Role of Heme in the Antifungal Activity of the Azaoxoaporphine Alkaloid Sampangine

14 Pages·2008·0.83 MB·English

EUKARYOTICCELL,Feb.2008,p.387–400 Vol.7,No.2
1535-9778/08/$08.00(cid:1)0 doi:10.1128/EC.00323-07
Copyright©2008,AmericanSocietyforMicrobiology.AllRightsReserved.
Role of Heme in the Antifungal Activity of the Azaoxoaporphine
(cid:1)
Alkaloid Sampangine †
Ameeta K. Agarwal,1‡* Tao Xu,1‡ Melissa R. Jacob,1 Qin Feng,1 Michael C. Lorenz,2
Larry A. Walker,1,3 and Alice M. Clark1,4
NationalCenterforNaturalProductsResearch,1DepartmentofPharmacology,3andDepartmentofPharmacognosy,4Schoolof
Pharmacy,UniversityofMississippi,University,Mississippi38677,andDepartmentofMicrobiologyandMolecularGenetics,2
TheUniversityofTexasHealthScienceCenteratHouston,Houston,Texas77030
Received30August2007/Accepted2December2007
Sampangine, a plant-derived alkaloid found in the Annonaceae family, exhibits strong inhibitory activity
D
againsttheopportunisticfungalpathogensCandidaalbicans,Cryptococcusneoformans,andAspergillusfumiga-
o
tus. In the present study, transcriptional profiling experiments coupled with analyses of mutants were per- w
formedinanefforttoelucidateitsmechanismofaction.UsingSaccharomycescerevisiaeasamodelorganism, n
weshowthatsampangineproducesatranscriptionalresponseindicativeofhypoxia,alteringtheexpressionof lo
a
genes known to respond to low-oxygen conditions. Several additional lines of evidence obtained suggest that d
theseresponsescouldinvolveeffectsonheme.First,thehem1(cid:1)mutantlackingthefirstenzymeintheheme e
d
biosynthetic pathway showed increased sensitivity to sampangine, and exogenously supplied hemin partially
f
rescued the inhibitory activity of sampangine in wild-type cells. In addition, heterozygous mutants with ro
deletions in genes involved in five out of eight steps in the heme biosynthetic pathway showed increased m
susceptibility to sampangine. Furthermore, spectral analyses of pyridine extracts indicated significant accu- h
mulation of free porphyrins in sampangine-treated cells. Transcriptional profiling experiments were also tt
p
performed with C. albicans to investigate the response of a pathogenic fungal species to sampangine. Taking :
/
intoaccounttheknowndifferencesinthephysiologicalresponsesofC.albicansandS.cerevisiaetolowoxygen, /e
significant correlations were observed between the two transcription profiles, suggestive of heme-related c
.
defects. Our results indicate that the antifungal activity of the plant alkaloid sampangine is due, at least in a
s
part,toperturbationsinthebiosynthesisormetabolismofheme. m
.
o
r
g
The azaoxoaporphine alkaloid sampangine (Table 1) be- noquinonemoietyinitsstructure(Table1)thatisalsopresent
/

longs to the aporphine family of alkaloids, which have been in the closely related marine alkaloid ascididemin, which was o
n
reported to show antibacterial, antifungal, antiviral, antipara- similarlyreportedtogeneratereactiveoxygenspecies,leading
A
sitic, and antitumor activities (e.g., 11, 12, 23, 27, 28, 49, 63). toDNAdamageintumorcells(48).However,transcriptional p
Theproductionofsampangineisassociatedwithspecieswithin profilingexperimentsconductedwithascididemininM.tuber- ril
theplantfamilyAnnonaceae(custard-applefamily),including culosis indicated that the profile generated by this compound 1
2
Duguetia hadrantha and Cananga odorata (51, 57, 60). Like clustered with the profiles of known iron-scavenging agents, ,

2
otheraporphinealkaloids,sampangineisofparticularinterest andsubsequentexperimentsconfirmedthatascididemininhib-
0
asapotentialtherapeuticagent,asitexhibitsstronginhibitory ited M. tuberculosis growth through iron depletion (9). In the 1
9
activityagainstthehumanfungalpathogensCandidaalbicans, case of ascididemin, oxidative stress and DNA damage are
b
Cryptococcusneoformans,andAspergillusfumigatus(51,57).It thereforelikelytorepresentsecondaryeffectsoftheinhibitor y

also displays activity against the bacterium Mycobacterium tu- causedbydepletionofcellulariron.Thus,whethertheoxida- g
u
berculosisandthemalariaparasitePlasmodiumfalciparumand tive stress associated with the antitumor properties of sam- e
s
has activity against human cancer cells, including malignant panginereflectsitsprimarymechanismofactionremainstobe t
melanomacellsandHL-60leukemiacells(32,33,51,57). determined.
For both antimicrobial and antitumor properties, little is Further characterization of the biological activities of sam-
known concerning sampangine’s molecular mechanism of ac- panginewillbeusefulnotonlyinfacilitatingitspharmacolog-
tion.Kluzaetal.(33)havereportedthatsampangineinduces ical development but also in understanding its importance in
apoptosisinhumanHL-60leukemiacellsviathegenerationof theproducingplantspecies,whereitislikelytoplayarolein
reactive oxygen species. This has been attributed to the imi- chemical defense. In the present study, we have conducted
transcriptional profiling experiments followed by analyses of
mutants in an effort to gain insight into its mechanism of
action. Using Saccharomyces cerevisiae as a model, we show
*Correspondingauthor.Mailingaddress:NationalCenterforNat-
uralProductsResearch,SchoolofPharmacy,UniversityofMississippi, that sampangine produces a transcriptional response sugges-
University, MS 38677. Phone: [phone]. Fax: [phone]. tiveofhypoxia,causingsignificantchangesintheexpressionof
E-mail:[email protected]. genes known to respond to low-oxygen conditions. Interest-
†Supplemental material for this article may be found at http://ec
ingly,aspreviouslyobservedwithhumanHL-60leukemiacell
.asm.org/.
‡A.K.A.andT.X.contributedequallytothiswork. lines (33), sampangine also induced an oxidative stress re-
(cid:1)Publishedaheadofprinton21December2007. sponseinS.cerevisiaecells.Furtherexperimentsrevealedthat
387
388 AGARWAL ET AL. EUKARYOT.CELL
TABLE 1. Structureofsampangineandtheinvitroantifungal TABLE 2. S.cerevisiaeandC.albicansstrainsusedinthisstudy
activityofsampanginecomparedtothatofamphotericinBa
Sourceor
Strain Description
reference
S.cerevisiaestrains
S288C MAT(cid:8)SUC2malmelgal2 ATCC
CUP1flo1flo8-1
JK93d(cid:8)a MAT(cid:8)his4HMLaleu2-3,112 J.Heitman
rme1trp1ura3-52
BY4743 MATa/(cid:8)his3(cid:3)1leu2(cid:3)0 OpenBiosystems
met15(cid:3)0ura3(cid:3)0lys2(cid:3)0
BY4741 MATahis3(cid:3)1leu2(cid:3)0met15(cid:3)0 OpenBiosystems
ura3(cid:3)0
yMH339b MATaura3(cid:3)0his3(cid:3)200leu2(cid:3)0 Unpublished
Sampangine AmphotericinB lys2-128HAP1 data
Species hem1(cid:3)::kanMX
IC50 MIC IC50 MIC HEM1/hem1(cid:3) HEM1/hem1(cid:3)inBY4743 OpenBiosystems
HEM2/hem2(cid:3) HEM2/hem2(cid:3)inBY4743 OpenBiosystems
C.albicans 0.2 3.1 0.3 0.6 HEM3/hem3(cid:3) HEM3/hem3(cid:3)inBY4743 OpenBiosystems
C.glabrata 1.0 3.1 0.3 3.1 HEM4/hem4(cid:3) HEM4/hem4(cid:3)inBY4743 OpenBiosystems D
CAC...fknuremuosfieogiraimtuasns (cid:10)200...0805 660...2205 000...663 610...226 HHHHEEEEMMMM11112345////hhhheeeemmmm11112345(cid:3)(cid:3)(cid:3)(cid:3) HHHHEEEEMMMM11112345////hhhheeeemmmm11112345(cid:3)(cid:3)(cid:3)(cid:3)iiiinnnnBBBBYYYY4444777744443333 OOOOppppeeeennnnBBBBiiiioooossssyyyysssstttteeeemmmmssss own
yap1(cid:3)/yap1(cid:3) yap1(cid:3)/yap1(cid:3)inBY4743 OpenBiosystems lo
aBroth microdilution assays were performed according to CLSI guidelines sod2(cid:3)/sod2(cid:3) sod2(cid:3)/sod2(cid:3)inBY4743 OpenBiosystems a
(52,53).ThemediausedwereRPMIforA.fumigatusandCandidaspp.and cin5(cid:3)/cin5(cid:3) cin5(cid:3)/cin5(cid:3)inBY4743 OpenBiosystems d
SabourauddextroseforC.neoformans.Thetemperatureofincubationwas37°C TXSc001 pRS426inJK93d(cid:8) Thisstudy ed
forCandidaspp.and30°CforC.neoformansandA.fumigatus.Numbersarein TXSc025 pRS426-CIN5inJK93d(cid:8) Thisstudy
micrograms/milliliterandareaveragevaluesfromduplicateexperiments.The fr
strainsusedwereC.albicansATCC90028,C.glabrataATCC90030,C.kruseii C.albicansstrains om
ATCC6258,A.fumigatusATCC90906,andC.neoformansATCC90113. SC5314a URA3/URA3 21
BWP17a ura3::(cid:9)imm434/ura3::(cid:9)imm 74 h
434his1::hisG/his1::hisG t
t
arg4::hisG/arg4::hisG p
these effects are likely due to an alteration in heme metabo- KRC1c hem1(cid:3)::URA3/hem1(cid:3)::ARG4in 5 :/
/
lism. Results obtained using the important human fungal BWP17 e
c
pathogenC.albicanswereconsistentwiththoseobtainedusing aReceivedfromJosephHeitman(DukeUniversity). .a
S. cerevisiae when known differences in the physiological re- bReceivedfromMarkHickman(HarvardUniversity). s
cReceivedfromMartinBard(IndianaUniversity). m
sponses of these two fungal species to low-oxygen conditions
.
o
weretakenintoaccount.Collectively,ourresultssuggestthat r
g
heme plays an integral role in the antifungal activity of sam- /

