Survival in the Presence of Antifungals

15 Pages·2007·0.54 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.282,NO.45,pp.32935–32948,November9,2007
©2007byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Survival in the Presence of Antifungals
GENOME-WIDEEXPRESSIONPROFILINGOFASPERGILLUSNIGERINRESPONSETO
SUBLETHALCONCENTRATIONSOFCASPOFUNGINANDFENPROPIMORPH*□S
Receivedforpublication,July17,2007,andinrevisedform,August29,2007 Published,JBCPapersinPress,September5,2007,DOI10.1074/jbc.M705856200
VeraMeyer‡§1,RobbertA.Damveld§2,MarkArentshorst§,UlfStahl‡,CeesA.M.J.J.vandenHondel§,
andArthurF.J.Ram§
Fromthe‡DepartmentofMicrobiologyandGenetics,InstituteofBiotechnology,BerlinUniversityofTechnology,
Gustav-Meyer-Allee25,D-13355Berlin,Germanyandthe§DepartmentofMolecularMicrobiology,InstituteofBiology,
LeidenUniversity,Wassenaarseweg64,2333ALLeiden,TheNetherlands
How yeast cells respond to cell wall stress is relatively well teristicsduringdevelopmentalprocessesinfungidependupon
understood;however,howfilamentousfungicopewithcellwall the temporal regulation and spatial localization of cell wall
damageislargelyunexplored.Herewereportthefirsttranscrip- components and thereby ordered cell wall deposition (1–3).
tome analysis of Aspergillus niger exposed to the antifungal Moreover, cell wall rearrangements that guarantee the struc-
compoundscaspofungin,aninhibitorof(cid:1)-1,3-glucansynthesis, turalintegrityofthecellwallareofvitalimportancetowith-
and fenpropimorph, which inhibits ergosterol synthesis. The standenvironmentalstressconditionssuchasosmoticstressor
presence of sublethal drug concentrations allowed A. niger to thepresenceofantifungalsubstancescompromisingcellwall
adapt to the stress conditions and to continue growth by the and/orcellmembraneintegrity(4–6).Topreventcelllysisand
establishmentofnewpolarityaxesandformationofnewgerm to ensure cell survival, fungi have developed mechanisms to
tubes.Bycomparingtheexpressionprofilebetweencaspofun- sensecellsurfacestressandtorespondtothesestressesviaa
gin-exposedandnonexposedA.nigergermlings,weidentifieda remodelingofthecellwall(seealsoreviewsinRefs.1,7,8).
totalof172responsivegenesoutof14,509openreadingframes The composition of fungal cell walls and the mechanisms
presentontheAffymetrixmicroarraychips.Among165up-reg- involved in ensuring cell surface integrity have been studied
ulatedgenes,mainlygenespredictedtofunctionin(i)cellwall mostintensivelyinthemodelyeastSaccharomycescerevisiae.
assemblyandremodeling,(ii)cytoskeletalorganization,(iii)sig- ThecellwallofS.cerevisiaeconsistsofamoderatelybranched,
naling, and (iv) oxidative stress response were affected. Fen- flexible (cid:1)-1,3-glucan network to which to its external face
propimorph modulated expression of 43 genes, of which 41 (cid:1)-1,6-glucanchainsareboundwhichinturnarelinkedtoGPI
showedenhancedexpression.Here,genespredictedtofunction mannoproteins.Attheinnersideofthe(cid:1)-1,3-glucannetwork,
in(i)membranereconstruction,(ii)lipidsignaling,(iii)cellwall chitinchainsareattached(reviewedinRef.9).Uponcellwall
remodeling,and(iv)oxidativestressresponsewereidentified. stress,thecellwallbecomesreinforcedbyamassiveincreaseof
Northernanalysesofselectedgeneswereusedtoconfirmthe thechitincontentinthelateralwall(9–12)andbyincreased
microarrayanalyses.Theresultsfurthershowthatexpressionof incorporationofcertaincellwallproteinsinthecellwall(12–
the agsA gene encoding an (cid:2)-1,3-glucan synthase is up-regu- 14).Atleastthreesignalingpathways,thePkc1p-Slt2psignaling
lated by both compounds. Using two PagsA-GFP reporter pathway(alsonamedcellwallintegrity(CWI)3pathway),the
strainsofA.nigerandsubjectingthemto16differentantifungal general stress response pathway mediated by Msn2p/Msn4p,
compounds, including caspofungin and fenpropimorph, we and the Ca2(cid:1)/calcineurin pathway have been shown to be
could show that agsA is specifically activated by compounds involvedinthecellwallcompensatoryresponseofS.cerevisiae
interferingdirectlyorindirectlywithcellwallbiosynthesis. (15). Moreover, genome-wide surveys and large scale pheno-
typic analyses, aiming at an integrated view of pathways
involvedincellwallassemblyandintegrityofS.cerevisiae,have
Thefungalcellwallisadynamicstructurethatisessentialfor furthercontributedtotheunderstandingofitscellwallbiology.
sustaining cell morphology and for protection against life- AssummarizedbyLesageandBussey(1),fivelevelsofregula-
threateningenvironmentalconditions.Morphologicalcharac- tioncontributetoacontrolledcellwallassemblyandthereby
coordinate cell morphogenesis in yeast as follows: (i) the cell
wall synthetic machinery, (ii) surface signaling, (iii) cell cycle
*TheworkperformedattheKluyverCentreforGenomicsofIndustrialFer- regulation,(iv)cellpolarization,and(v)thesecretorymachin-
mentationwassupportedbytheNetherlandsGenomicsInitiativeandbya erycoupledwithproteinrecyclingthroughendocytosis.
grantfromtheDutchFoundationforTechnicalResearch.Thecostsofpub-
In contrast to yeast, information about cell wall biology in
lication of this article were defrayed in part by the payment of page
charges.Thisarticlemustthereforebeherebymarked“advertisement”in filamentousfungiandthemechanismsimportantformaintain-
accordancewith18U.S.C.Section1734solelytoindicatethisfact.
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplementalTablesS1andS2. 3Theabbreviationsusedare:CWI,cellwallintegrity;GPI,glycosylphospha-
1Towhomcorrespondenceshouldbeaddressed.Tel.:[phone];Fax: tidylinositol;GFP,greenfluorescentprotein;8-Br-cAMP,8-bromo-cAMP;
49-30-31472922;E-mail:[email protected]. MAP,mitogen-activatedprotein;ROS,reactiveoxygenspecies;BI,Branch-
2Presentaddress:DSMFoodSpecialties,A.Fleminglaan1,2600MADelft,The ingIndex;PtdIns(4,5)P ,phosphatidylinositol4,5-biphosphate;TOR,target
2
Netherlands. ofrapamycin.
NOVEMBER9,2007•VOLUME282•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 32935
This is an Open Access article under the CC BY license.
