofrapamycin.
The genomic FRR1 locus was cloned by screening a sub- Protein-drug-protein interactions were monitored by measur-
genomic library from strain H99 with the FRR1 cDNA as a ing expression of the GAL4-lacZ reporter gene by assaying
probe.A5.1-kbgenomicclonespanningtheFRR1openread- b-galactosidase activity (Fig. 4). Rapamycin-dependent inter-
ing frame was obtained. Sequence analysis revealed that the actions between wild-type C. neoformans TOR1 and FKBP12
FRR1 coding sequence is punctuated by five introns. All five weredetected.Incontrast,theSer1862LeumutantTOR1pro-
introns were small (96, 89, 50, 53, and 52 bp) and contained tein failed to interact with FKBP12-rapamycin (Fig. 4). Thus,
consensussplicedonor,acceptor,andbranchsites. the Ser1862Leu mutation confers rapamycin resistance in C.
FKBP12-rapamycin binds to wild-type TOR1 but not to a neoformans by preventing FKBP12-rapamycin binding to
rapamycin-resistant TOR1 mutant. Previous studies have TOR1.
VOL.19,1999 RAPAMYCIN ACTION IN C. NEOFORMANS 4105
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FIG. 2. SequencesoftheC.neoformansFRR1geneandFKBP12protein.TheDNAsequenceoftheC.neoformansgenomicFRR1locus,encodingtheFKBP12 a
protein,isdepicted.ThesequencewasdeterminedfromcDNAandgenomicclonesasdescribedinResultsandinMaterialsandMethods.The59and39untranslated s
regionsandintronicsequencesareinlowercase,andintronsarenumberedinorderofoccurrencefrom59to39.ExonsoftheFRR1openreadingframeareinuppercase, m
withthetranslatedproteinsequencebelowtheDNAsequenceinuppercasesingle-letteraminoacidabbreviations.Consensussplicedonor(GTNNGY),branch .o
(CTRAY),andacceptor(YAG)sitesareunderlined.Startandstopcodonsareinboldface. r
g
/

o
n
DisruptionoftheC.neoformansFRR1genebyhomologous restrictionsiteintheFRR1openreadingframe(Fig.5A).The
D
recombination. Because FKBP12 is not required for growth frr1::ADE2 disruption allele was introduced by biolistic trans- e
but is required for rapamycin and FK506 toxicity in S. cerevi- formation into strain M049, an isogenic Dade2 mutant of the c
e
siae, our hypothesis was that C. neoformans mutants lacking pathogenic C. neoformans isolate H99. Adenine-prototrophic m
FKBP12wouldbeviableandwouldberapamycinandFK506 transformants were selected on synthetic medium lacking ad- b
resistant.WetestedthisbydisruptingtheC.neoformansFRR1 enineandsubsequentlyscreenedforresistancetorapamycin(1 e
genebyhomologousrecombination. mg/ml) or FK506 (1 mg/ml at 37°C). Fourteen of 60 ADE1 r 2
TheC.neoformansFRR1genewasdisruptedbyinsertingthe prototrophic transformants analyzed (23%) were rapamycin 1
,
ADE2 selectable marker by blunt-end ligation into an RsrII and FK506 resistant. PCR and Southern blot analysis con- 2
0
1
8

b
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g
u
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s
t
FIG. 3. The C. neoformans FKBP12 protein has marked sequence identity with FKBP12 proteins from other organisms. The alignment of the human (64),
Drosophilamelanogaster(accessionno.U41441),Neurosporacrassa(68),C.albicans(25),S.cerevisiae(34),andC.neoformansFKBP12proteinsisdepicted.Theoverall
levelsofaminoacididentityareshownaspercentages.Aminoacidresiduesthatformthehydrophobicrapamycin-andFK506-bindingpocketofFKBP12arein
boldface.Thearrowindicatestheconservedtryptophanresidueinthefrr1-3mutant.