pangine. timesgreaterthanthestandardprotocol,wasusedinordertomimicmicroarray on
cultureconditions.
A
MATERIALSANDMETHODS FordeterminationofIC50valuesinlarge-scalecultures,anovernightculture p
(startedfromasinglecolony)inlateexponentialphasewasusedtoinoculate50 r
Strainsandmedia.S.cerevisiaeandC.albicansstrainsusedinthisstudyare mlofSDmediumtoanopticaldensityat600nm(OD600)of0.1(cellcountof il 1
listed in Table 2. Synthetic dextrose (SD) medium, containing 0.67% yeast (cid:6)2(cid:5)106CFU/ml).Multiplecultureswerestartedforeachdruginordertotest 2
nitrogenbasewithoutaminoacidsand2%dextrose,wasusedtogrowwild-type four to five different drug concentrations. The cultures were incubated in an ,
S.cerevisiaeS288CandC.albicansSC5314strains.Themediumwasbuffered environmentalshaker(200rpm)andallowedtorecoverfromstationaryphase 2
0
with0.165M3-[N-morpholino]propanesulfonicacid(MOPS),andthepHwas until an OD600 of 0.2 was reached. Sampangine was then added at two- to 1
adjustedwithNaOHto7.0forS.cerevisiaeandto4.5forC.albicanstomaintain fourfold serial dilutions into each culture. Two rounds of experiments were 9
yeastmorphology.Syntheticcomplete(SC)medium,consistingofSDmedium conducted,withabroadrangeofsampangineconcentrationstestedinthefirst b
supplemented with complete supplement mixture (Qbiogene, Inc., Carlsbad, roundandanarrowrangetestedinduplicateexperimentsinthesecondround. y