GlobalResponsesofA.nigertoAntifungals
ing cell surface integrity is sparse. Although there are indica- compounds that affected the morphology of A. niger. Caspo-
tionsthatarchitecturalprinciplesidentifiedinS.cerevisiaemay fungin, known as an inhibitor of (cid:1)-1,3-glucan synthesis in S.
also be valid for filamentous fungi (9, 16), remarkable differ- cerevisiae(36),andfenpropimorph,reportedasaninhibitorof
encesdoexistbothinthecompositionofthecellwallaswellas S. cerevisiae ergosterol biosynthesis (37), were selected, as
therelativeamountsofthecomponents.Whereasthepresence applicationofthesecompoundstoA.nigerresultedinmorpho-
of (cid:1)-1,6-glucan in S. cerevisiae is undisputed, its presence in logicalalterations.Bothcompoundswereappliedatsublethal
filamentousfungiiscontroversiallydiscussedand,ifpresent,is concentrationstoA.niger,andglobalexpressionprofilingwas
onlyinminoramounts(17).Thecellwalloffilamentousfungi performed,aimedatthefollowing:(i)identificationofcellular
alsocontainspolymersthatarenotpresentintheS.cerevisiae responsesinvolvedincellintegrityandadaptationtogrowth-
cellwallsuchas(cid:1)-1,4-glucans,(cid:2)-1,3-glucans,andgalactoman- inhibitory conditions, (ii) identification of drug-specific
nans(18,19).Moreover,thedistributionofpolymers,suchas responsesandtherebyfirstinsightsintotheirmodeofactionin
chitin, varies markedly between yeast and filamentous fungi A.niger,(iii)identificationofgeneswhoseproteinproductsare
(20).Thecompensatoryreactionsinresponsetocellwallstress importantfortheestablishmentandmaintenanceofpolarized
infilamentousfungiwerefirstanalyzedinAspergillusniger.It growth,and(iv)preventionofsecondarydrugeffectsornon-
has been shown that the cell wall stress response of A. niger specificresponsesrelatedtocelldeath.Theexperimentalsetup
involvesinducedexpressionofagsA,encodingaputative(cid:2)-glu- ofthestudyinvolvedtheuseofyoung,unbranchedgermlingsas
cansynthase(21).Inaddition,theRlmAtranscriptionfactoris, alteration of their morphology can easily be monitored and
similarlytoitsS.cerevisiaehomologueRlm1p,requiredforthe quantifiedbymicroscopicmeansandbecausegermlingsrepre-
up-regulation of cell wall stress-induced genes (4). Further- sent a more homogeneous cell population compared with
more,thecellwallstressresponseofA.nigeris,likeinS.cerevi- mycelialhyphae.
siae,alsoaccompaniedbyincreasedchitindeposition,suggest-
EXPERIMENTALPROCEDURES
ing that part of the remodeling mechanism via the CWI
pathwayisconservedamongfungi(22). Strains, Growth Conditions, and Antifungal Compounds—
Overthepastyears,evidenceforaclosecorrelationbetween The A. niger strains N402 (wild type, laboratory collection),
cellwallassemblyandcellmorphologyinfilamentousfungihas RD6.47 (38), and JvD1.1 (this study) were used. The strains
beenaccumulating.Severalstudieshaveshowntheimportance were grown at 37°C (unless otherwise stated) in minimal
of chitin synthesis in determining hyphal morphology. For medium(39)orcompletemedium(CM),consistingofminimal
example,AspergillusnidulansandAspergillusoryzaestrains,in medium(MM)supplementedwith1%yeastextractand0.5%
which several chitin synthase genes have been disrupted, are casaminoacids.Fermentationmedium(FM)wascomposedof
hyperbranched(23,24).Anarrestinpolarizedgrowthandthe 0.75%glucose,0.45%NH Cl,0.15%KH PO ,0.05%KCl,0.05%
4 2 4
induction of (sub)apical branches have been reported for A. MgSO ,0.1%saltsolution(39),and0.003%yeastextract.The
4
niger when treated with the antifungal protein AFP, most pH of FM was adjusted to pH 3. Caspofungin was purchased
recentlyshowntobeaninhibitorofchitinsynthaseactivityin from Merck (Cancidas(cid:2)) and fenpropimorph from Sigma,
A. niger (25). Likewise, inhibition of (cid:1)-glucan synthesis in respectively. AFP was isolated and purified from Aspergillus
AspergillusfumigatusandA.oryzaebypneumocandinsorbya giganteusculturesasdescribedinRef.40.Allotherantifungal
mutationin(cid:1)-1,3-glucansynthasegeneinNeurosporacrassa compoundsweremadeavailablebyBASF.
causes considerable changes in morphology, such as swollen ScreeningforMorphologicalChangesInducedbyAntifungal
germ tubes and highly branched hyphal tips (26, 27). Finally, Compounds—5(cid:2)105conidiaofstrainN402wereinoculatedin
inhibitionofthecross-linkingofglycanfibersbytheantifungal Petridishescontaining5mlofliquidMMsupplementedwith
agentcalcofluorwhitecausesanarrestofpolarizedgrowthand 0.003%yeastextract.Priortoinoculation,twocoverslipswere
swellingofhyphaltipsinA.niger(21).Remarkably,inhibition placedontothebottomofthePetridishes.Sporeswereallowed
of polarized growth of filamentous fungi has not only been to germinate for 5 h at 37°C, andsmall germ tubes became
describedasaconsequenceofdirectcellwallperturbationsbut visibleinmorethan90%ofthespores.Compoundswereadded
alsoforconditionsthatratherindirectlyaffectcellwallbiosyn- at various concentrations, whereas the negative control was
thesis. For example, interference with the assembly of the supplemented with the same volume of H O. The following
2
cytoskeleton(28),cAMP-dependentproteinkinasesignaling(29– range of concentrations was tested: caspofungin, 1 ng/ml to
31),calciumsignaling(32),plasmamembraneintegrity(33),and 12.5 (cid:3)g/ml; AFP, 0.2–0.9 (cid:3)g/ml; fenpropimorph, 0.5–60
withthesecretorymachinery(34)causedapparentmorphological (cid:3)g/ml; myriocin, 30–200 (cid:3)g/ml; 8-Br-cAMP, 1–10 mM; caf-
changes (see also Ref. 27). However, the underlying molecular feine,1–10mM.Afterfurthercultivationfor1hat37°C,germ-
mechanismsandtheinterconnectionsofthedifferentpathways lings that were adherent to the coverslips were analyzed by
withcellwallassemblyarefarfrombeingunderstood. microscopy(seebelow).Fromatleast50germlingspersample,
TherecentsequencingandannotationofthegenomeofA. themorphologywascharacterizedasbeingeitherunbranched
niger (35) and the availability of the Affymetrix microarray (germlings with a single germ tube) or branched (germlings
technologyforA.nigernowmakeitfeasibleforthefirsttimeto withapicaland/orsubapicalbranches).A“BranchingIndex(BI)”
studythemechanismsinvolvedinensuringcellsurfaceinteg- wascalculatedthatwasdefinedasfollows:BI(cid:3)((cid:4)branchedgerm-
rityanditscorrelationwithpolarizedgrowthinthisbiotechno- lings)(cid:2)((cid:4)branched(cid:1)unbranchedgermlings)(cid:5)1.
