*Author ([email protected]) for correspondence M5S 1A8. Toronto,Genetics, University of Medical SciencesBuilding, Toronto, Canada, Molecular of Malaysia, Pulau Pinang, Malaysia. **Presentaddress:Department University, Providence, RI02912,USA. crosstalk, stresspathwayinterferenceandabiologicaltransistor. by multiplemechanismsthatincludesignallingandchemical to fungus–hostinteractionsduringdiseaseprogression,aremediated additive effects uponC.albicans. stress resistance,andthatsomecombinatorialstressesexertnon- become apparentthatchangesincarbonsourcestronglyinfluence combinations ofstresses(ratherthanindividualstresses).Ithas by alternativecarbonsourcesandsimultaneousexposureto pathogen occupiescomplexanddynamichostnichescharacterised of glucose-growncellstoindividualstresses.However, 144 attributed. use, distribution and reproductioninany medium provided thatisproperly theoriginalwork Attribution License(http://creativecommons.org/licenses/by/3.0), unrestricted permits which theCreative Commons of distributed undertheterms This isanOpenAccessarticle ¶ ‡ Sciences, Foresterhill, AberdeenAB252ZD, UK. Medical Aberdeen,Instituteof MedicalSciences, University of School of occupies awiderangeofdivergent nicheswithinthehost.It Candida albicans stress, Stressadaptation,Carbonmetabolism Oxidative stress,NitrosativeOsmoticCationic KEY WORDS:Candidaalbicans,Fungalpathogenicity, Heatshock, albicans across diverseenvironmentalniches.Ourunderstandingof warm-blooded animals,comparedwithotheryeaststhatarefound C. albicans Schizosaccharomyces pombe. benign modelyeasts number ofregulatorsinC.albicans rewiring ofsomestressregulatorycircuitrysuchthattherolesa include evolutionarilyconservedmodules.However, therehasbeen attenuates virulence.Stresssignallingpathwaysin responses tolocalenvironmentalstresses,theinactivationofwhich the pathogenicityofC.albicans.Fitnessattributesincluderobust infections. A batteryofvirulencefactorsandfitnessattributespromote compromised, (thrush). Additionally, whentheimmunesystembecomesseverely immune defencesareperturbeditcausesmucosalinfections is carriedbymanyindividualsasaharmlesscommensal,butwhen Candida albicans Delma S.Childers,ShahidaShahanaandMichelleD.Leach** Iuliana V. Ene Alistair J.P. Brown*,SusanBudge,DespoinaKaloriti,AnnaTillmann, MetteD.Jacobsen,ZhikangYin, Stress adaptationinapathogenicfungus REVIEW © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,144-155doi:10.1242/jeb.088930 Biology Center, Nebraska-Lincoln, Lincoln,NE68588-0662,USA. University of of humans of Introduction: Introduction: ABSTRACT Present address: Department of MolecularMicrobiologyandImmunology, of Brown Present address:Department Present address:InstitutPerubatan andPergigian Termaju, Universiti Sains stress signallingisbasedprimarilyonthe as anopportunisticpathogenobligatelyassociatedwith Candida albicans albicans Candida C. albicans ‡ , IrynaBohovych is amajorfungalpathogenofhumans.Thisyeast is amajorfungalpathogenofhumansthat often causeslife-threateningsystemic Saccharomyces cerevisiae This reflectsthespecificevolutionof § Present address:Nebraska Redox These effects, whicharerelevant – an opportunistic pathogen – anopportunistic have divergedrelativetothe § , DoblinSandai in vitro C. albicans in vivo responses ¶ , StavroulaKastora,JoannaPotrykus,ElizabethR.Ballou, and this C. a complexparasexualcycle. thought tobeconstitutivelydiploid(Hickmanetal.,2013),displays spores. Rather, thisdiploidyeast,which untilveryrecentlywas C. albicans defined bytheirpackagingofsexualsporesintoanascusstructure, and