CA)wasusedforgrowingS.cerevisiaedeletionmutantsobtainedfromOpen Theculturesweregrowntolateexponentialphase(17hforS.cerevisiaeand9h g
u
Biosystems(Huntsville,AL).ForC.albicans,allmutantstrainsandtheiriso- for C. albicans), and a final OD600 was measured using an Ultrospec 2000 e
genic wild-type strains were grown in YPD medium (1% yeast extract, 2% spectrophotometer(AmershamBiosciences,Piscataway,NJ).TheIC values s
peptone,2%dextrose)supplementedwithuridine(YPD(cid:1)Urimedium)at80 weredeterminedtobe1.17(cid:2)g/mlforS.cerevisiaeand6.13(cid:2)g/mlforC.50albicans t
(cid:2)g/ml.Thehem1(cid:3)strainsofS.cerevisiae(yMH339)andC.albicans(KRC1) (datanotshown).
weremaintainedinYPDmediumorYPD(cid:1)Urimediumsupplementedwith200 Cellcultureanddrugexposureformicroarrayexperiments.Asinglecolonyof
(cid:2)g/ml (S. cerevisiae ) or 50(cid:2)g/ml (C. albicans ) of(cid:4)-aminolevulinate (ALA; S.cerevisiaeorC.albicanswasinoculatedinto25mlofSDmediumandgrown
YPD(cid:1)ALAorYPD(cid:1)Uri(cid:1)ALA). overnightinanenvironmentalshaker(200rpm)untillateexponentialphase.
Allreagents,includingketoconazole,ergosterol,hemin,ALA,anddimethyl Theculturewasusedtoinoculate50mlofSDmediumtoanOD of0.1.Six
600
sulfoxide(DMSO),wereobtainedfromSigma(St.Louis,MO).Stocksolutions independent50-mlculturesweregrownforeachexperiment,threefortreated
ofketoconazoleandsampangineweremadeinDMSO.Ergosterolstocksolution and three for untreated samples. The cultures were allowed to recover from
waspreparedinTween80/ethanol(1:1[vol/vol]).A6.5mg/mlstocksolutionof stationaryphaseuntilanOD of0.2wasreached.Sampanginewasthenadded
600
hemin was prepared by dissolving in 0.1 M NaOH at 37°C, followed by the toeachcultureataconcentrationequivalenttotheIC value(1.17(cid:2)g/mlforS.
50
addition of 1 M TrisHCl, pH 7.5, to a final concentration of 0.1 M and an cerevisiaeand6.13(cid:2)g/mlforC.albicans).Controlculturesweresimultaneously
adjustmentofthefinalpHto7.0withHCl. treatedwith0.25%dimethylsulfoxide(DMSO).Thecultureswereallowedto
IC determinations for microarray experiments. In all microarray experi- growuntilanOD of0.5wasreached((cid:6)3hforS.cerevisiaeand(cid:6)1.5hforC.
50 600
ments,S.cerevisiaestrainS288CandC.albicansstrainSC5314wereused.All albicans).Cellswereharvestedbycentrifugation,flashfrozeninliquidnitrogen,
experimentswereconductedat30°CforS.cerevisiaeand37°CforC.albicans. andstoredat(cid:7)80°C.
For50%inhibitoryconcentration(IC )determinationsinsmall-scalecultures, RNApreparation.TotalRNAwasisolatedusingaQiagenRNeasyMidikit
50
brothmicrodilutionassayswereperformedaccordingtoClinicalandLaboratory (Qiagen,Valencia,CA)accordingtothemanufacturer’sinstructions,withthe
StandardsInstitute(CLSI,formerlyNCCLS)protocols(52),withthemodifica- followingmodifications:frozencellsweregroundintoapowderwithamortar
tionthattheinoculumsizewas2(cid:5)106CFU/ml.Thiscelldensity,whichis(cid:6)200 andpestleinliquidnitrogentofacilitatecelldisruption.Thecellpowderwas
VOL.7,2008 MECHANISM OF ANTIFUNGAL ACTIVITY OF SAMPANGINE 389
resuspendedin4mloflysisbufferprovidedinthekitandhomogenizedfor2min brothmicrodilutionassayswereperformedasdescribedaboveexceptthat180(cid:2)l
at30-sintervalsat25,000rpmusingaPT3100polytron(BrinkmannInstruments, oftheinoculumwasaddedtoamicroplatecontaining10(cid:2)lofsampangineand
Westbury,NY).TheRNAconcentrationandpurityweredeterminedspectro- 10(cid:2)lofeitherergosterolorhemin.Theconcentrationsofergosterolandhemin
photometricallybymeasuringtheabsorbanceat230,260,280,and320nm.The usedwere10(cid:2)g/mland32.5(cid:2)g/ml,respectively.ThegrowthofS.cerevisiaeand
purityandintegrityoftheRNAwereconfirmedbyagarosegelelectrophoresis. C.albicanscellswasnotinhibitedattheseconcentrations,asdeterminedbypilot
Microarrayanalysis:samplepreparation,hybridization,anddataanalysis. checkerboardassays.
ForS.cerevisiae,targetpreparationwasperformedaccordingtotheGeneChip Todeterminethesensitivitiesofvariousmutantstrainstosampangine,agar-
expressionanalysisprotocol(Affymetrix,SantaClara,CA).AOne-Cycletarget baseddroptestassayswereperformed.OvernightculturesatanOD of3.0
600
labelingandcontrolreagentkit(Affymetrix)wasusedforfirst-strandandsec- wereusedtoprepare1:5serialdilutionsintheappropriatemedia,andtheywere
ond-strandcDNAsynthesis,biotin-labeledcRNAsynthesis,cRNAfragmenta- spottedin3-(cid:2)lamountsontoagarplates.Plateswereincubatedfor2to3days
tion,andhybridization.TenmicrogramsoftotalRNAwasusedforfirst-strand attherequiredtemperature.FortestingtheS.cerevisiaedeletionmutantsob-
cDNAsynthesis.ThequantityofcRNAgeneratedwasdeterminedspectropho- tainedfromOpenBiosystems(Table2),overnightculturesfromsinglecolonies
tometrically,andthequalityofthecRNAandfragmentedcRNAwasanalyzed ofthemutantsandthewild-typestrainBY4743weregrowninSCbroth(plus200
onanagarosegel.FifteenmicrogramsoffragmentedcRNAwashybridizedtoa (cid:2)g/mlG418forthemutantsonly),dilutedtoanOD of3.0,andusedindrop
600
GeneChip yeast genome S98 array (Affymetrix) in a GeneChip hybridization testassaysonSCagarplatescontainingDMSOor1.25(cid:2)g/mlor2.0(cid:2)g/mlof
oven640.Washingandstainingwithstreptavidinphycoerythrinwereperformed sampangine.
using a GeneChip fluidics station 450, and scanning was performed using a Fortestingthehem1(cid:3)mutantofS.cerevisiae,overnightculturesofhem1(cid:3)
GeneChipscanner3000.Imageanalysis,scaling,andprobeset-leveldataanalysis mutantandtheparentstrain(BY4741)weregrowninYPD(cid:1)ALAbroth(200 D
wereperformedusingthedefaultparametersofGeneChipOperatingSoftware (cid:2)g/ml)andYPDbroth,respectively.Thecultureswerewashedthreetimesin o
version1.1(GCOS;Affymetrix).Thereportfilegeneratedbythesoftwarewas sterile distilled water, and cells from the mutant strain were resuspended in w
usedtoassessthequalityofeachexperiment,toensurethatthescalingfactor YPD(cid:1)ALAbroth(8(cid:2)g/ml),whilecellsfromtheparentstrainwereresus- n
was similar across the arrays, and to ensure that the values obtained from pendedinYPDbroth.Thecellswereallowedtogrowwithshakingfor(cid:6)5h.The lo
housekeepingcontrolsaswellasthespike-incontrolswerewithintherecom- cellsweredilutedtoanOD of3.0andusedindroptestassaysonYPD(cid:1)ALA a
600 d
mendedlimits. (8(cid:2)g/ml)agarplatescontainingeitherDMSOor1.25(cid:2)g/mlofsampangine. e
ForC.albicans,targetpreparationwasperformedaccordingtotheaminoallyl Plateswereincubatedfor2daysat30°C.Thehem1(cid:3)/(cid:3)strainofC.albicanswas d
labelingprotocolofTheInstituteforGenomicResearch(http://www.tigr.com). tested in a similar manner, except the medium used was YPD (cid:1) Uri, the f
r
First-strandsynthesiswasperformedwith15(cid:2)goftotalRNA.ThecDNAwas concentrationofALAusedforovernightcultureswas50(cid:2)g/ml,andthecon- o
coupledwithCy3dyeorCy5dye,andthelabeledtargetwasquantitatedbyusing centrationofsampangineusedwas5(cid:2)g/ml. m
aspectrophotometer.Equalamounts((cid:6)500ngofcDNA[each])oftheCy3-and Pyridinespectrumanalysis.Pyridinespectralanalysiswasperformedaccord- h
Cy5-labeledtargetsampleswerehybridizedtoC.albicansglassarrays.Thearrays ingtothemethodsdescribedbyFalk(16).OvernightculturesofS.cerevisiaeor tt
consistedof(cid:6)6,40070-meroligonucleotides(Qiagen)thatwereprintedonglass C.albicansgrowninSDbrothwereusedtoinoculatefreshculturesatanOD600 p:/
slides (Microarrays, Inc., Nashville, TN). Arrays were hybridized and washed of0.1.Afteronedoubling,sampanginewasaddedtoeachcultureataconcen- /
e
according to the protocol outlined in the Corning UltraGAPS coated slides trationequivalenttotheIC50value(1.17(cid:2)g/mlforS.cerevisiaeand6.13(cid:2)g/ml c
instruction manual. Arrays were scanned using a ScanArray 5000 scanner for C. albicans). Control cultures were simultaneously treated with 0.25% .a
(PerkinElmer,Wellesley,MA),andimageanalysiswasperformedusingQuant- DMSO.Thecultureswereallowedtogrowfor(cid:6)4doublings.Equalnumbersof s
m
Arrayversion3.0software(PerkinElmer).Thequalityofeachexperimentwas cellsfromthecorrespondingtreatedanduntreatedcultureswereharvestedby
.
assessed using the Scatter Plot feature of QuantArray (Cy5 signal intensity centrifugationandwashedoncewithsteriledistilledwater.Thecellpelletswere o
plottedagainstCy3signalintensityofallspots)toensurethatthemajorityof resuspendedinalkalinepyridine(pyridine:0.1NNaOH,2:1[vol/vol]),andincu- rg
spotswereevenlydistributedclosetothe1:1identityline. batedfor10mininthedark.Celllysateswereclearedbycentrifugationand /
DifferentiallyexpressedgeneswereidentifiedusingBRBArrayToolsversion analyzedspectrophotometrically.Twoindependentexperimentswereperformed o
n
3.2.3softwaredevelopedbyRichardSimonandAmyPengLam(http://linus.nci withindependentlygrowncultures.
A
.nih.gov/BRB-ArrayTools.html).ForS.cerevisiae,datafromCHPfilesgenerated Detectionofcarbonylatedproteins.AnOxyBlotproteinoxidationdetection
p
withGCOSsoftware(background-correctedandnormalizedsignalvaluesafter kitfromMillipore(Billerica,MA)wasusedtodetectcarbonylatedproteins.The r
probe set-level data analysis) were uploaded into BRB Array Tools. For C. kitprovidesreagentsfortheimmunodetectionofcarbonylgroupsintroduced il
1
albicans,datageneratedusingQuantArraysoftware(background-correctedsig- intoproteinsthathaveundergoneoxidativemodification.Carbonylatedproteins 2
nalintensities)wereuploadedandwerenormalizedusingtheLowessalgorithm weresuccessfullydetectedwiththekitinatrialexperimentperformedwithS. ,