logically important filamentous fungus. To get first insights Construction of GFP Reporter Strains—The reporter strain
into these processes, in this study we screened for antifungal containing the PagsA-H2B-GFP-TtrpC reporter construct
32936 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER45•NOVEMBER9,2007
GlobalResponsesofA.nigertoAntifungals
(RD6.47) has been described previously (38). The reporter TABLE1
strain with a cytoplasmically expressed GFP under control of Primersusedinthisstudy
theagsApromoter(PagsA-GFP-TtrpC)wasconstructedasfol- Openreading Gene Primerpairs(sequence5(cid:3)-to3(cid:3)-oriented)
framecode
lows.PlasmidPagsA-GFP-TtrpCwasconstructedbyligationof
An09g04010 chsC Forward,TGGTGATCATACGCTCTCCAAA
a 2-kb SalI-NcoI fragment, containing PagsA from PagsA-
Reverse,GAACGAACCAAGGAACGCAC
uidA-TtrpC (21), into an SalI-NcoI opened PagsA-H2B:: An03g05940 gfaA Forward,TCAGTCTGGTGAAACAGCCG
GFP-TtrpC vector, thereby removing the PagsA-H2B and Reverse,GGGTAGTGCCTCGTCAACAAGA
An12g10200 Forward,GGCAGCTATGGCAACTACCAAG
replacing it with PagsA to give pJD1. For the Agrobacterium- Reverse,CTCGTCACGCTTGTACGCCT
mediatedtransformation,thePagsA-GFP-TtrpCconstructwas An18g03740 mkkA Forward,CCTCGATGTGGATGATTTGGAT
Reverse,TCTCGGACCATCGAATACCG
insertedintoabinaryvector.The(cid:6)3-kbHindIIIfragmentcon- An01g03350 Forward,GCGCCTGGAGCAGTTCTACAT
tainingPagsA-GFP-TtrpCfrompJD1wasclonedintoaHindIII Reverse,GGCGTATCCGAAGAAGAGCAT
An01g07000 Forward,CCATCACCATTGGCCTACCTT
openedpTAS5vectortogivepTAS5-PagsA-GFP-TtrpC.The Reverse,CCAGAGAAGATCGCCAAAGGA
pTAS5vectorconsistsofthebinaryvectorpSDM14(41)with An03g06410 Forward,TGACTTTCCTGGAGAAGGCG
Reverse,CTTCGCCGGTAAAGTAGCCC
thehygromycinexpressioncassettefrompAN7.1(42)inserted An01g14200 Forward,GAGGTCGAGTCTCACAACGATG
between the borders. pTAS5-PagsA-GFP-TtrpC was trans- Reverse,GGTGGCATGACCAACCGATA
formedtoA.nigerstrainN402usingtheAgrobacteriumstrain
LBA1100.Hygromycin-resistanttransformantsweresubjected RNAExtraction,ExpressionProfiling,andNorthernAnalysis—
toSouthernanalysistoconfirmcompleteintegration.Genomic Total RNA was isolated from homogenized mycelial samples
DNAwasisolatedaccordingtoRef.43anddigestedwithPstIor usingTRIzolreagent(Invitrogen).RNAqualitycontrol,label-
SstIItodeterminethecopynumber.StrainJvD1.1,containing ing,microarrayhybridization,andscanningwereperformedat
multiple copies ((cid:4)2) integrated in the genome (data not ServiceXS(Leiden,TheNetherlands).Briefly,RNAqualitywas
shown),wasselectedasareporterstrain. verifiedusingAgilentBioanalyzer“LabonChip”system(Agi-
ScreeningofAntifungalCompoundsinGlassBottomMicro- lent Technologies, Palo Alto, CA). Processing, labeling, and
titer Plates—The PagsA-GFP (JvD1.1) and PagsA-H2B-GFP hybridizationofcRNAtoA.nigerAffymetrixGeneChipswere
(RD6.47)reporterstrainswereusedtoscreenantifungalcom- performed according to Affymetrix protocols for “Eukaryotic
pounds for their ability to induce the cell wall integrity of A. Target Preparation” and “Eukaryotic Target Hybridization.”
niger.Conidia(2(cid:2)104)fromthereporterstrainswereinocu- For washing and staining, the protocol “Antibody Amplifica-
lated in each well of 96-well optical glass bottom microtiter tion for Eukaryotic Targets” was followed. Hybridized probe
plates(Nuncart)in100(cid:3)lof2(cid:2)CMandgrownfor6hat37°C. arrayslideswerescannedwithaG2500AGeneArrayScanner
Aftersporegermination,100(cid:3)lofa2-folddilutionseriesfor (AgilentTechnologies)ata3-(cid:3)mresolutionandawavelength
eachantifungalcompoundwasaddedtoindividualwells.The of570nm.AffymetrixMicroarraySuitesoftwareMAS5.0was
effectofeachcompoundwastestedforatleastsevendifferent usedtocalculatesignalsandpvaluesandtosettheabsolutecall
concentrations. After adding the antifungal solution, the flagofthealgorithm,whichindicatesthereliabilityofthedata
microtiterplateswereincubatedforanadditional3hat30°C. pointsaccordingtoP(present),M(marginal),andA(absent).
Afterdiscardingthemediumbyinvertingthemicrotiterplate, Microarrayanalysesforeachcondition(control,caspofungin-
germlings that adhered to the bottom of each well were ana- treatedgermlings,andfenpropimorph-treatedgermlings)were
lyzed by microscopy (see below). As a positive control, strain performedoncellsobtainedfromtwoindependentbioreactor
MA26.1,containingPgpdA-H2B-GFP-TtrpCsinglecopyatthe cultivations (biological duplicate). The complete set of tran-
pyrGlocuswasused.4StrainN402wasusedasanegativecon- scriptional raw data is available as supplemental Table S1.
trol.AcquiredimageswereanalyzedforbothgrowthandGFP ExpressiondatawereanalyzedusingtheprogramGeneSpring
levels. 7.3.(AgilentTechnologies).Fornormalization,defaultsettings
BioreactorCultivation—Freshlyharvestedconidia(5(cid:2)109) wereused(50thpercentileperchip,medianpergene).Genes
fromstrainN402wereusedtoinoculate5litersofFM.Culti- weredefinedasdifferentiallyexpressediftheirexpressionlev-
vationswereperformedinaBioFlo3000bioreactor(NewBrun- els varied at least 1.5-fold in the caspofungin- (or fenpropi-
swickScientific)usinganagitationspeedof250rpm.Temper- morph)-treatedsamplescomparedwiththecontrolandifthe
ature(37°C)andpH(setto3)werecontrolledon-lineusingthe differencewasstatisticallysignificant(Student’sttest,pvalue
program NBS Biocommand. Aeration was performed via the cutoffof0.05).
headspaceuntilthedissolvedoxygentensiondroppedto40% Northernanalysesusingeach5(cid:3)gofRNAfromthesixcon-
andwasthenswitchedtospargeraeration.After5hofcultiva- ditionswereperformedasdescribedearlier(4).RNAsamples
tion,caspofunginorfenpropimorph(dissolvedin5mlofdis- were balanced according to their content of the 18S mRNA
tilled H O) or 5 ml of distilled H O (negative control) were (datanotshown).PCRampliconsobtainedbyusingdifferent
2 2
added. After an additional hour of cultivation, 400 ml of the primerpairsaslistedinTable1werelabeledbyrandomprimer
culturebrothwerequicklyharvestedviafiltration,andmycelial labelingusing32P-labeleddATP(AmershamBiosciences)and
samples were immediately frozen using liquid nitrogen. In usedasprobesforNorthernanalysis.Hybridizationswerecar-
addition, samples were taken for microscopic analysis (see riedoutaccordingtothemanufacturer’sinstructions(Amer-
below)andcalculationoftheBIvalue. shamBiosciences).
Microscopy—Pictures of A. niger germlings were captured
4M.Arentshorst,unpublishedstrain. usinganAxioplan2(Zeiss)equippedwithaDKC-5000digital
NOVEMBER9,2007•VOLUME282•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 32937
GlobalResponsesofA.nigertoAntifungals
TABLE2
Theeffectofcaspofungin(50ng/ml)andfenpropimorph(50(cid:3)g/ml)
onbranchingefficiencyofA.nigergermlings
Morethan200germlingswerecountedforeachbioreactorruninordertodeter-
minetheBIforasingleexperiment.Foreveryduplicateexperiment,ameanBIwas
calculated.—,negativecontrol;CA,caspofungin;FP,fenpropimorph.