sharks.Furthermore,althoughascomycetesaregenerally distance representsgreaterseparationthanexistsbetweenhumans million years)(Galaganetal.,2005),andthelatterevolutionary related to 2007). However, inevolutionaryterms and Sudbery, 2002;Enjalbertetal.,2006;NobleandJohnson, paradigms againstwhich Schizosaccharomyces pombe which includesthemodelyeasts system. pathogenic behaviourandalsoitspropertiesasanexperimental animals (Odds, 1988). Therefore,itisnotsurprising thatthisfungus hence isthoughttobeobligately associatedwithwarm-blooded not beenassociatedwithany particular environmentalnicheand can adapteffectively todiversehostniches. al., 2012b).Fromthefungalperspective,itisclearthat of effective antifungaldrugs(PfallerandDiekema,2010;Brownet infections, theresultantdelaysintreatment,andlimitedchoice al., 2012b),reflectingthechallengesindiagnosingsystemicfungal azoles, polyenesandechinocandins(Oddsetal.,2003a;Brown despite theavailabilityofspecialisedantifungaldrugssuchas patient groups(Perlrothetal.,2007).Thishighmorbidityexists bloodstream infections,overhalfofwhichcanbefatalinsome Candida brain (PfallerandDiekema,2007;CalderoneClancy, 2012). colonisation ofinternalorgans suchasthekidney, liver, spleenand surgery), thefunguscansurviveinbloodstream,leadingto (such asneutropenicpatientsundergoing chemotherapyortransplant In individualswhoseimmunesystemsareseverelycompromised example inHIV patients(Sobel,2007;RevankarandSobel,2012). antibiotic usageorwhenimmunedefencesareperturbed,for vaginal infections(thrush)whenthemicrofloraisdisturbedby and Clancy, 2012).However, C.albicans most healthyindividuals(Odds,1988;Calderone,2002;Calderone of theskin,oralcavity, andgastrointestinal andurogenitaltractsof normally existsasaharmlesscommensalorganism inthemicroflora largely byitsclonalbehaviourasapathogen. 2007). Indeed,itsrecentevolution appearstohavebeendriven structure ispredominantlyclonal (Cowenetal.,2002;Odds contributed totherecentevolution of (Forche etal.,2008).Whileparasexualrecombinationcould have is followedbychromosomelosstoreturnthediploid state before itmatestoformtetraploids(NobleandJohnson,2007). This epigenetic switchtomatingcompetentcells(theopaqueform) homozygosis atthematingtypelocus( The evolutionaryhistoryof Candida albicans S. cerevisiae is thefourthmostcommoncauseofhospital-acquired has notbeenobservedtoundergo meiosistogenerate ( circa C. albicans is amemberoftheascomycetephylum, 150 millionyears)and . Thesebenignmodelyeastsprovide C. albicans Candida albicans Saccharomyces cerevisiae is oftencompared(Berman C. albicans,thepopulation C. albicans MTL) andthenundergo an frequently causesoraland has establishedbothits Candida albicans is onlydistantly S. pombe must undergo C. albicans (>400 and has

The Journal of Experimental Biology in have hadasignificantimpactontheevolutionofstressadaptation al., 2009;Bakeret2012;Sandai2012).Thesechanges cerevisiae transcriptional andpost-transcriptionalcircuitriesrelativeto Nikolaou etal.,2009)aswellevolutionaryrewiringof attributes associatedwithitspathogenicity(Butleretal.,2009; has undergone therapidevolutionofvirulencefactorsandfitness REVIEW that stressgenes areinducedwhenthefunguscomes incontactwith First, numerousgenome-wideexpression profileshavedemonstrated importance ofstressadaptation forthevirulenceof microenvironment. Two maintypesofevidencehavehighlighted the physiological fitnessof in metabolicandstressadaptation andactbytuningthe termed ‘fitnessattributes’ (Brown,2005),include functionsinvolved interacting directlywiththehost.Thesefactors,whichhave