4106 CRUZ ET AL. MOL.CELL.BIOL.
FKBP12mutations.Toaddressthis,theFRR1genewasPCR
amplified from genomic DNA isolated from the rapamycin-
FK506-resistant C. neoformans mutant strains C20F1, C20F2,
and C21F3 that were isolated from the congenic serotype D
MATa strain JEC20 and MATa strain JEC21 (48). A PCR
product of the expected size was obtained from the C21F3
mutant, whereas larger PCR products were produced from
mutant strains C20F1 and C20F2 (Fig. 6C). The FRR1 gene
from the C21F3 mutant strain was cloned and sequenced,
revealingasingleaminoacidsubstitution,Trp60Arg(frr1-3),in
the conserved tryptophan residue at the base of the FKBP12
hydrophobic drug-binding pocket (33, 69, 70) (Fig. 3). Two
independent PCR amplifications of the FRR1 gene from the
C21F3mutantwerecarriedout,confirmingthatthemutation
D
ispresentingenomicDNAandisnotaPCRartifact. o
PCR, Southern blot analysis (Fig. 6B and C), and DNA w
sequenceanalysis(datanotshown)revealedthattheFRR1loci n
in the C20F1 (frr1-1) and C20F2 (frr1-2) rapamycin-FK506- lo
a
resistantmutantstrainshaveundergoneinsertionsoftwodif- d
ferentDNAsequences.An;780-bpsequencehasinsertedin e
d
the C20F2 mutant, and ;2,200 bp has been inserted in the
f
C20F1 mutant. Because these were spontaneous mutations, ro
FIG. 4. Arapamycin-resistantTOR1mutationpreventsFKBP12-rapamycin this finding suggests that an unusual genomic instability is m
bfuinsidoinngp.rTotheeinGsAwLer4e(DcoBe)x-pTrOesRse1dFwRitBhdthoemGaiAnLw4i(ldA-Dty)p-eFKanBdPS1e2rf1u8s6io2Lnepuromteuitnanint occurring in this human fungal pathogen, which may involve h
thetwo-hybridhoststrainSMY4(fpr1TOR1-3),withorwithoutrapamycin(1 mobilizationofnoveltransposableelements(22a). tt
mg/ml).b-GalactosidaseactivitywasmeasuredbyCPRGassay,andthevalues WesternblotanalysiswithantiserumtoS.cerevisiaeFKBP12 p:
weredeterminedintriplicate. showed that these rapamycin-resistant mutant strains, includ- //m
ing the C21F3 (frr1-3) mutant containing the Trp60Arg c
FKBP12pointmutant,allfailtoexpressFKBP12protein(Fig. b
.
firmedthatthewild-typeFRR1locushadbeenreplacedbythe 6D). On the other hand, the C21F2 mutant strain, which is a
s
frr1::ADE2 disruption allele through homologous recombina- resistanttoFK506butnottorapamycin,containedawild-type m
tionwithoutectopicintegrationinallfourtransformantsthat FKBP12geneandexpressedFKBP12;inthisFKR1-1mutant .
o
were analyzed (Fig. 5B). Thus, FKBP12 is not essential for strainacalcineurinmutationmayberesponsibleforthedom- r
g
viabilityinC.neoformans. inantFK506-resistantphenotype. /

To confirm that disruption of the FRR1 gene abrogates Phenotypic analysis of C. neoformans mutants lacking o
n
FKBP12expression,totalproteinswereisolatedfromisogenic FKBP12.InadditiontorapamycinandFK506resistance(Fig.