Bioreactorrun Compound BI MeanBI
% %
1 — 14.7 13.7(cid:8)1.0
2 12.7
FIGURE1.TheeffectofselectedantifungaldrugsonthemorphologyofA. 3 CA 43.4 43.7(cid:8)0.7
niger.Caspofungin(CA,5ng/ml),fenpropimorph(FP,5(cid:3)g/ml),andAFP(0.9 4 44.1
(cid:3)g/ml)wereaddedto1(cid:2)105/mlsporesgrownfor5hinMMat37°C.After 5 FP 33.2 30.5(cid:8)2.7
1hoffurtherincubation,germlings(n(cid:3)50–100)weremicroscopicallyana- 6 27.8
lyzed.BIisgiveninparentheses.NC,negativecontrol.Bar,10(cid:3)m.
medium(FM)wereused.Duringbioreactorruns,thedissolved
camera(Sony).Bothlight(usingDICsettings)andfluorescence oxygentensionwasfollowedandusedasanindicationforequal
images(usingGFPsettings)wereobtainedwitha(cid:2)40objec- growth behavior between the different experiments (data not
tive.ForGFPimages,afixedexposureof2swasused.Images shown).After5hoftotalcultivation,caspofunginorfenpropi-
were processed using Adobe Photoshop 6.0 (Adobe Systems morphwereadded,andthecultivationswerecontinuedforan
Inc.). additionalhour,afterwhichsamplesweretakenfordetermina-
tionoftheBIvalueandfortranscriptomicanalysis.Usingthis
RESULTS
experimentalsetup,weobservedthata10-foldincreasedcon-
Screening for Morphology-affecting Compounds—To select centrationofbothcaspofunginandfenpropimorphwasneces-
suitablecompoundsthataffectthemorphologyofA.niger,we sarytosignificantlyaffectthemorphologyofA.nigergermlings
have screened various substances proposed to interfere with when compared with the screening experiment described
fungalcellwallsynthesis(caspofungin,AFP),ergosterolsynthe- above(Table2anddatanotshown).Ontheonehandthiscan
sis (fenpropimorph), sphingolipid synthesis (myriocin), and beexplainedbythehighersporetiterusedforbioreactorinoc-
cAMP-dependent protein kinase signaling (8-Br-cAMP and ulation and on the other hand by different cultivation condi-
caffeine).Thesecompoundswereappliedindifferentconcen- tions used in both experiments. In addition to inducing the
trationsto5(cid:2)105A.nigergermlingsgrownoncoverslipsin5 formationof(sub)apicalbranches,bothcaspofunginandfen-
mlofminimalmedium,andtheireffectongermtubeelonga- propimorphwereobservedtoinducetheestablishmentofnew
tionandbranchingwasmicroscopicallyfollowed.Morpholog- polarityaxesthatstartedfromthesporeandthusresultedinthe
icalalterationswereonlyobservedinresponsetocaspofungin, formation of new germ tubes. In particular, the amount of
AFP,andfenpropimorph(Fig.1),whereasnosignificanteffect spores displaying three or four germ tubes, which are usually
wasobservedwhengermlingsweretreatedwithaseriesofcon- rarelyobservedinA.niger,wassignificantlyincreasedbyboth
centrations of myriocin, 8-Br-cAMP, or caffeine (data not antifungals(Fig.2).Thisobservationindicatedthatthegerm-
shown). In Fig. 1, the morphological changes provoked by lings may counteract the disturbance of existing polarity
caspofungin,AFP,andfenpropimorpharedepicted.Tipswell- growthsitesbytheformationofnewpolaritysites.
ing and an increase in (sub)apical branching were observed Global Gene Expression Responses to Caspofungin and
aftertreatmentofthegermlingswithaminimalconcentration Fenpropimorph—Affymetrix microarray chips representing
of5ng/ml,5(cid:3)g/ml,and0.9(cid:3)g/mlforcaspofungin,fenpropi- 14,509 open reading frames of A. niger were hybridized with
morph,andAFP,respectively.Tojudgeandquantifytheeffect RNAsamplesfromeachoftwobiologicalreplicatesofcaspo-
onmorphology,aBIwasdeterminedthatgivesthepercentage fungin-, fenpropimorph-, and nontreated samples, respec-
of germlings displaying (sub)apical branches (n (cid:7) 50). As tively,asdescribedunder“ExperimentalProcedures.”Follow-
showninFig.1,thestrongesteffectsonbranchingwereexerted ing normalization to account for deviations in hybridization
by caspofungin and fenpropimorph, where the BI value was intensity,genesshowingatleast1.5-foldchange(pvaluecutoff
about7-foldhighercomparedwiththenegativecontrol.Appli- of0.05)inexpressionlevelwereconsideredtobedifferentially
cation of higher drug concentrations did not significantly expressed.
increasetheBIbutresultedinthepresenceofdeadgermlings Atotalof172genesweredifferentiallyexpresseduponexpo-
(datanotshown). suretocaspofungin,165ofwhichshowedincreasedexpression
MorphologicalResponsestoCaspofunginandFenpropimorph— and7genesdecreasedexpression.Incomparison,atotalof43
Caspofungin and fenpropimorph were selected for further geneswasfoundtoberesponsivetotreatmentwithfenpropi-
analysis as their effect on morphology was more prominent morph,41ofthesewereup-regulatedand2weredown-regu-
comparedwithAFP.Thescreeningassaydescribedabovewas lated (supplemental Table S2). The modulated genes were
repeatedinlargescaleusingacultivationofA.nigersporesina functionally classified according to FunCat (44) as shown in
bioreactor(workingvolumeof5liters).Usingsuchanexperi- Table3.Thecategorywiththelargestnumberofknowngenes
mental design, we wanted to ensure controlled and equal thataremodulatedbybothantifungalsisthecategoryinvolved
growthconditionsbetweentreatedandnontreatedgermlings inmetabolism.
and thereby reliable expression data. An increased starting Gene Expression Responses to Caspofungin—Caspofungin
inoculum(1(cid:2)106spores/ml)andaslightlydifferentminimal hasbeenshowntobeapotentinhibitorof(cid:1)-1,3-glucansynthe-
32938 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER45•NOVEMBER9,2007
GlobalResponsesofA.nigertoAntifungals
(An18g06820/GfaA, An03g05940/GfaB, and An12g07840/
GnaA), chitin formation (An09g04010/class III chitin synthase
ChsB), (cid:2)-glucan synthesis (An09g03100/AgtA), (cid:1)-1,3-glucan
remodeling (An03g05290/BgtB, An10g00400/GelA), cross-link-
age of chitin to (cid:1)-1,6-glucan (An07g07530/CrhB, An07g01160/
ChrC), GPI anchor processing (An14g03520/DfgC), protein
mannosylation (An03g01090/HocA, An18g06500/Sec53, and
An16g04330/DpmA),andgenesencodingputativecellwallpro-
teins(An12g10200andAn14g01820).Genesinvolvedinsignaling
cascadesensuringcellwallintegritywerealsoup-regulatedsuchas
An16g04200/RhoB (similar to Rho2 GTPase of Schizosaccharo-
mycespombe,regulatorof(cid:2)-1,3-glucansynthesis),An10g00490/
Rho-GAP,andAn18g03740/MkkA(similartoS.cerevisiaeMAP
kinasekinase2involvedinCWIsignaling).