been pathogenicity. and diseaseprogression,therebyenhancing factors aretemporallyandspatiallyregulatedduringcolonisation host immuneresponses(Pietrellaetal.,2010).Theseand other invasion, providenutrientstosupportfungalgrowthandmodulate (Naglik etal.,2003;Schaller2005)thatenhancetissue hydrolytic enzymesincludingproteases,lipasesandphospholipases A.J.P.B., unpublished). Candidaalbicans 2012), andwhichareinducedduringrenalinfection(J.P. and which areessentialforvirulence(Almeidaetal.,2009;Citiulo et al., additional factorsinvolvedinironandzincassimilation,someof colonisation (Liuetal.,2011). iron (Almeidaetal.,2008;Almeida2009)andtobrain 2007), contributingtotheassimilationofessentialmicronutrient invasin bypromotingtheinvasionofendothelialcells(Phanetal., Hoyer etal.,2008).Oneoftheseadhesins,Als3,alsoactsasan adhesins thatpromoteadherencetohosttissues(Staabetal.,1999; 2012). Initialcolonisationismediatedbyfamiliesofcellsurface contribute totissuepenetration(Sherwoodetal.,1992;Brand, Hyphae alsodisplaythigmotropicresponsesthatappearto whereas thefilamentousformsarebettersuitedtopenetratetissue. al., 2003).Yeast formsarethoughttopromotedissemination, contribute tothevirulenceof transitions betweenyeast,pseudohyphalandhyphalgrowthforms (Odds etal.,2003b).Forexample,reversiblemorphogenetic those fungalfactorsthatinteractdirectlywithhostcomponents pathogenicity of revealed anarmouryofvirulencefactorsthatpromotethe (Noble andJohnson,2007).Nevertheless,theseapproacheshave rather thangeneticstrategiestoexaminethevirulenceofthisfungus have hadtorelymainlyongenomicandmolecularapproaches, the experimentaldissectionof Additional factorspromotethevirulenceof The lossofabonafidesexualcyclehashadmajorimpacton AKstress-activatedproteinkinase reactiveoxygenspecies reactivenitrogenspecies reactivechlorinespecies SAPK ROS RNS MAP kinase RCS MAP kinase mitogen-activatedproteinkinase heatshockprotein MAPKKK heatshockelement MAPKK greenfluorescentprotein MAPK HSP HSE GFP List of abbreviations List of C. albicans (Ihmels etal.,2005;Martchenko2007;Lavoie (Brown etal.,2012a). C. albicans.Virulence factorshavebeendefinedas C. albicans C. albicans C. albicans Candida albicans cells totheirlocalhost (Lo etal.,1997;Saville pathobiology. Researchers also secretesfamiliesof C. albicans C. albicans C. albicans. expresses without S. (Leach etal.,2012a). Followingthermaladaptation, Hsp90interacts transistor asitmodulatestheactivity ofclientregulatoryproteins shock protein90(Hsp90)has beendescribedasamolecular activated inanHsf1-dependent fashion(Nichollsetal.,2009).Heat and Gozalbo,2003;Leachet al., 2011). The expression, whichisrequiredfor resistancetothermalstress(Roig their relevancetoinfection. section istosummarisekeystresssignallingpathways,highlighting et al.,2010;Brown2012a).Therefore,thepurposeofthis et al.,2006;Alonso-Monge2009b;Brown2009;Smith albicans have beenevolutionarilytunedtohostniches.Stresssignallingin This isconsistentwiththeideathatstressresponsesin with modelyeasts(Jamiesonetal.,1996;Nikolaou2009). is relativelyresistanttophysiologicallyrelevantstressescompared 2004; Ramsdaleetal.,2008;Cheetham2007),and regulators havediverged (Enjalbertetal.,2003;Nicholls 1). However, therolesofsomethese Nikolaou etal.,2009)(Fig. evolutionarily conservedin cerevisiae Stress signallingpathwaysarerelativelywellcharacterisedin individual stressescommonlystudied respond tomultipleenvironmentalinputs,ratherthanthe host niches(mentionedabove),inwhich review –stressadaptationinthecontextofcomplexanddynamic discussed