availableinBRBArrayTools.Forbothdatasets,signalvalueswerelogtrans- cerevisiaecellsthatwereexposedto1mMHO inearlylogphasefor3h(data 2
2 2 0
formedandfilteredusingthedefaultparametersavailabletoremoveprobesets notshown).Todetectoxidativestressduetosampangine,anovernightculture 1
or spots with very low signal values, missing signal values, and similar signal ofS.cerevisiaewasgrowninSDbroth(MOPSbuffered;pH7.0)andusedto 9
valuesacrossallthearraysinagivenexperiment.Differentiallyexpressedgenes inoculatefreshmediumatanOD600of0.1.Afteronedoubling,eithersampang- b
wereidentifiedbyperformingatwo-classcomparisonbetweenuntreatedand ine(1.17(cid:2)g/ml)orDMSO(0.25%)wasaddedtothecultures.Thecellswere y

treatedclassesusingthedefaultstatisticalparametersavailableinBRBArray allowedtogrowandwereharvestedbycentrifugationatonedoubling(2.5h)and g
Tools,andgeneswithaPvalueof(cid:1)0.001wereconsideredtobesignificant. twodoublings(5h)aftertreatment.Five-milliliteraliquotsofcellswerewashed u
e
Gene annotations were obtained from the Saccharomyces Genome Database twotimeswithsteriledistilledwater,andthepelletswerestoredat(cid:7)80°C.The s
(SGD)ortheCandidaGenomeDatabase(CGD).TheGeneOntology(GO) experimentalconditions,includingmedia,temperature,aeration,andconcen- t
TermFinderandGOSlimMappertoolsinSGDorCGDwasusedtodistribute trationofsampangine,wereidenticaltotheconditionsusedinthetranscript
thegenesintoGO-basedcategories,andoverrepresentedGOtermswereiden- profilingexperiments.Twoindependentexperimentswereperformedwithinde-
tifiedforeachdataset. pendentlygrowncultures.
SensitivityassayswithS.cerevisiaeandC.albicansstrains.Brothmicrodilu- Proteinextractswerepreparedbytreatingthecellpelletswith50(cid:2)lofY-PER
tion assays were performed according to CLSI guidelines (52). An overnight reagent(PierceBiotechnology,Inc.,Rockford,IL)containing1%(vol/vol)beta-
culturestartedfromasinglecolonywasdilutedintheappropriatemediumafter mercaptoethanoland10mMphenylmethanesulfonylfluoride,accordingtothe
comparisontothe0.5McFarlandstandardtoaffordafinalinoculumof(cid:6)1(cid:5)104 manufacturer’sinstructions.Theproteinconcentrationwasdeterminedusingthe
CFU/ml. After dilution, 190 (cid:2)l of the inoculum was added to a 96-well flat- Bradfordassay.Twentymicrogramsofproteinfromeachextractwasderivatized
bottommicroplate(Corning,Inc.,Corning,NY)containing10(cid:2)lofsampangine with2,4-dinitrophenylhydrazine(DNPH)inthepresenceof6%sodiumdodecyl
atvariousconcentrations.Sampanginewaspreparedasastocksolutionof400 sulfate(SDS),accordingtotheinstructionsintheOxyBlotkitmanual.Identical
(cid:2)g/mlinDMSO,whichwastwofoldseriallydilutedinDMSO,followedbya negativecontrolreactionswereperformedforeachextracttogeneratenonderi-
fivefolddilutionintheappropriatemedium.Thestartingconcentrationofsam- vatizedsamples.ThesampleswereseparatedbySDS-polyacrylamidegelelec-
pangineinthefinalassaywas4(cid:2)g/ml.Themicroplateswerereadat600nmprior trophoresisfollowedbyWesternblotting.Theblottedmembranewasincubated
to and after incubation using a BioTek Powerwave XS microplate reader withprimaryantibody(1:145dilution),specifictotheDNPmoiety,followedby
(BioTekInstruments,Inc.,Winooski,VT).ForS.cerevisiae,thetemperatureof incubation with horseradish peroxidase-conjugated secondary antibody (1:275
incubationwas30°C,andthetimeofincubationwas48h.ForC.albicans,the dilution). Chemiluminescent detection was performed with reagents from an
temperatureofincubationwas37°C,andthetimeofincubationwas24h. ECLWesternblottingsystem(AmershamBiosciences).Toensureequalloading
Totestwhetherergosterolorhemincouldalterthesensitivitytosampangine, andintactproteins,apolyacrylamidegelwasrunwithanaliquotofthederivat-
390 AGARWAL ET AL. EUKARYOT.CELL
izedandnonderivatizedsamplesandstainedwithGelCodeBluestainreagent lationofgenesrequiredformitochondrialrespirationanden-
(PierceBiotechnology). ergymetabolismregulatedbythetranscriptionfactorsHap1p
Multicopylibraryscreening.S.cerevisiaestrainJK93d(cid:8)wastransformedwith
andHap2/3/4/5p,theupregulationofgenesrequiredforsterol
amulticopygenomicDNAlibraryreceivedfromJ.Heitman(DukeUniversity),
whichwasconstructedinthe2(cid:2)vectorpRS426.Transformantswereselectedon homeostasisandcellwallfunctionundertheregulationofthe
SC-Ura(SCminusUra)agarplates.Atotalof(cid:6)1(cid:5)106colonieswereobtained transcriptionfactorUpc2p,andtheupregulationofgenesreg-
andpooledinSC-Urabroth.Glycerolstocks(15%[vol/vol]finalglycerolcon- ulatedbythetranscriptionfactorRox1p.Allofthesetranscrip-
centration)werepreparedfromaliquotsofthepooledmaterialandstoredat tionfactorsareknowntoregulategeneexpressioninresponse
(cid:7)80°C.Thepreparationofglycerolstocksfrompooledtransformantseliminates
to altered heme and/or cellular oxygen levels. Many of the
theneedtoperformahigh-efficiencyyeasttransformationforeachscreening.To
screenthelibraryagainstsampangine,aglycerolstockwasthawedonice,diluted genes responding to sampangine are also regulated by these
toadensityof1(cid:5)106cells/ml,andplatedon10SC-Uraagarplates(150by15 transcriptionfactors(Table3).
mminsize)with0.3mlofdilutedcellsperplate.After2daysofincubationat Severalgenesthatwererepressedbysampanginetreatment
30°C,thetransformantswerepooledanddilutedtoadensityof1(cid:5)106cells/ml,
are regulated by the heme-dependent transcription factors
and0.3mlofcellswasplatedontoeachof10SC-Uraagarplates(150by15mm
insize)containing4(cid:2)g/mlsampangine.Atthisconcentrationofsampangine, Hap1pand/orHap2/3/4/5p(Fig.1andTable3).Themajority
yeastcellscarryingvectoralone(strainTXSc001)platedatasimilarcelldensity ofthesegenesareactivatedbythesetranscriptionfactorsun-
showednogrowthupto5days.Atotalof50transformantsthatsurvivedon der aerobic growth conditions (reviewed in references 35, 78, D
sampangineplateswereobtainedafter5daysofincubationat30°C.Plasmid and80),andrepressedunderanaerobicconditions(8,36,40, o
DNAwasisolatedfromthetransformantsusingaQIAPrepSpinMiniPrepkit w
41, 58, 65, 67). The downregulation of these “aerobic” genes
(Qiagen)withthemodificationthatcelllysiswasperformedwithP1bufferinthe n
presenceofacid-washedglassbeads(Sigma)inabeadmill(Retsch,Inc.,New- during anaerobiosis is consistent with the fact that many of lo
town, PA). The plasmids were transformed into Escherichia coli DH5(cid:8) cells, them encode cytochromes, cytochrome-related oxidoreducta- a
d
amplified,andpurifiedusingstandardprotocols.TheplasmidDNAwasdigested ses, and NADH-dependent dehydrogenases that play impor- e
to check for the presence of inserts and to identify duplicate clones. Twelve d
tantrolesinmitochondrialelectrontransportandrespiration.
clones had no inserts or showed indications of missing restriction sites and f
rearrangements. Of the remaining 38 clones, 14 different restriction patterns Thus,sampanginetreatmentappearstoblocktheHap1p-and ro
were obtained upon digestion with EcoRI. Clones representing each pattern Hap2/3/4/5p-dependentexpressionofgenesnormallyinduced m
were partially sequenced using T7 and T3 primers, and the genes present in undertheaerobicconditionsemployedinthepresentwork.It h
themwereidentifiedusingSGD.All14clonescontainedtheCIN5genepresent shouldbenotedthattheHAP1geneintheS.cerevisiaeS288C tt
p
in genomic fragments of various lengths. Clones carrying only the full-length
CIN5 open reading frame were retransformed into JK93d(cid:8) cells, and their strainusedinthisstudyisinterruptedbyaTy1elementinthe ://
resistancetosampanginewasconfirmedindroptestagarassays,asdescribed 3(cid:11)endofitsopenreadingframe(datanotshown),resultingin ec
above. One of the confirmed S. cerevisiae strains carrying pRS426-CIN5 was a hypomorphic allele that is partially functional under anaer- .a
designatedTXSc025. obicconditions,thusretainingsomeactivityinthisstrain(19). s
Profilingdataaccessionnumber.Thetranscriptionalprofilingdatadescribed m
Sampanginetreatmentalsoresultedintheinductionofsev-
inthisarticlehavebeendepositedinNCBI’sGeneExpressionOmnibus(GEO; .o
http://www.ncbi.nlm.nih.gov/geo/)andareaccessiblethroughGEOseriesacces- eral genes known to be regulated by the transcription factor rg
sionno.GSE10104. Upc2p(Fig.1andTable3).Allofthesegeneswerepreviously /