The presence of sublethal concentrations of caspofungin
allowedA.nigertoadapttoitsinhibitoryeffectinsuchaway
thatthefungussurvivedandcontinuedgrowthbytheestablish-
ment of new polarity axes and formation of new germ tubes
(Fig.3).Wethusexpected,besidesidentifyinggenesinvolvedin
FIGURE2.Caspofunginandfenpropimorphprovoketheestablishment cellwallmaintenance,toalsoidentifygenescodingforproteins
ofnewpolarityaxes.A,microscopicimagesofgermlingsthatwerehar-
vested from the bioreactor cultivations (see “Experimental Procedures”). having a function in cell growth regulation and cell polarity.
ImagesweretakenusingDICsettings;Bar10(cid:3)m.B,atleast200germlingsper Indeed,threegenesinvolvedingrowthcontrolwereup-regu-
singlefermentationrunwereanalyzedforthenumberofgermtubespresent
latedasfollows:An17g02350showingstrongsimilaritytothe
andforthepresenceofsubapicalbranches.Means(cid:8)S.D.werecalculated
fromeachtwoindependentfermentationruns.CA,cellstreatedwithcaspo- humanRas-relatedGTPaseRhebknowntobeakeycomponent
fungin; FP, cells treated with fenpropimorph; NC, negative control, cells oftheTORsignalingpathway(45),An13g00100(similartothe
treatedneitherwithcaspofunginnorwithfenpropimorph.AandB,N,num-
S. cerevisiae cell-cycle checkpoint protein kinase Dun1), and
berofgermtubespergermling;SAB,germlingswithsubapicalbranches.
An14g00010 corresponding to the secretion related Rab-
TABLE3 GTPaseSrgAshowntobeimportantforefficientsecretionand
Functionalcategoriesinducedbycaspofungin(CA)and maintenanceofpolarityinA.niger(46).
fenpropimorph(FP)
Furthermore, genes for which a function in cytoskeleton
Anannotatedlistofallgenes,includingfoldchange,pvalue,andclassification,can
befoundinsupplementalTableS2. organizationandmaintenancehasbeenestablishedforeukary-
No.ofgenes otic organisms were up-regulated as follows: (i) An08g06410
Functionalcategory responsiveto andAn18g06590showinghomologytoactin-bindingproteins
CA FP importantfortheintegrityofcorticalactinpatchesandactin-
Metabolism 41 26 dependent endocytosis in S. cerevisiae and S. pombe; (ii)
Energy 3 1
CellcycleandDNAprocessing 1 An01g03770displayinghomologytomicrotubule-basedmotor
Transcription 3 proteins;(iii)An16g03000andAn18g03900withhighhomol-
Proteinsynthesis 11
Proteinfate 17 4 ogytosubunitsoftheprefoldincomplexinvolvedinthefolding
Cellulartransportandtransportmechanism 5 2 oftubulinandactin;(iv)An05g00810withhomologytotubu-
Cellularcommunication 7 1
lin-specificchaperones;and(v)An01g13120predictedtobea
Cellrescue,defense,andvirulence 3 1
Regulationof/interactionwithcellularenvironment 1 ADP-ribosylation factor-like 2 of the Ras superfamily of
Transportfacilitation 4
GTPases, which has been shown to be important for tubulin
Unclassifiedproteins 70 5
stabilityanddynamicsinhumancells(47).
Caspofunginalsoinducedexpressionofgenespredictedto
sisinS.cerevisiae(36).As(cid:1)-1,3-glucanisthecentralcellwall function in lipid metabolism and signaling as follows:
polysaccharidetowhichothercellwallcomponentsofS.cer- An02g01180, coding for diacylglycerol pyrophosphate phos-
evisiae, such as (cid:1)-1,6-glucan, chitin, and mannoproteins, are phataseDpp1andAn02g13220predictedaslysophospholipase
cross-linked (1), the inhibition of (cid:1)-1,3-glucan synthesis by LplB.Moreover,agenecodingforageranylgeranyltransferase
caspofungincausescellwalldisorganizationandcelllysisinS. type II (An13g01040) involved in prenylation of proteins and
cerevisiae. One of the compensatory responses in yeast therebyinthemembranetargetingandinteractionofthemod-
describedtothepresenceofcaspofunginistheinductionofthe ifiedproteins(48)showedincreasedexpression.Remarkably,a
CWI pathway (69, 106), including induced expression of cell largenumberofsignalingproteinssuchasGTPasesofthesub-
wallproteinencodinggenesandcellwallremodelingenzymes. families Rho, Rac, Rab, and Rap require this modification for
Inagreementwiththis,weobservedanup-regulationofseveral their cycling between intracellular membrane compartments
A. niger genes involved in cell wall assembly and remodeling and hence their activity (48, 49). Increased expression of
(Table4).Genesthatwereup-regulatedincludedgenescodingfor An13g01040mightthusprobablyreflectahigherdemandfor
proteinsinvolvedinUDP-glucosesynthesis(An02g07650/PgmB relocalization/recycling of GTP-binding proteins in response
and An12g00820/UgpA), UDP-N-acetylglucosamine synthesis tocaspofungintreatment.
NOVEMBER9,2007•VOLUME282•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 32939
GlobalResponsesofA.nigertoAntifungals
TABLE4
Selectedcaspofungin-responsivegenesorderedintodifferentbiologicalprocesses
Openreadingframecode Gene Foldchange pvalue (Predicted)proteinfunction
Cellwallsynthesis
An14g01820 (33.14)a 0.020 CellwallproteinrelatedtophiA
An12g10200 (23.03)a 0.000 Cellwallproteinwithinternalrepeats,hypothetical
An03g05940 gfaB (7.98)a 0.051 Glutamine:fructose-6-phosphateamidotransferase
An09g04010 chsB 3.08 0.007 ChitinsynthaseclassIII
An12g07840 gnaA 3.18 0.004 Glucosamine-6-phosphateN-acetyltransferase
An10g00400 gelA (3.05)a 0.029 (cid:1)-1,3-Glucanosyltransferase
An07g07530 crhB 2.58 0.030 GPI-anchoredglucanosyltransferase
An18g06820 gfaA 2.32 0.008 Glutamine:fructose-6-phosphateamidotransferase
An18g06500 2.12 0.020 Phosphomannomutase(Sec53)
An09g03100 agtA 2.10 0.016 GPI-anchored(cid:2)-glucanosyltransferase
An07g01160 crhC 2.03 0.030 GPI-anchoredglucanosyltransferase
An03g01090 hocA (2.03)a 0.046 (cid:2)-1,6-Mannosyltransferase
An01g04650 2.00 0.032 Nucleosidediphosphate-sugarepimerase
An02g07650 pgmB 1.95 0.036 Phosphoglucomutase
An16g04330 dpmA 1.85 0.032 Mannosephosphodolicholsynthase
An03g05290 bgtB 1.77 0.040 (cid:1)-1,3-Glucanosyltransferase
An12g00820 ugpA 1.64 0.048 UTP-glucose-1-phosphateuridylyltransferase
An14g03520 dfgC 2.40 0.017 Endomannanase
An02g14500 2.21 0.009 GPI-anchoredcellwallprotein
CWIsignaling
An16g04200 rhoB 2.32 0.007 GTPase(Rho2-related)
An10g00490 rapA 2.16 0.039 Rho-GAP(ScSag7-related)
An18g03740 mkkA 2.03 0.009 MAPkinasekinase
Cellgrowthandpolarity
An17g02350 3.04 0.007 GTP-bindingprotein(Rheb-related)
An14g00010 srgA 2.51 0.012 GTPase
An13g00100 1.91 0.035 Serine/threonine-proteinkinase(Chk2-related)
Cytoskeleton
An01g03770 5.87 0.001 Dyneinlightchain
An16g03000 (2.97)a 0.037 SubunitoftheGim/prefoldinprotein
An01g13120 (2.64)a 0.026 ADP-ribosylationfactorfamilyprotein
An05g00810 2.60 0.016 Tubulin-specificchaperone(Rbl2-related)
An18g03900 2.16 0.027 Prefoldinsubunit2
An08g06410 2.13 0.016 Actin-likeproteinARP2
An18g06590 1.89 0.023 ARP2/3complexsubunit1A
Lipidmetabolism
An08g10110 2.88 0.011 Lipidtransferprotein
An02g01180 dppA (2.62)a 0.044 Diacylglycerolpyrophosphatephosphatase
An13g01040 1.84 0.043 Rabgeranylgeranyltransferase
An02g13220 lplB 1.77 0.025 Lysophospholipase
aValuesgiveninparenthesesasgeneshaveanAbsentflaginthecontrolexperiment.