here.Thisprovidestheplatformformainthemeofthis model yeasts.A briefoverviewofthesemechanismswillbe albicans elaboration ofstress-adaptiveresponses,theirregulationin Cheetham etal.,2011). Significantprogresshasbeenmadeinthe et al.,1998;Alonso-Monge1999;Hwang2002; Hog1, thecatalaseCat1orsuperoxidedismutaseSod1(Wysong stress functionssuchasthestress-activatedproteinkinase(SAPK) in mousemodelsofinfectionisattenuatedbytheinactivationkey 2007; Miramónetal.,2012).Second,thevirulenceof diagnostic greenfluorescentprotein(GFP)fusions(Enjalbertetal., stress responseshasbeenconfirmedbysinglecellprofilingusing Fradin etal.,2005),andtheniche-specificinductionofspecific or neutrophils(Rubin-Bejeranoetal.,2003;Lorenz2004; functions areinducedwhencellsphagocytosedbymacrophages the host.Forexample,oxidative,nitrosativeandheatshock Indeed, in proteins, therebypromotingcellularadaptationtothethermalinsult. gene inductionleadstotherefoldingordegradationofdamaged 1988; JakobsenandPelham,Holmberg etal.,2001). interaction thatisconservedinothereukaryotes(Sorger andPelham, elements (HSEs)intheirpromoters(Nichollsetal.,2009), an of target heatshockprotein ( albicans Sarge etal.,1993;Wu, 1995).Inresponsetoacuteheatshock, humans andisessentialforviability(Sorger andPelham,1988; factor Hsf1(Nichollsetal.,2009).isconservedfromyeasts to albicans, asinotheryeasts,isdrivenbytheheatshocktranscription Lindquist, 1993;FederandHofmann,1999).Theresponse in target aggregatedordamagedproteinsfordegradation(Parselland (HSPs), manyofwhichpromotethefoldingclientproteinsor involves theinductionofadefinedsetheatshockproteins The heatshockresponseisubiquitousinnature.Ineukaryotes,it Overview of stress adaptation mechanisms stress in of Overview Heat shock has beendescribedinanumberofrecentreviews(Chauhan Hsf1 becomesphosphorylatedandinducestheexpression and theirdivergence fromthecorrespondingpathwaysin and C. albicans The JournalofExperimentalBiology(2014)doi:10.1242/jeb.088930 S. pombe.A numberofthekeyregulators are heat shockinducespolyubiquitin ( HSP) genesviacanonicalheatshock C. albicans in vitro C. albicans (Butler etal.,2009; . HSP90 C. albicans C. gene isalso C. albicans C. albicans C. albicans cells must UBI4) HSP 145 C. C. C. C. S.

The Journal of Experimental Biology of stressadaptationmechanismsin response toheatshock,andHsp90thendownregulatesHsf1(see‘Overview synthesis ofthebiologicaltransistorHsp90(green)isactivatedbyHsf1in C. albicans functions in (red) andtranscriptionfactors(blue)contributetotheregulationofstress conserved mitogen-activatedproteinkinase(MAPK)signallingmolecules 146 REVIEW encoding glycerol biosyntheticenzymes(Smith etal.,2004; which inturnmediatestheactivation oftarget genesincluding those phosphorylation andnuclear accumulation oftheSAPKHog1, turgor pressure(KühnandKlipp,2012).Thistriggers the which causesrapidwaterloss, areductionincellsizeandlossof Exposure toNaClorKClimposes osmoticandcationicstress, albicans physically withHsf1todownregulatetheheatshockresponsein Ste11, MAPKKK;Hst7,MAPKK;Cek1,MAPK. MAPKKK; Mkk1,MAPKK;Mkc1,MAPK.Mating/invasivegrowthpathway: MAPK/stress-activated proteinkinase(SAPK).Cellintegritypathway:Bck1, MAPK kinase(MAPKKK);Pbs2,(MAPKK);Hog1, regulator; SODs,superoxidedismutases.Hog1signallingpathway:Ssk2, pathway: Cap1,AP-1bZIP transcriptionfactor;Skn7;putativeresponse cluster transcriptionfactor;Yhb1,nitricoxidedioxygenase.Oxidativestress Hsf1, heatshocktranscriptionfactor. Nitrosativestresspathway:Cta4,zinc an integratednetwork.Heatshockpathway:Hsp90,heatprotein90; represented aslinearpathways(forsimplicity),butmostprobablyoperatein