shown to be induced under anaerobic conditions in various o
n
microarraystudieswithS.cerevisiae(8,36,40,41,58,65,67).
RESULTS A
Interestingly, two of the Upc2p-induced genes listed in Table p
r
Transcriptome response to sampangine in S. cerevisiae. 3,AUS1andDAN1,areinvolvedintheuptakeofextracellular il
Sampanginedisplayspotentantifungalactivity,comparableto sterol (73). Two additional sampangine-induced genes not 1
2
thatofthewidelyuseddrugamphotericinB,againstthefungal known to be regulatory targets of Upc2p, SUT1, and SUT2 ,

2
pathogens C. albicans, Candida glabrata, Candida kruseii, C. (listedunder“SterolTransport”inFig.1),arealsoinvolvedin 0
neoformans,andA.fumigatus(Table1).Inordertounderstand steroluptake(2,54).ThegeneSUT1wasalsoreportedtobe 1
9
thecellulareffectsofsampangineinfungalcells,wemadeuse upregulated under low-oxygen conditions by both Tai et al.
b
of S. cerevisiae, an established model organism that has been (65)andTerLindeetal.(67).Anincreasedexpressionlevelof y

extensively used for elucidating the molecular targets of anti- steroluptakegenesinS.cerevisiaeunderanaerobicconditions g
u
fungalandtherapeuticcompounds(reviewedinreferences56 is associated with the phenomenon of “aerobic sterol exclu- e
s
and64).Atranscriptionalprofilingstudywasconductedusing sion” (45). Under aerobic conditions, S. cerevisiae cells fulfill t
S. cerevisiae cells that were treated with a concentration of their sterol requirements via de novo synthesis of ergosterol,
sampangine that resulted in 50% growth inhibition (1.17 (cid:2)g/ while under anaerobic conditions, sterol biosynthesis is com-
ml). Genes that were significantly differentially expressed be- promised and the cells are dependent upon sterol uptake for
tween the treated and untreated cells (P value, (cid:1)0.001) were survival. Interestingly, sterol uptake can occur aerobically in
identified as described in Materials and Methods. A total of heme-deficient mutants (e.g., 22, 43), indicating a close rela-
204genesshowedexpressionchangesinresponsetosampang- tionship between oxygen, heme, and sterol homeostasis in S.
ine,with129genesshowingincreasedexpressionand75genes cerevisiae.
showing decreased expression. Data for a few selected genes Rox1p is a heme-dependent repressor of anaerobic genes
areshowninFig.1tohighlighttheoverallbiologicalresponse (reviewed in references 35, 78, and 80), and several Rox1p-
tosampangine. regulated anaerobic genes were induced in the presence of
Theglobaltranscriptionalresponsetosampanginerevealed sampangine(Fig.1andTable3).Allofthesegeneshavealso
several similarities to that shown by S. cerevisiae cells grown beenpreviouslyreportedtobeinducedunderanaerobiccon-
under anaerobic conditions in previous studies (8, 36, 40, 41, ditions in S. cerevisiae (8, 36, 40, 41, 58, 65, 67). The genes
58, 65, 67). In these studies, the most consistent gene expres- ARE1 (sterol metabolism; 71), HEM13 (heme biosynthesis;
sion responses to anaerobiosis appeared to be the downregu- 31), and ATF1 (synthesis of unsaturated fatty acids; 17) all
VOL.7,2008 MECHANISM OF ANTIFUNGAL ACTIVITY OF SAMPANGINE 391
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a
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o
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A
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il
1
2
,

2
0
1
FIG. 1. DistributionofgenesrespondingtosampangineinS.cerevisiae.Dataareshownforasubsetofgenesthatweresignificantlyinduced 9
orrepressed(Pvalue,(cid:1)0.001)inthreebiologicalreplicatesamples.DatawereorganizedintovariousbiologicalprocessesusingGOTermFinder b
y
andGOSlimMappertoolsinSGD.GOtermsshownarethosethatwereconsideredsignificantlyoverrepresentedbytheanalyses.Theterms“cell
g
wallorganization”and“otherprocesses”wereincludedseparatelytohighlightadditionalgenesthatarerelevanttotheoverallbiologicalresponse
u
tosampangine.Forsimplicity,notall“childterms”withina“parentterm”areshown.Numbersinparenthesesrepresentaveragechangeingene e
expression.Positivevaluesindicateinduction,andnegativevaluesindicaterepression.AcompletelistofallsignificantgenescanbefoundinTable s
t
S1inthesupplementalmaterial.
encodeproteinsthatplayimportantrolesinheme-dependent oxygencanbeattributedtothefactthatmanyiron-containing
pathways. proteins play important roles in oxygen-dependent reactions
Treatmentwithsampangineresultedinthedownregulation involvingheme,suchashemebiosynthesis,sterolmetabolism,
ofnumerousgenesinvolvedinirontransportandhomeostasis lipiddesaturation,andmitochondrialrespiration(reviewedin
(Fig. 1 and Table 3) that are known to be regulated by the reference30).
iron-sensingtranscriptionfactorAft1p(reviewedinreference Overall, the transcriptional response seen for S. cerevisiae
30).Interestingly,Laietal.(40)havereportedthedownregu- cellsfollowingexposuretosampangineinthepresentworkis
lation of genes required for iron homeostasis (FRE1, UTR1, highlyreminiscentofthatobservedforS.cerevisiaecellsgrown
FTR1, and FET3 ) inS. cerevisiae in response to anaerobiosis. under conditions of reduced oxygen availability. One notable
Crisp et al. (13) have also shown that the inhibition of heme distinctionisthatsampanginetreatmentresultedintheinduc-
biosynthesis results in decreased transcription of iron uptake tion of genes involved in responses to oxidative stress, which
genes in S. cerevisiae. This interplay between iron, heme, and are typically observed to be downregulated under low-oxygen
392 AGARWAL ET AL. EUKARYOT.CELL
TABLE 3. Distributionofsampangine-respondinggenesinS.cerevisiaebasedontheirpotentialregulationbyknowntranscriptionfactors
Regulator Targetgenes Biologicalprocess References
Hap1p,Hap2/3/4/5p COX4,COX5A,COX6,COX12,COX13,COX15, Respirationandelectron 35,66,a68,
QCR2,QCR7,QCR9,QCR10,CYC1,DLD1, transport 78,80
RIP1,PET10,NDE1,NDI1
Upc2p DAN1,DAN2,DAN4,TIR1,TIR3,TIR4 Cellwallorganization 1,66,a73
DAN1,AUS1 Steroltransport
HES1,UPC2 Lipidmetabolism
PAU1,PAU3,PAU4,PAU6,PAU7 Otherprocesses
Rox1p ATF1,ARE1 Lipidmetabolism 1,35,36,
DAN1,TIR1,TIR3 Cellwallorganization 66,a68
ANB1,HEM13,AAC3 Otherprocesses
Aft1p FRE1,FRE4,FRE5,FET3,FTR1,FIT2,FIT3, Ironiontransport 30,62,66,a
ARN1,ARN2,ENB1,SIT1 76,77 D
ARN2,SIT1,TIS11,HMX1,CCC2,YLR126C Ironionhomeostasis o
w
aDescribestheYEASTRACTdatabase,whichisacuratedrepositoryofmorethan27,800regulatoryassociationsbetweentranscriptionfactorsandtargetgenesin n
S.cerevisiae.Targetgeneslistedabovefallintothecategoryof“documentedassociation”inthedatabase,basedonexperimentalevidenceofatleastoneofthe lo
following:(i)changeinexpressionoftargetgeneduetoadeletion(ormutation)inthegeneencodingthetranscriptionfactorand(ii)bindingofthetranscriptionfactor a
tothepromoterregionofthetargetgene. d
e
d