with homology to the S. cerevisiae ergosterol pathway were
responsive to fenpropimorph treatment (Table 5) as follows:
An08g05400(homologueofErg10pthatcatalyzesthefirstand
rate-limiting step in ergosterol biosynthesis), An03g06410
(Erg25phomologue;pvalue0.06),An01g07000(Erg24phomo-
logue), An01g03350 (Erg2p homologue), and An15g00150
(Erg3p homologue; p value 0.06), indicating that fenpropi-
morphalsotargetsergosterolsynthesisinA.niger.
Ergosterol is most abundant in the plasma membrane and
secretoryvesiclesofS.cerevisiae(50),anditsmetabolismhas
FIGURE 3. Northern analysis of selected genes that were identified been shown to be closely linked with the biosynthesis of
ascaspofungin-responsive(A)orfenpropimorph-responsive(B)genes.
sphingo- and phospholipids (51–53). Further support for the
RNAsamplesextractedforthemicroarrayexperimentswereusedforhybrid-
izations.Each5(cid:3)gofRNAwasloadedontoeachlaneandhybridizedwith linkbetweenergosterolbiosynthesisandotherlipidsandfatty
differentprobesasindicated.Controlhybridizationswith18SRNAandactin acidbiosyntheseswasprovidedbytheobservationofincreased
probes confirmed equal loading (data not shown). CA, cells treated with
caspofungin;FP,cellstreatedwithfenpropimorph;C,control. transcriptlevelsofthreegenespredictedtofunctioninsphin-
golipidsynthesisasfollows:An01g10030displayinghomology
Gene Expression Responses to Fenpropimorph—Fenpropi- to the S. cerevisiae sphinganine hydroxylase Sur2p as well as
morph belongs to the morpholine fungicides and interferes An01g14200 and An01g14190 coding for sphingolipid (cid:2)-hy-
withergosterolbiosynthesisinS.cerevisiaebyinhibitingsterol droxylaseScs7p.Furthermore,ageneencodinganinositol-1-P
C-14reductase(ERG24gene)andsterolC-8isomerase(ERG2 synthase (An10g00530; homologous to S. cerevisiae Ino1p)
gene) (37). We thus assumed that among the A. niger genes showed increased expression. Perturbation of ergosterol bio-
up-regulated by fenpropimorph, a considerable number of synthesisinA.nigerwasalsoaccompaniedbyalterationofthe
genesshouldbeconnectedtolipidmetabolismandespecially cellularfattyacidmetabolism.Sixgenespredictedtofunction
toergosterolbiosynthesis.Indeed,fivegenesencodingproteins inperoxisomalfattyacid(cid:1)-oxidationwereup-regulatedasfol-
32940 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER45•NOVEMBER9,2007
GlobalResponsesofA.nigertoAntifungals
TABLE5
Selectedfenpropimorph-responsivegenesorderedintodifferentbiologicalprocesses
Openreadingframecode Gene Foldchange pvalue (Predicted)proteinfunction
Cellwallsynthesis
An01g11010 crhD 1.51 0.024 GPI-anchoredglucanosyltransferase
Cellgrowthandpolarity
An13g02780 (2.90)a 0.008 Aldolaseandadducinheaddomain
An04g02340 2.07 0.047 Kinesinlightchain
An14g02370 1.71 0.014 Apyrase,nucleosidediphosphatase(Ynd1-related)
Lipidmetabolism
An03g06410 (24.04)a 0.062 C-4methylsteroloxidase(Erg25-related)
An17g01150 (16.65)a 0.022 Acyl-CoAdehydrogenase
An18g05210 (10.70)a 0.002 Peroxisomaldehydratase
An01g07000 10.56 0.002 C-14sterolreductase(Erg24-related)
An08g05400 (5.41)a 0.012 Acetyl-CoAacetyltransferase(Erg10-related)
An08g07520 5.24 0.001 3-Oxoacyl-(acyl-carrier-protein)reductase
An15g01280 (5.22)a 0.006 Peroxisomal(cid:9)3,(cid:9)2-enoyl-CoAisomerase
An16g04520 4.13 0.004 3-Oxoacyl-(acylcarrierprotein)reductase
An14g04050 (4.04)a 0.045 Pyridoxamine-phosphateoxidaseactivity
An16g05340 3.60 0.001 Enoyl-(acylcarrierprotein)reductase
An18g01590 2.97 0.018 Mitochondrialcarnitineacetyltransferase
An04g00740 2.92 0.003 Sterolcarrierprotein
An14g00990 (2.92)a 0.005 Peroxisomalmultifunctional(cid:1)-oxidationprotein
An01g14200 2.75 0.012 Sphingolipid(cid:2)-hydroxylase(Scs7-related)
An01g14190 2.37 0.018 Sphingolipid(cid:2)-hydroxylase(Scs7-related)
An01g03350 2.21 0.038 C-8sterolisomerase(Erg2-related)
An15g00150 2.01 0.062 C-5steroldesaturase(Erg3-related)
An01g10030 1.72 0.039 Sphinganinehydroxylase(Sur2-related)
An10g00530 1.62 0.028 Myoinositol-1-phosphatesynthase
aValuesgiveninparenthesesasgeneshaveanAbsentflaginthecontrolexperiment.
lows: An11g00400 and An17g01150 both coding for an acyl- Thismightbereflectedbythelowernumberofup-regulated
CoA dehydrogenase mediating the first committed step of genes that putatively play a role in cell polarity of A. niger as
(cid:1)-oxidation of fatty acids, two fatty acid dehydrogenases follows: An04g02340 (low homology to kinesin light chain),
(An18g05210, An14g00990), one fatty acid isomerase An14g02370(apyrase,requiredforGolgiN-andO-glycosyla-
(An15g01280), and a sterol carrier protein 2 (An04g00740), tioninS.cerevisiae),andAn13g02780exhibitingsimilarityto
whichfunctionsinvitroasachaperoneforacyl-CoAoxidase (cid:2)-adducin,acrucialassemblyfactorofthespectrin-actinmem-
(54) and was recently shown to bind to fatty acids (55). The brane skeleton in higher eukaryotes (58). However, as ergos-
mainproductoffattyacid(cid:1)-oxidationisacetyl-CoAthatmight terol and sphingolipid metabolism have been shown to be
serveasprecursorfordenovosynthesisofergosterol(substrate important for protein secretion and for the establishment of
forErg10p)andfattyacids(substrateforthefattyacidsynthase cell polarity in yeast and filamentous fungi (33, 59, 60) might
multiprotein complex), which in turn provides fatty acids for suggest that some lipid genes mentioned above could also be
sphingolipid and phospholipids biosynthesis. In agreement, involvedinpolaritycontrolofA.niger.