D
FRR1wild-typeandfrr1mutantstrains,fractionatedbySDS– 5C),weaddressedseveralotherpossiblephenotypesconferred e
15% PAGE, and transferred to a nitrocellulose membrane. by FKBP12 mutations in C. neoformans. No differences in c
e
PolyclonalantibodiesraisedagainsttheyeastFKBP12protein growthratesinliquidorsolidorinrichorminimalmediawere m
(17) were used to detect the C. neoformans FKBP12 protein. observed between wild-type and frr1 mutant strains; in con- b
By Western analysis, this polyclonal antiserum recognizes a trast, S. cerevisiae FKBP12 mutant strains exhibit a modest e
r
12-kDaproteinthatispresentinextractsfromwild-typeFRR1 growthdefectinrichmedia(34).C.neoformansFKBP12mu- 2
strains and missing in frr1 mutant strains (Fig. 6D). These tant strains also had no growth defect at low (24°C) or high 1
,
findings confirm that the FRR1 gene has been disrupted by (37°C) temperature, exhibited no novel auxotrophic require- 2
homologousrecombination,thatthesizeoftheFKBP12pro- ments, grew normally on different carbon sources (glucose, 0
1
tein is in agreement with that predicted from the FRR1 gene galactose,andglycerol),werenotresistantorhypersensitiveto
8
sequence, that the FKBP12-encoding gene is unique in C. cycloheximide, and had no defects in the melanin or capsule
b
neoformans, and that a rabbit polyclonal antiserum against S. virulencefactors(datanotshown). y

cerevisiae FKBP12 cross-reacts with the C. neoformans In addition, FKBP12 was not required for mating, either g
FKBP12homolog. unilaterally(MATafrr1orMATafrr1mutantstrainscrossedto ue
frr1::ADE2 mutants lacking FKBP12 were resistant to the MATa or MATa FRR1 wild-type strains) or bilaterally (frr1 s
t
toxic effects of FK506 and to rapamycin (Fig. 5C). In plate mutant crossed to an frr1 mutant), since abundant filaments,
assays, the MIC of either FK506 or rapamycin was 0.1 to 1.0 basidia, and viable basidiospores were produced. Thus,
mg/ml in FRR1 wild-type strains and .100 mg/ml in the FKBP12 is not required for response to pheromone, cell fu-
frr1::ADE2mutantstrainslackingFKBP12.QuantitativeMIC sion,filamentation,nuclearmigration,basidiumformation,nu-
and minimum fungicidal concentration determinations in liq- clearfusion,meiosis,orsporulation(datanotshown).FKBP12
uid culture further reveal that frr1 mutants are completely wasnotrequiredforproduction,secretion,orresponsetothe
resistanttothetoxiceffectsofbothdrugs(datanotshown;see mating pheromone MFa1, because conjugation tubes were
alsoreferences48and49).Thus,FKBP12mediatesFK506and observed to the same extent when a plasmid expressing the
rapamycinactioninC.neoformans. MFa1 pheromone was introduced into a MATa frr1 mutant
Spontaneous FKBP12 mutations confer rapamycin resis- strainandintotheisogenicMATaFRR1wild-typestrain(data
tance in C. neoformans. In previous studies, we identified re- not shown). Finally, FKBP12 was not required for haploid
cessive mutations that render C. neoformans rapamycin and fruiting, because filaments were observed to similar extents
FK506 resistant (48) (Fig. 6A). Because both drugs are toxic whenapGAL7-STE12expressionplasmidwasintroducedinto
when bound to FKBP12, we hypothesized that the FK506- aMATafrr1mutantstrainandintotheisogenicMATaFRR1
rapamycin resistance phenotype might be attributable to wild-type strain (data not shown). In summary, FKBP12 is
VOL.19,1999 RAPAMYCIN ACTION IN C. NEOFORMANS 4107
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FIG. 5. DisruptionoftheC.neoformansFRR1geneencodingFKBP12.(A)Diagramofthefrr1::ADE2genedisruption.TherestrictionmapofthegenomicFRR1 u
e
geneisshown.TheFRR1genewasdisruptedbyinsertingablunt-endedADE2geneintoanRsrIIrestrictionsite.R,EcoRI;H,HindIII.(B)Confirmationofthe s
frr1::ADE2disruptionbySouthernanalysis.GenomicDNAfromtheisogenicFRR1wild-typestrainM049(H99Dade2)andfourfrr1::ADE2disruptionmutantstrains t
was cleaved with EcoRI (R) and HindIII (H), electrophoresed in a 0.8% agarose gel, and transferred to nitrocellulose. The membrane was hybridized to a
random-primed32P-labelled700-bpgel-purifiedfragmentspanningtheFRR1gene.PositionsofDNAsizemarkersareshownontheleft.NotethatthereisaHindIII
siteintheADE2marker;hence,twofragmentsarisefromthefrr1::ADE2allele.Inaddition,integrationoftandemcopiesofthefrr1::ADE2disruptionalleleresults
inmoreintensehybridizationwiththefrr1::ADE2allelethanwiththewild-type(WT)locus.(C)Anfrr1::ADE2mutantstrainisrapamycinandFK506resistant.