f
r
o
conditions in S. cerevisiae (e.g., 41). Oxidative stress-related Intriguingly, we also observed that wild-type S. cerevisiae m
genes induced by sampangine included SRX1, AAD4, GPX2, cells grown on agar plates in the presence of high concentra- h
t
AAD6, GTT2, CCP1, TSA2, CTA1, and PRX1 (Fig. 1). These tions of sampangine accumulated a red pigmentation after 3 tp
genes encode various peroxidases, catalases, and oxidoreduc- daysofgrowth(Fig.2C).Thisresultissuggestiveofapossible :/
/
tases that play a role in defense responses to oxidative stress. disruptioninhemebiosynthesis,resultingintheaccumulation e
c
However, this result is consistent with a previous report doc- of heme biosynthetic precursors, such as porphyrins, in the .a
umentingtheproductionofreactiveoxygenspeciesinhuman cells.Forexample,asimilarphenotypehasbeenobservedfor sm
cancer cells following exposure to sampangine (33). The oxi- the pop1 mutants of Saccharomycopsis lipolytica, which pro- .
o
dativestressresponsetosampangineisdiscussedinmorede- ducered-pigmentedcoloniesduetotheaccumulationofpro- r
g
tailbelow(seeDiscussion). toporphyrinIX,theimmediateprecursorofhemeintheheme /
Role of heme in the activity of sampangine against S. cer- o
biosynthetic pathway (7). In addition, mutants of S. cerevisiae n
evisiae.AnaerobicresponsesinS.cerevisiaeareregulatedatthe
withpartialdefectsinhemebiosynthesisalsoaccumulatepor- A
levelofgeneexpression,andhemeplaysapivotalroleinthis p
phyrins and exhibit red fluorescence under UV light (34, 69).
r
regulation (reviewed in reference 35). When oxygen levels Tofurtherinvestigatethenatureofthepigmentaccumulating il
decrease, heme synthesis declines, and transcription factors 1
inS.cerevisiaecellsexposedtosampangine,wecomparedthe 2
such as Hap1p, Hap2/3/4/5p, Rox1p, and Upc2p are directly ,
absorptionspectraofalkalinepyridineextractspreparedfrom 2
affected,resultingintheinductionofanaerobicgenesandthe wild-type cells treated with sampangine to those treated with 0
downregulation of aerobic genes. Given the important role 1
DMSOsolventalone.Comparedwithsolvent-treatedcells,the 9
playedbyhemeintheregulationofoxygen-responsivegenes,
sampangine-treatedcellsexhibitedincreasedabsorbance,with b
wewerethereforealsointerestedindeterminingwhetherthe y
amaximumatapproximately400nm(Fig.2D),characteristic
inhibitory effects of sampangine involved perturbations in g
oftheSoretpeakoffreeporphyrins(16).Theseresultssuggest u
hememetabolism. e
that sampangine exposure in S. cerevisiae might result in the s
To examine the potential involvement of heme, we first t
testedthesusceptibilitytosampangineofahem1(cid:3)mutantofS. accumulation of heme biosynthetic intermediates, arising ei-
ther through the inhibition of a heme biosynthetic enzyme or
cerevisiae, which has a deletion in the gene encoding the en-
throughtheinhibitionofsecondaryprocessesthatregulatethe
zyme ALA synthase that catalyzes the first step in the heme
biosyntheticpathway(70).Thehem1(cid:3)mutantshoweddramat- pathway.
Anadditionalindicatorofpotentialhemeinvolvementisthe
icallyincreasedsensitivitytosampanginecomparedtothecor-
responding wild-type strain (Fig. 2A). Since this mutant re- observationthatS.cerevisiaecellsgrownunderaerobiccondi-
quires ALA for survival, we included ALA in the medium; tions become dependent upon exogenous sterol for survival
however, in order test the effect of heme deficiency on sam- under conditions of heme deficiency (e.g., see references 22
pangine sensitivity, a low concentration of ALA was used (8 and 43). Therefore, we determined whether the presence of
(cid:2)g/ml). We also tested whether the presence of exogenous exogenous sterol would alter the sensitivity of S. cerevisiae to
hemewouldalterthesensitivitytosampangineinwild-typeS. sampangine.TheIC ofsampangineincreasedfrom0.2(cid:2)g/ml
50
cerevisiaecells.Thepresenceofexogenouslysuppliedhemein to0.55(cid:2)g/ml,andtheMICincreasedfrom0.5to2.0(cid:2)g/ml,in
theformofheminincreasedtheIC forsampanginefrom0.2 thepresenceofergosterol(Fig.2E),indicatingthatergosterol
50
(cid:2)g/ml to 0.6 (cid:2)g/ml and the MIC from 0.5 (cid:2)g/ml to 2.0 (cid:2)g/ml reduced the susceptibility of S. cerevisiae cells to sampangine.
(Fig.2B). Thisresultindicatesthat,justlikehemebiosyntheticmutants,
VOL.7,2008 MECHANISM OF ANTIFUNGAL ACTIVITY OF SAMPANGINE 393
wild-type S. cerevisiae cells treated with sampangine have the
abilitytoimportexogenoussterolunderaerobicconditions.
To further explore the effect of sampangine on heme me-
tabolism, heterozygous mutants of S. cerevisiae carrying dele-
tions in genes involved in each step of the heme biosynthetic
pathwaywerealsoanalyzed.Thishaploinsufficiencyapproach
hasbeensuccessfullyusedinseveralstudiestodeterminethe
moleculartargetsofvariousclinicallyrelevantdrugs(e.g.,see
references4,20,and46).AscanbeseeninFig.3,mutantswith
heterozygous deletions in the HEM2 and HEM4 genes, in-
volvedinthesecondandfourthstepsinthehemebiosynthetic
pathway, showed a distinct increase in susceptibility to sam-
pangine compared to the wild-type strain, and smaller but
clearly discernible increases in sensitivity were also observed
formutantswithheterozygousdeletionsintheHEM1,HEM13, D
andHEM14genes.Theseresultsprovidefurtherevidencethat o
w
the inhibitory activity of sampangine against S. cerevisiae is
n
likely associated with effects on heme biosynthesis or heme- lo
requiringpathways. a
d
Sampangine induces oxidative stress in S. cerevisiae. As e
d
mentioned above, in addition to the induction of anaerobic

f
genes,sampanginetreatmentalsoresultedintheupregulation ro
of a variety of genes associated with responses to oxidative m
stress. The latter observation is consistent with previous re- h
t
portsdocumentingtheoccurrenceofoxidativestressinhuman t
p
cancer cells following exposure to this compound (33). To :/
/
confirm if similar effects occur in S. cerevisiae cells, we moni- e
c
toredproteincarbonylationlevelsafterexposuretosampang- .a
ine.Oxidativestressresultsintheformationofcarbonylgroups s
m
onproteinsduetotheoxidationofspecificaminoacids,which
.
o
canbedetectedbyderivatizationtoDNP-hydrazonefollowed r
g
byWesternanalysisusingaDNP-specificantibody(42).Using /

the same experimental conditions as were used in the tran- o
n
scriptional profiling experiments, S. cerevisiae cells were ex-
A
posedtosampangine(1.17(cid:2)g/ml)foronedoubling(2.5h)and p
r
twodoublings(5h).Asacontrol,cellswerealsotreatedwith il
DMSOsolventalone.AscanbeseeninFig.4A,sampangine- 1
2
treatedcellscontainedincreasedlevelsofcarbonylatedprotein ,