three genes coding for proteins involved in fatty-acid syn- ValidationofTranscriptomeDatabyNorthernAnalysis—To
thase reactions showed increased expression (An08g07520, confirmthechangesingeneexpressiondetectedbytheexpres-
An16g04520, and An08g07520). Interestingly, a gene coding sion profiling, Northern analyses were performed using the
forhexosetransporter(firstandrate-limitingstepofglycolysis; sameRNAsamplesasusedinthemicroarrayexperiments.For
An15g03940)andcitratesynthase(importantfortheshuttleof cellstreatedwithcaspofungin,fourgenespredictedtofunction
acetyl-CoA from mitochondria to the cytosol; An15g01920) in cell wall biosynthesis and integrity were selected
werealsoup-regulated,probablyhintingatthepossibilitythat (An09g04010/chsCandAn03g05940/gfaA,An12g10200/hypo-
the cytosolic demand for acetyl-CoA necessary for fatty acid theticalcellwallprotein,andAn18g03740/mkkA).Inthecase
biosynthesisisaccommodatedbyusingglycolysisasalternative of the fenpropimorph-treated samples, four genes coding for
sourceforacetyl-CoA. proteinsputativelyinvolvedinlipidbiosynthesiswereselected
Apart from genes connected to lipid metabolism, genes (An01g03350/ERG2homologue),An01g07000/ERG24homo-
involvedinensuringcellwallintegritywerealsoup-regulated logue, An03g06410/ERG25 homologue, and An01g14200/
uponfenpropimorphtreatment:An01g1010(CrhD)displaying SCS7homologue).AsshowninFig.3,theresultsoftheNorth-
strongsimilaritytotheS.cerevisiaecellwallproteinCrh1,most ernhybridizationsareingoodagreementwiththemicroarray
recentlyshowntofunctioninthecross-linkageofchitinchains data. Genes that showed high/low levels of induction in the
to(cid:1)-1,6-glucan(56)andAn03g05000predictedasZIPfamily expression profiling also showed signals of strong/moderate
zinctransporterwithhomologytotheS.cerevisiaeYke4p.This induction in the Northern experiment (e.g. An03g06410 and
protein is important for balancing zinc levels between the An18g03740).
cytosolandthesecretorypathwayinyeast(57). agsAExpressionIsSpecificallyInducedbyCompoundsAffect-
Inresponsetofenpropimorph,newpolargrowthsiteswere ingCellWallIntegrity—Theexpressionprofilinginthisstudy
developed by A. niger germlings, although to a lesser extent revealedthatthegenecodingfortheregulatorof(cid:2)-1,3-glucan
whencomparedwiththecaspofungin-treatedsamples(Fig.3). synthesis (Rho2-GTPase, An16g04200) and agtA (GPI-an-
NOVEMBER9,2007•VOLUME282•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 32941
GlobalResponsesofA.nigertoAntifungals
chored(cid:2)-glucanotransferase,An09g03100)wereup-regulated and subsequently to the activation of the cell wall salvage
upon caspofungin treatment. We have shown previously that pathway.
theagsAgenecodingfor(cid:2)-1,3-glucansynthase(An04g09890) The second group of compounds includes chitosan and
is strongly induced in response to compounds that interfere epoxiconazole.ThesecompoundsinhibitedgrowthofA.niger
with cell wall or cell membrane integrity of A. niger such as andgaveamoderateinductionofGFPexpression.Thesecom-
calcofluorwhite,SDS,caspofungin,andAFP(21,25),suggest- poundshavebeenreportedtohaveaneffectwhentheintegrity
ingthat(cid:2)-1,3-glucansynthesismightbegenerallyinvolvedin of the cell wall was impaired, suggesting an indirect effect of
securingcellsurfaceintegrity.Inthisstudy,theagsAgenewas thesecompoundsoncellwallbiosynthesis(65,66).Thethird
unexpectedly not found among the significantly up-regulated groupofcompoundsconsistingofhydrogenperoxide(Fig.4D),
genes.Acloserlookatthetranscriptomicdatarevealed,how- pyraclostrobin,benomyl,andcycloheximideshowedaninhibi-
ever,thatagsAwasnotexpressedinthecontrolexperimentbut tionofgrowthatoneormoreconcentrationsusedbutshowed
strongly expressed when A. niger germlings were exposed to no induction of GFP expression. To our knowledge, none of
bothcaspofunginandfenpropimorph(pvalue(cid:7)0.05,seesup- thesecompoundswerereportedsofartoaffectdirectlyorindi-
plemental Table S2), implying that regulation of agsA gene rectly cell wall biosynthesis. Into the last group, compounds
weresortedthatdidnothaveanyeffectoneithergrowthorGFP
expressionisactuallyunderthecontrolofstressconditionsthat
expression such as myriocin and nikkomycin. Although
affecttheintegrityoftheplasmamembraneand/orthecellwall.
nikkomycinwasshowntointerferewithchitinsynthesisinS.
To further support this conclusion, we used two A. niger
cerevisiae(67),LiandRinaldi(68)showedthatA.nigerwasnot
reporter strains, containing either a cytoplasmically (strain
sensitive to nikkomycin concentrations (minimal inhibitory
JvD1.1)ornuclear(strainRD6.47)targetedgfpgeneunderthe
concentration (cid:7)64 (cid:3)g/ml). These results are consistent with
controloftheagsApromoter.Bothstrainswereexposedto16
ourfindingthatnikkomycinhadnoeffectoneithergrowthor
antifungal compounds (including caspofungin and fenpropi-
GFPexpression.
morph)thattargetdifferentcellularprocesses,andtheireffect
Taken together, the data show that the agsA promoter is
on growth and agsA expression was monitored by light and
specifically activated by compounds interfering directly with
fluorescence microscopy (Table 6). Based on their effects, we
cellwallbiosynthesisorbycompoundsinhibitingplasmamem-
havedividedthecompoundsintofourgroups.Thefirstgroup
branefunctionortheproteinsecretionmachinery,therebydis-
ofcompoundsincludescalcofluorwhite,caspofungin,tunica-
turbingcellwallbiosynthesismoreindirectly.
mycin, spiroxamine, fenpropimorph, terbinafine, fludioxonil,
and cyprodinil. These compounds inhibited growth and pro- DISCUSSION
vokedhighexpressionoftheGFPreporter.Inresponsetocal-
Growthanddevelopmentoffungiaswellastheirabilityto
cofluor white, fungal growth became inhibited, and aberrant
withstandinternalturgorpressureandtosurviveenvironmen-
hyphalmorphologysuchastipswellingaswellasaclearinduc-
tal stress conditions depend on maintaining the integrity of
tion of GFP expression was observed (Fig. 4A). Similarly, the
theircellsurface.Asafirststeptowardacomprehensiveunder-
inductionofagsAinresponsetothepresenceofcaspofungin
standing of the regulatory network(s) of A. niger involved in
wasalsoobservedinbothreporterstrains,confirmingprevious
maintenanceofcellsurfaceintegrity,weexaminedinthisstudy
results (21) and the results of the expression profiling in this
the global gene expression profile of A. niger in response to
study (Fig. 4B). When the reporter strains were stressed with
treatmentswithcaspofunginandfenpropimorph.