Wild-typeFRR1strainM049andanisogenicfrr1disruptionmutantlackingFKBP12weregrownfor72honYPDmediumcontaining1mgofrapamycinorFK506
permlat37°C.
dispensable for normal vegetative growth and growth during ployedarabbitmodelofcryptococcalmeningitis(50).Rabbits
stress and for physiological and differentiation events during were immunosuppressed with corticosteroids and inoculated
haploidfilamentationandmating. intrathecally with isogenic FRR1 wild-type and frr1 mutant
FKBP12isnotrequiredforC.neoformansvirulence.Given strains. Survival of C. neoformans cells was determined by
thatFKBP12isthetargetoftwopotentantifungaldrugsthat removal of CSF and quantification of CFU by serial dilution
bind and inhibit enzymatic activity, we tested if FKBP12 is and plating. As shown in Fig. 7A, both the wild-type and
involvedinC.neoformansvirulence.Forthispurpose,weem- FKBP12 mutant strains persisted in the CSF of the immuno-
4108 CRUZ ET AL. MOL.CELL.BIOL.
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FIG. 6. Spontaneous frr1 mutations confer rapamycin and FK506 resistance in C. neoformans. (A) FRR1 wild-type (H99, JEC20, and JEC21) and isogenic s
frr1::ADE2,frr1-1,frr1-2,frr1-3,andFKR1-1mutantstrainsweregrownonYPDmediumcontainingrapamycin(1mg/ml),FK506(1mg/ml),orCsA(100mg/ml).Rand t
S,drugresistantandsensitive,respectively.(B)SouthernanalysisofgenomicDNAfromisogenicwild-typeFRR1andfrr1rapamycin-FK506-resistantmutantstrains.
GenomicDNAwascleavedwithEcoRI(R),HindIII(H),orPstI(P),electrophoresedina0.8%agarosegel,transferredtonitrocellulose,andhybridizedtoa700-bp
FRR1geneprobe.PositionsofDNAsizemarkersareshownontheleft.(C)PCRamplificationofthefrr1mutantlocusinstrainsC20F1,C20F2,andC21F3.Genomic
DNAwasPCRamplifiedwithapairofprimersdirectedagainsttheN-terminalregionoftheFKBP12gene(primers1and2)oragainsttheC-terminalregionofthe
FKBP12gene(primers3and4).ThelongerPCRproductsinlanes5and6resultingfromstrainsC20F1andC20F2wereclonedandsequenced,revealingnovelDNA
sequencesof;2,200and;780bpthathaveinsertedintotheFKBP12locusinstrainsC20F1andC20F2andareflankedbyFKBP12genesequenceinbothcases.Lane
M,markers.(D)Rapamycin-resistantmutantsfailtoexpressFKBP12.Proteinextractsfromwild-typeandisogenicrapamycin-resistantmutantsweresubjectedto
SDS-PAGE,transferredtoanitrocellulosemembrane,andprobedwithrabbitpolyclonalantiserumagainstyeastFKBP12.StrainswereFRR1wild-typeserotypeA
strainH99andtheisogenicfrr1::ADE2mutantstrain,wild-typeFRR1serotypeDstrainJEC20andtheisogenicfrr1-1(C20F1)andfrr1-2(C20F2)mutantstrains,and
wild-typeFRR1serotypeDstrainJEC21andtheisogenicFKR1-1(C21F2)andfrr1-3(C21F3)mutantstrains.Onehundredmicrogramsofproteinfromthesame
extractswasanalyzedbyWesternblottingwithantiserumagainsttheC.neoformanscyclophilinAprotein(CypA)asaloadingcontrol.
suppressedanimalhostforthedurationoftheexperiment(14 differences were not statistically significant, and CSF yeast
days); although there were initial moderate reductions in the counts were similar by 2 weeks of infection. By comparison,
colony counts of the frr1 mutant strain compared to the wild several other mutations (gpa1, cna1, and nmt1) have a pro-
type in CSF obtained on days 4, 7, and 10 of infection, these foundimpactonC.neoformanssurvival,withsustainedreduc-
VOL.19,1999 RAPAMYCIN ACTION IN C. NEOFORMANS 4109
(Fig. 7B). Thus, in two different animal model systems,
FKBP12 mutant strains were as virulent as the isogenic wild-
typeparentalstrain.Theseobservationsareinaccordwiththe
findingthatFKBP12mutationsconferrednodefectsinknown
virulence factors of C. neoformans, including prototrophy,
growthat37°C,capsule,melanin,ormating.