2
compared with control treatments, demonstrating the occur- 0
renceofoxidativestress.Inaddition,thesignalswerestronger 1
9
at 5 h, indicating that the amount of protein carbonylation in
b
responsetosampangineincreasedovertime. y

WealsomadeuseofS.cerevisiaemutantscarryingdeletions g
u
ingenesplayingcriticalrolesincellularresponsestooxidative e
s
stress, which exhibit hypersensitivity to chemical agents pro- t
motingtheformationofreactiveoxygenspecies.Mutantswith
deletions in YAP1, which encodes a transcription factor re-
quiredforoxidativestresstolerance,aswellasSOD2encoding
FIG. 2. RoleofhemeintheactivityofsampangineinS.cerevisiae. mean (SEM). (C) S. cerevisiae S288C colonies grown on YPD agar
(A)Dilutions(fivefold)ofwild-type(BY4741)andhem1(cid:3)(yMH339) werestreakedonSDagarmediumcontainingvariousconcentrations
strainsgrownasdescribedinMaterialsandMethodswereinoculated of sampangine and incubated for 3 days at 30°C. The 0 represents
ontoYPD(cid:1)ALA(8(cid:2)g/ml)agarmediumandincubatedfor2daysat mediumcontainingsolvent(DMSO).(D)Absorptionspectraofalka-
30°C. (cid:7)SMP, medium containing solvent (DMSO); (cid:1)SMP, medium linepyridineextractspreparedfromS.cerevisiaeS288Ccellstreated
containing sampangine at 1.25 (cid:2)g/ml. (B) Results shown are from withsampangine(solidline)orwithDMSOsolvent(dashedline).(E)
broth microdilution assays performed in triplicate using S. cerevisiae Resultsareshownfrombrothmicrodilutionassaysperformedintrip-
strain S288C in the presence of various concentrations (Conc.) of licate with S. cerevisiae strain S288C in the presence of various con-
sampanginewith32.5(cid:2)g/mlhemin((cid:1)hemin)orwithouthemin((cid:7)he- centrationsofsampanginewith10(cid:2)g/mlergosterol((cid:1)Ergo)orwith-
min). Percent growth is shown as the mean (cid:12) standard error of the outergosterol((cid:7)Ergo).Percentgrowthisshownasthemean(cid:12)SEM.
394 AGARWAL ET AL. EUKARYOT.CELL
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FIG. 3. Effectofsampangineonthegrowthofheterozygousmutantsharboringdeletionsinhemebiosyntheticgenes.Wild-type(BY4743), /e
HEM1/hem1(cid:3), HEM2/hem2(cid:3), HEM3/hem3(cid:3), HEM4/hem4(cid:3), HEM12/hem12(cid:3), HEM13/hem13(cid:3), HEM14/hem14(cid:3), and HEM15/hem15(cid:3) strains c
weregrownasdescribedinMaterialsandMethods.Dilutions(fivefold)werepreparedfromeachculture,inoculatedontoSCagarmedium,and .a
incubatedfor3daysat30°C.(cid:7)SMP,mediumcontainingsolvent(DMSO);(cid:1)SMP,mediumcontainingsampangineat2.0(cid:2)g/ml. s
m
.
o
r
g
superoxide dismutase, showed increased sensitivity to sam- anismbywhichCIN5overexpressionconfersresistancetovar- /

panginecomparedtothecorrespondingwild-typeparentstrain ioustoxinsisunclearatpresent.Interestingly,CIN5wasitself o
n
(Fig.4B).Takentogether,theresultsshowninFig.4indicate foundtobeupregulatedinresponsetosampangine(seeTable
A
thatsampangineexposureinducesoxidativestressinS.cerevi- S1inthesupplementalmaterial),suggestingthatthistranscrip- p
r
siaecells,aspreviouslyobservedforhumancancercells;thus, tionfactorcouldalsoberequiredforlong-termadaptationto il
thegenerationofoxidativestresspotentiallyrepresentsacom- theeffectsofhemedeficiencythatareimplicatedinsampang- 1
2
mon consequence of exposure to this compound in diverse ine’sactivityinthepresentstudy. ,

2
biologicalsystems. Transcriptome response to sampangine in C. albicans. The 0
OverexpressionofCIN5confersresistancetosampanginein mostfrequentlyencounteredCandidaspeciesinopportunistic 1
9
S.cerevisiae.Tofurtherexplorethecellularresponsestosam- fungalinfectionsofimmunocompromisedhostsisC.albicans,
b
pangine in S. cerevisiae, an overexpression screening was per- also considered to be the fourth-most-common cause of hos- y

formed using a genomic DNA library constructed in the 2(cid:2) pital-acquiredbloodstreaminfections,withamortalityrateof g
u
vector pRS426. Thirty-eight insert-containing sampangine-re- approximately40%(50,75).WhileS.cerevisiaeisanexcellent e
s
sistanttransformantswereobtainedfromaprimaryscreening model organism, it is nonpathogenic and does not provide t
of approximately one million library clones, and all resistant informationonpossiblefungalpathogenesis-relatedresponses
clones were subsequently identified as containing the CIN5 tosampangine,whichcouldproveusefulforitspotentialphar-
gene within their genomic inserts. One representative clone, macological development. Furthermore, sampangine displays
designated TXSc025, exhibiting reduced sensitivity to sam- potent activity against C. albicans (Table 1); therefore, tran-
panginerelativetotheemptyvector-containingclone,isshown scriptionalprofilingstudieswerepursuedforthisorganismas
in Fig. 5A. The corresponding deletion mutant of CIN5 was well.
similarlycomparedtowild-typeS.cerevisiaecells,anditexhib- AsdescribedabovefortheS.cerevisiaetranscriptionalpro-
itedmildhypersensitivitytothecompound(Fig.5B). filing studies, experiments were conducted using C. albicans
Cin5p,alsoknownasYap4p,belongstotheYap1pfamilyof cellstreatedwithaconcentrationofsampanginethatresultsin
transcriptionfactorsandhasbeenshowntobeinducedunder 50% growth inhibition (6.13 (cid:2)g/ml). Genes that were signifi-
various stress conditions, such as osmotic, heat, metal, and cantly differentially expressed between the treated and un-
oxidative stresses (55). The overexpression of Cin5p has also treatedcells(Pvalue,(cid:1)0.001)wereidentifiedasdescribedin
been shown to confer resistance to DNA-damaging agents, Materials and Methods. A total of 191 genes showed expres-
suchascisplatinandmethylmethanesulfonate(18),andtothe sionchangesinresponsetosampangine,with157genesshow-
antimalarial drug quinidine (14); however, the specific mech- ing increased expression and 34 genes showing decreased ex-
VOL.7,2008 MECHANISM OF ANTIFUNGAL ACTIVITY OF SAMPANGINE 395
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FIG. 4. RoleofoxidativestressintheactivityofsampangineinS.cerevisiae.(A)S.cerevisiaeS288Ccellsinearlyexponentialphaseweregrown /

inthepresenceorabsenceofsampangine(SMP)fortheindicatedamountoftime.Cellswereharvested,proteinextractswereprepared,and20 o
(cid:2)gofproteinfromeachextractwasderivatizedwithDNPH.ThederivatizedproteinswereseparatedbySDS-polyacrylamidegelelectrophoresis, n

blottedtopolyvinylidenedifluoridemembrane,anddetectedwithanti-DNPantibody.Astainedproteingelonwhichaliquotsofthesamesamples A
wereseparatedisshownontheright.(B)Dilutions(fivefold)ofwild-type(BY4743),yap1(cid:3)/(cid:3),andsod2(cid:3)/(cid:3)strainsgrownasdescribedinMaterials p
amneddiMumethcoondtsawineinregisnaomcpualantgeidneonatto1.S2C5(cid:2)agga/mrlm. edium and incubated for 2 days at 30°C. (cid:7)SMP, medium containing solvent (DMSO); (cid:1)SMP, ril 1
2
,