tunicamycin (inhibitor of protein N-glycosylation), swollen
Responsive Genes to Caspofungin—The category with the
hyphae and high GFP expression were visible (Fig. 4C). As
highest number of genes showing enhanced transcription in
N-glycosylation mutants in S. cerevisiae have been shown to
responsetocaspofunginisthegroupofgenesrequiredforcell
havedefectsincellwallintegrity(15,61),itisverylikelythatthe
wall biogenesis and maintenance. About 12% of the up-regu-
additionoftunicamycintoA.nigeralsoresultsinweakeningof
latedgenescanbeclassifiedintothiscategory,suggestingthat
the cell wall and activation of the cell wall integrity pathway.
the primary (or an important) response to caspofungin is to
The induction of the GFP reporter by the lipid synthesis dis- counteracttheinhibitoryeffectofcaspofunginon(cid:1)-1,3-glucan
turbingcompoundsspiroxamine,fenpropimorph,andterbin- synthesis by transcriptional activation of cell wall reinforcing
afinesuggeststhatdisturbanceoftheplasmamembraneinteg- genes.Caspofungininhibits(cid:1)-1,3-glucansynthesisinS.cerevi-
rity negatively affects the integrity of the cell wall and siaeandseveralAspergillispecies(7,36)andhasbeenshownto
substantiates the expression data with respect to fenpropi- (mainly)up-regulategenesinvolvedinthesynthesisofcellwall
morph.ActivationofagsA::gfpexpressionbyfludioxonil(acti- componentsandcellwallstrengtheningintheyeastsS.cerevi-
vator of the Hog1 osmotic signal transduction pathway in S. siaeandCandidaalbicans(69,70),implyingthatcaspofungin
cerevisiae)couldhintattheexistenceofacross-talkbetween triggersasimilarresponseinA.nigerasinyeasttoreinforcethe
thecellwallintegritypathwayandtheosmoticsignaltransduc- strengthofthecellwall.
tionpathwayinA.nigerasshownrecentlyforS.cerevisiae(62, Oneofthesignaltransductionpathwaysthatbecomesacti-
63).Interestingly,cyprodinil(interfereswithmethioninesyn- vated in S. cerevisiae in response to caspofungin is the CWI
thesisandsecretionofhydrolyticenzymes(64))alsoleadstoan pathway(69).Inbrief,theCWIpathwayofS.cerevisiaeconsists
up-regulationofthereporter,suggestingthatanefficientsecre- oftheplasmamembrane-localizedsensorproteins(Wsc1–4p
torypathwayisrequiredforpropercellwallbiosynthesis.Inter- andMid2p)thatmediatethecellwallstresssignalthroughthe
feringwithproteinsecretionmightleadtocellwallweakening Rho1-GTPaseandthePkc1pkinase.Pkc1pinitiatesaphospho-
32942 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER45•NOVEMBER9,2007
GlobalResponsesofA.nigertoAntifungals
TABLE6
Theeffectofselectedcompoundstestedforantifungalactivity
aTheconcentrationrangeoftheantifungalcompoundsisgivenin(cid:3)g/ml.2-Foldserialdilutionsgivingsevendifferentconcentrationsweretested.Thevalueofthehighestand
lowestconcentrationisshown.
bSchematicrepresentationoftheaveragegreenfluorescentproteinlevelsinbothreporterstrains(RD6.47andJvD1.1)growninthepresenceofdifferentcompoundconcentrations.The
leftcolumnrepresentsthelowestantifungalconcentrationused.Thenumbers0,1,and2representlow/basal,intermediate,andhighgreenfluorescentproteinlevels,respectively.
cTheeffectongrowthbasedonthehyphallengthinthemicroscopeimages.Thenumbers0,1,and2representno,intermediate,andhighgrowthinhibition.Noeffectongrowth
orongreenfluorescentproteinexpressionwasfoundwhenthesolventsMeSOorethanolwereused(datanotshown).
2
rylationcascadeinvolvingtheMAPkinasesBck1p,Mkk2p,and are present in the genome of A. niger (35), suggesting that
Slt2p. Slt2p finally phosphorylates the transcription factor thispathwayisnotonlyimportantforensuringcellintegrity
Rlm1p that induces expression of genes involved in cell wall in yeast but also in filamentous fungi. The results of this
reinforcement(71).AllcomponentsoftheyeastCWIpathway study provide indications that the CWI pathway becomes
NOVEMBER9,2007•VOLUME282•NUMBER45 JOURNALOFBIOLOGICALCHEMISTRY 32943
GlobalResponsesofA.nigertoAntifungals
activatedinA.nigerandisrequired
for adaptation to caspofungin-me-
diatedinhibitionofcellwallbiogen-
esis(Fig.5).First,An02g01180dis-
playing homology to diacylglycerol
pyrophosphate phosphatase is up-
regulated. This enzyme generates
diacylglycerol that has been shown
to be a physiological activator of
fungal Pkc1p homologues (72, 73).
Second, a homologue to the S.
pombe Rho2p (An16g04200/RhoB)
showed increased expression. In
S. pombe, the Rho2-GTPase has
been shown to stimulate (cid:2)-1,3-
glucan synthesis through activation
ofthePkc1phomologuePck2p(74).
Third, enhanced expression of
An18g03740/MkkA (homologous
to Mkk2p) is further indicative for
an involvement of the CWI path-
way. Finally, targets of the A. niger
RlmA transcription factor such as
gfaA (chitin synthesis) and agsA
((cid:2)-1,3-glucansynthesis(4))showed
enhancedexpression.
ThemotilityoftheS.cerevisiae
(cid:1)-1,3-glucan synthase Fks1p to-
ward the polar growth site is
strongly dependent on cortical ac-
tin patch movement which itself
requires the activity of the Arp2/3
complex(seeRef.75andreferences
therein). In this study, two com-
ponents of the Arp2/3 complex
(An08g06410 and An18g06590)
showed increased expression upon
caspofungin treatment. As other
proteins predicted to function in
actin and tubulin folding/stability
were up-regulated (An16g03000,
An18g03900, An05g00810, and
An01g13120) suggests that inhibi-
tion of (cid:1)-1,3-glucan synthesis may
alsoaffectactinstabilityinA.niger,
whichiscounteractedbytheinduc-
tionofgenesencodingforproteins
that assist in actin stabilization
andcytoskeletonmaintenance.The
induced expression of this class of
genes has not been observed in
studiesinyeast(S.cerevisiaeandC.
albicans(69,70))andmightthere-
FIGURE4.Microtiterplate-basedscreeningofantifungalsbymicroscopicanalysisofthemorphology
fore be related to the filamentous
andGFPexpression.StrainsRD6.47andJvD1.1werepre-grownfor6handsubsequentlystressedfor3hat
30°Casdescribedunder“ExperimentalProcedures.”Differentcompounds(calcofluorwhite,caspofungin, growthofA.niger.
tunicamycin,andhydrogenperoxide)wereused,A–D,respectively.Thecompoundswere2-foldserialdiluted. One pathway that has been
Thefinalconcentrationofthecompoundsisindicatedtotherightofeachimage.
described to be involved in actin
polarization in S. cerevisiae is the
32944 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER45•NOVEMBER9,2007