DISCUSSION
We have elucidated the mechanisms of action of the anti-
fungaldrugrapamycininC.neoformans.Previousstudiesdem-
onstratedthatrapamycinistoxictothisorganism,andgenetic
analysis of drug-resistant mutants provided evidence that an
FKBP12 homolog might mediate rapamycin action (48), as is
D
thecaseinS.cerevisiaeandinTlymphocytes.Toaddressthis o
hypothesis, we isolated the C. neoformans genes encoding w
FKBP12 (FRR1) and the target of the rapamycin kinase ho- n
molog(TOR1). lo
a
The C. neoformans FKBP12 homolog was identified with a d
novel two-hybrid screen for rapamycin-dependent protein e
d
binding to the C. neoformans TOR1 homolog. The C. neofor-
f
mans FKBP12 protein has marked identity with known ro
FKBP12proteins(Fig.3).DisruptionoftheFKBP12-encoding m
FRR1 gene did not result in a loss of viability and conferred h
bothrapamycinandFK506resistance(Fig.5),confirmingthat tt
p
FKBP12isrequiredforbothrapamycinandFK506antifungal :
/
action in C. neoformans. Three spontaneous rapamycin- /m
FK506-resistant mutants were found to harbor mutations in c
the FRR1 gene encoding FKBP12 (Fig. 6). One mutation re- b
.
sults from a single amino acid substitution, Trp60Arg, which a
s
destabilizes FKBP12. This tryptophan residue is conserved in m
allknownFKBP12proteins,liesatthebaseofthehydrophobic .
o
drug-binding/active-site pocket, and mediates rapamycin, r
g
FK506,andsubstratebindingtoFKBP12. /

PreviousstudiesonFKBP12functioninS.cerevisiaeprovide o
n
aframeworktoconsiderFKBP12functionsinotherorganisms.
D
Forexample,yeastmutantslackingFKBP12areviableandare e
rapamycin and FK506 resistant. On the other hand, yeast c
e
FKBP12 mutants exhibit only subtle additional phenotypes, m
including a modest growth defect, enhanced recovery from b
pheromone arrest, and alterations in the regulation of the e
r
metabolicenzymeaspartokinase(2,16,34).Bycomparison,C. 2
neoformans mutants lacking FKBP12 are also viable and are 1
,
FIG. 7. FKBP12isnotrequiredforvirulenceofC.neoformans.(A)Rabbits resistant to rapamycin and to FK506. Hence, in both organ- 2
isms, FKBP12 mediates drug action. On the other hand, 0
wtyepreeFiRmRm1uCno.snuepopforremssaendswMit0h49steexrporiedsssianngdFiKnBocPu1l2at(eEd)i(nwtriladt-hteycpaellMy0w4it9hAwDiEld2- FKBP12mutationsinC.neoformansconferrednogrowthde- 18
reconstituted)andtheisogenicfrr1::ADE2mutantstrainlackingFKBP12(F). fects during vegetative growth or under stress conditions and
b
CSFwasremovedondays4,7,10,and14followinginoculation,andthenumber had no effect on mating, responses to pheromone, filamenta- y
ofsurvivingC.neoformanscells(expressedasthemeanlog10CFUpermilliliter tion,meiosis,sporulation,orvirulence. g
ofCSFfromeightrabbitsateachtimepoint)wasdeterminedbyserialdilution u
andplatingonYPDmediumwithgrowthfor3daysat30°C.Errorbarsindicate InbothS.cerevisiaeandTlymphocytes,rapamycinbindsthe e
thestandarderrorofthemean.(B)Mice(10each)wereinjectedinthelateral FKBP12protein,formingaprotein-drugcomplexthatinhibits s
tail vein with 107 cells of the FRR1 wild-type strain H99 or the isogenic theTORkinases.Toaddresswhethersuchamechanismalso t
frr1::ADE2mutantstrainlackingFKBP12.Survivalwasmonitoredandplotted
explainsrapamycinantifungalactioninahumanpathogen,we
withrespecttotime.