2
0
pression.DataforafewselectedgenesareshowninFig.6to YAH1,MET1),homologsofwhichinS.cerevisiaeareknownto 1
9
highlighttheoverallbiologicalresponsetosampangine. be involved in heme A and siroheme biosynthesis (6, 25).
b
The majority of genes upregulated by sampangine in C. Furthermore, the induction of several additional genes in C. y

albicans are involved in mitochondrial functions related to albicanscouldpotentiallybeaccountedforbyperturbationsin g
u
sulfur metabolism, heme metabolism, respiration, electron heme metabolism. For example, the induction of genes in- e
s
transport, mitochondrial protein synthesis, mitochondrial or- volved in sulfur metabolism could be related to the fact that t
ganization and biogenesis, and protein targeting to the mito- some of the early steps in methionine biosynthesis are cata-
chondrion (Fig. 6). While these results suggest fundamental lyzed by heme-containing enzymes, such as those encoded by
differencesintheoveralltranscriptomeresponsesofS.cerevi- MET10 and ECM17 (Fig. 6) (47). A few additional heme-
siae and C. albicans to sampangine (as discussed further be- related genes that were induced include genes that are in-
low), striking similarities were also observed. First, genes in- volved in the import of cytochrome precursors into the mito-
volved in ergosterol biosynthesis were downregulated in both chondria (e.g., TOM22 and TOM40, listed under “Protein
organisms: ERG5, ERG20, ERG28, and CYB5 in S. cerevisiae TargetingtoMitochondrion”inFig.6)(72)andintheattach-
(Fig.1)andERG6andERG25inC.albicans(seeTableS2in mentofhemetocytochromec(e.g.,CYC3,listedunder“Res-
the supplemental material). Second, as seen for S. cerevisiae, pirationandElectronTransport”inFig.6)(15).Thus,signif-
severalgenesinvolvedinironhomeostasisweredownregulated icant parallels appear to exist between the transcriptome
in C. albicans; these included FTH1, RBT5, SIT1, and HMX1 responses to sampangine in C. albicans and in S. cerevisiae,
(seeTableS2inthesupplementalmaterial).Third,theheme suggesting that sampangine exposure could similarly affect
biosynthetic gene HEM13 was upregulated in both organisms heme-relatedpathwaysinthetwoorganisms.
(Fig. 1 and 6). A few additional genes involved in heme me- Role of heme in the activity of sampangine against C. albi-
tabolism were upregulated in C. albicans (COX10, COX15, cans. As described above for S. cerevisiae, additional experi-
396 AGARWAL ET AL. EUKARYOT.CELL
D
FIG. 5. EffectofCIN5overexpressionanddeletiononsusceptibilitytosampangine.(A)JK93d(cid:8)cellscontainingvectorpRS426(TXSc001)and o
w
aCIN5-overexpressingplasmid(TXSc025)wereinoculatedontoSC-Uraagarmediumandincubatedfor5daysat30°C.(B)Dilutions(fivefold) n
ofwild-type(BY4743)andcin5(cid:3)/(cid:3)strainswereinoculatedontoSCagarmediumandincubatedfor2daysat30°C.(cid:7)SMP,mediumcontaining lo
solvent(DMSO);(cid:1)SMP,mediumcontainingsampangineat4(cid:2)g/ml(A)and1.25(cid:2)g/ml(B). a
d
e
d

f
ments were performed to further examine the possibility that reduced the susceptibility of S. cerevisiae to sampangine, sug- r
o
heme-relatedeffectswerealsoinvolvedinsampangine’sactiv- gestingthatexposuretosampanginemimicshemedeficiency, m
ityagainstC.albicans.First,asseenforS.cerevisiae(Fig.2A), aconditionwhichallowstheuptakeofexogenoussterol.(iii)A h
ahem1(cid:3)/(cid:3)mutantofC.albicansshowedincreasedsensitivity hem1(cid:3) mutant of both S. cerevisiae and C. albicans exhibited tt
p
to sampangine (Fig. 7A). As in the S. cerevisiae experiment, increasedsensitivitytosampangine,andexogenoushemepro- :/
/
ALA was added to the medium at a low concentration (8 vided as hemin reduced the sensitivity to sampangine in both e
c
(cid:2)g/ml) in order to test the effect of heme deficiency on sam- organisms. (iv) Upon prolonged exposure to high concentra- .
a
panginesensitivity.Second,wetestedwhetherthepresenceof tionsofsampangine,bothorganismsproducedredpigmenta- s
m
exogenoushemewouldalterthesensitivitytosampangineinC. tion which can be attributed to increased levels of heme bio-
.
o
albicans cells. The presence of exogenously supplied heme in synthetic precursors (free porphyrins) in the cells, based on
r
g
0th.0e25fo(cid:2)rmg/molfthoe0m.2in(cid:2)ign/cmrela(sFeidg.t7hBe).ICT5h0irdo,fassamobpsaenrgviendefofrromS. scpereecvtirsoiapehomtoumtaenttrsicwaintahlyhsiesteorfoazlykgaoliunsedpeyrleidtiionnesexintragcetsn.e(sv)inS-. o/
n
cerevisiae(Fig.2C),whenC.albicanscellsweregrownonagar volvedinfiveoutofeightstepsinthehemebiosyntheticpath-
A
plates in the presence of high concentrations of sampangine wayshowedincreasedsensitivitytosampangine. p
for3days,thecellsaccumulatedareddishpigmentation,sug- Whileourresultsstronglysuggestthattheantifungalactivity ril
gestingthattherewasanaccumulationofporphyrininterme- of sampangine most likely involves a disruption in heme me- 1
2
diates(Fig.7C).Thisaccumulationwasfurtherconfirmedbya tabolismorfunction,theexactmechanismbehindthiseffectis ,

2
comparative analysis of pyridine spectra from sampangine- unclearatthistime.Onepossibilityisthatsampanginedirectly
0
treated and untreated C. albicans cells. Sampangine-treated inhibitstheactivityofoneoftheenzymesinthehemebiosyn- 1
9
cells revealed an absorption peak at (cid:6)400 nm, indicative of theticpathway.However,itisunlikelythatsampangineinhibits
b
freeporphyrins(Fig.7D). the enzyme ALA synthase (the first committed step in heme y

Wealsotestedtheeffectofergosterolonthesusceptibilityof biosynthesis), given that exogenously supplied ALA had no g
u
C.albicanstosampangine.IncontrasttoS.cerevisiae(Fig.2E), apparenteffectonsensitivitytosampangine(datanotshown). e
s
ergosterol did not alter the sensitivity of C. albicans cells to In addition, the production of red pigmentation in the pres- t
sampangine(Fig.7E).Thisresultisinagreementwiththefact enceofsampangine,indicatingtheaccumulationofporphyrin
thathememutantsofS.cerevisiaerelyonexogenoussterolfor intermediates,wouldbeconsistentwithpathwayinhibitionoc-
growth (22, 43), while a hem1(cid:3) mutant of C. albicans cannot curringatalaterstep.
growonexogenouslysuppliedsterol(5). Sampangine could also interfere with heme metabolism
through indirect mechanisms, such as causing a reduction in
available iron or the misdirection of biosynthetic intermedi-
DISCUSSION
ates, since heme biosynthesis in S. cerevisiae occurs as a mul-
Usingacombinationofgenomicandgeneticapproaches,we tisteppathwayspatiallyseparatedbetweenthecytosolandthe
haveshownthathemeplaysanimportantroleintheantifungal mitochondria(37).Hemebiosynthesisistightlycoupledtoiron
activity of the plant alkaloid sampangine. This conclusion is availability due to the requirement of iron by the enzyme
based on several lines of evidence obtained from the present ferrochelatase,whichcatalyzesthefinalstepinthehemebio-
work. (i) The transcriptome response of S. cerevisiae to sam- synthetic pathway (38). Two mitochondrial iron transporters,
pangine is indicative of a hypoxia-like response, which is Mrs3pandMrs4p,havebeenshowntoplayanimportantrole
knowntobemediatedbyhemethroughthetranscriptionfac- intherapidtransportofironintothemitochondriaforheme
tors Hap1p, Hap2/3/4/5p, Rox1p, and Upc2p. (ii) Ergosterol biosynthesis in S. cerevisiae (79). Interestingly, MRS4 is in-