isolated and characterized the C. neoformans TOR1 gene,
whichencodesan;280-kDaproteinthathasmarkedidentity
withS.cerevisiaeTOR1andTOR2andthemammalianmTOR
tions of yeast counts by .10,000-fold during similar experi- homolog (Fig. 1). In the yeast two-hybrid system, the C. neo-
ments(3,42,48). formansFKBP12andTOR1proteinsphysicallyinteractonlyin
We also tested the possible effects of the frr1 mutation on thepresenceofrapamycin(Fig.4).Aspontaneousrapamycin-
virulence in the murine model of cryptococcal infection. resistantmutantthatresultsfromapointmutationintheFRB
Groupsof10micewereinjectedinthelateraltailveinwith107 domain of TOR1 (Ser1862Leu) prevents FKBP12-rapamycin
cellsoftheisogenicFRR1wild-typestrainexpressingFKBP12 binding to TOR1 (Fig. 4). Analogous mutations in the FRB
or the frr1 mutant strain lacking FKBP12. Survival was moni- domain of mTOR and the S. cerevisiae TOR1 and TOR2
toredandplotted.Inthismurinemodelsystem,thewild-type conferrapamycinresistancebysimilarmechanisms(10,15,17,
andfrr1mutantstrainswereequallyvirulent,resultingin50% 35,43,63).
mortalitybyday13to17and100%mortalitybyday33to36 Ourstudiesdemonstratethatrapamycinantifungalactionis
4110 CRUZ ET AL. MOL.CELL.BIOL.
mediated by the formation of FKBP12-rapamycin complexes nosuppressive rapamycin analogs are available. Fourth, rapa-
thatinhibitaTORkinasehomolog.Thesestudiesextendour mycin has broad-spectrum antifungal activity against several
understandingoftherapamycinmechanismofactionfromthe humanpathogens,includingC.albicans,C.neoformans,andA.
ascomycetousyeastS.cerevisiaetotheevolutionarilydivergent fumigatus(48,73).
pathogenic basidiomycete C. neoformans. Hence, the mecha- Insummary,ourstudiesdefinethemolecularmechanismof
nism of action and targets of rapamycin are conserved from rapamycinantifungalactioninthefungalpathogenC.neofor-
nonpathogenicyeaststopathogenicfungiandhumans.Given mans, reveal a novel mechanism of spontaneous mutagenesis
that rapamycin has potent antifungal activity against other in a pathogen, and suggest that nonimmunosuppressive anti-
human-pathogenic fungi and yeasts, such as C. albicans and fungal rapamycin analogs have potential as novel antifungal
Aspergillus fumigatus, FKBP12 and TOR homologs are likely agents.
conservedtargetsofrapamycininthesepathogens(25).
PreviousstudiesonrapamycinactionandtheTORkinases
inbuddingandfissionyeastsprovideaframeworktoconsider ACKNOWLEDGMENTS
the physiological roles of TOR in other organisms. The S.
We thank Andrew Alspaugh, N. Shane Cutler, Arturo Verrotti, D
cerevisiaeTOR1andTOR2proteinsshareaconservedrolein JamesWalker,andPingWangforcommentsonthemanuscript,Phil o
regulationoftranslation(4,23).InhibitionofthisTORfunc- James for yeast strain PJ69-4A, and Cristl Arndt for technical assis- w
tionbyFKBP12-rapamycinunderliestheeffectsofrapamycin tance. n
oninducingG orG cellcyclearrest(4,33)andthestimula- This work was supported in part by RO1 grants AI41937 and lo
1 0 a
tion of starvation-related events, including vacuole-mediated AI39115(toM.E.C.,J.R.P.,andJ.H.),andsupplementAI41937-S1(to d
proteindegradation(47)andmeiosis(75).TheroleofTORin M.C.C.andJ.H.)fromtheNIAIDandbyK01awardCA77075from e
regulatingtranslationhasbeenconservedfromS.cerevisiaeto the NCI (to M.E.C.). Joseph Heitman is a Burroughs Wellcome d
humans(4,5).GiventhatrapamycinistoxictoC.neoformans Scholar in Molecular Pathogenic Mycology and an associate investi- fro
gatoroftheHowardHughesMedicalInstitute.
andmediatesitseffectsviaFKBP12-dependentinhibitionofa m
TOR homolog, the TOR proteins may also play an essential h
translationfunctioninC.neoformans.TheS.cerevisiaeTOR2 REFERENCES tt
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