Conserved stressregulatorsin 1. Fig. Osmotic andcationicstress has beenmaintainedin undergone evolutionaryrewiring(seebelow),heatshockregulation acute heatshocks. maintenance ofthermalhomeostasis,notmerelyforadaptation to al., 2011). Clearly, theHsf1–HSE reguloniscriticalforthe activation isessentialforthevirulenceof such asthosesuffered byfebrile patients.ThisexplainswhyHsf1 regulon appearstobeactivatedevenduringslowthermaltransitions an answertothisconundrum(Leachetal.,2012c).TheHsf1–HSE dynamic regulationofHsf1duringthermaladaptationhasprovided albicans activation oftheheatshockresponseattenuatevirulence of imposed inthelaboratory. Interestingly, mutationsthatblockthe host andisgenerallyshelteredfromtheacuteheatshocksthatare Presumably thefungusoccupiesthermallybuffered nichesinthe obligate associationwithwarm-bloodedanimals(Odds,1988). Chaperones Thermal Significantly, whileotherconservedstressregulatorycircuitshave stress Hsp90 Hsf1 Lahe l,21b Fg 1). (Leach etal.,2012b)(Fig. (Nicholls etal.,2011). Mathematicalmodellingofthe ’). Hsf1andHsp90operateinanautoregulatorycircuit,whereby C. albicans Nitrosative stress Yhb1 Cta4 & thioredoxin Glutaredoxin Glutathione (see ‘Overviewofstressadaptationmechanismsin systems C. albicans Cap1 Oxidative Stress adaptation stress Skn7 Virulence Trehalose catalase SODs C. albicans (Nicholls etal.,2009)despiteits Candida albicans. accumulation Osmotic/ cationic Glycerol stress ’). Thesepathwaysare Hog1 Pbs2 Ssk2 C. albicans Cell wallremodelling Cell walldamage Mck1 Mkk1 Bck1 Evolutionarily (Nicholls et Ste11 Cek1 Hst7 C. C. established unambiguouslyin this MAPKmoduleinresponsetoosmoticstresshavenotbeen 1).However, theupstreamregulatorsthatactivate al., 2007)(Fig. kinase (MAPKKK),Ssk2(Aranaetal.,2005;Cheetham (MAPKK) Pbs2,whichinturnisactivatedbyasingleMAP kinase MAP kinase(MAPK)isactivatedbytheMAP kinase yeasts (Nikolaouetal.,2009;Smith2010).In protein (MAP)kinasepathwayinvolvedinosmo-adaptationother 2004). stresses areHog1dependent(SanJoséetal.,1996;Smith successful adaptationof Glycerol biosyntheticgeneinduction,glycerolaccumulationandthe the restorationofturgor pressureandtheresumptionofgrowth. Enjalbert etal.,2006).Thisleadstotheaccumulationofglycerol, the cellwallof Calcofluor WhiteandCongoRed areoftenusedtoexertstressupon Antifungal drugssuchascaspofungin andchemicalssuchas 0 mll mmol 600 certain hostniches.First,NaClconcentrationscanapproach osmotic andcationicstressadaptationplaysignificantroles in Eisman etal.,2006).Nevertheless,severalobservationssuggest that Monge etal.,2003;Alonso-Monge2009a;Smith 2004; affects cellwallfunctionality(Alonso-Mongeetal.,1999;Alonso- modulates cellularmorphogenesis,influencesmetabolismand functions. Hog1isrequiredforadaptationtootherstresses, adaptation becauseHog1hasbeenshowntoexecuteadditional is notattributablesimplytothelossofosmoticorcationicstress (Alonso-Monge etal.,1999;Cheetham2011). However, this osmotic adaptationislikelyto be requiredinmanyhostniches. experimentalists tendtoimpose addition toitsroleinadaptationtheacuteosmoticshocks that and themaintenanceofwaterbalance(Klippetal.,2005), in this regulatoryapparatusinmediatingcellularosmo-homeostasis osmotic stressadaptationin Santos etal.,2002;Fang,2004).Third,mathematicalmodelling of K 1997; Zhangetal.,2004).Second, Cell wallstress histidine kinasesin (Nikolaou etal.,2009),aswellproteinsthatarerelatedto Candida albicans response toabroadrangeofstresses,includingosmoticstress. activates Hog1signallingviatheMAPKKKsSsk2andSsk22in The Sln1phospho-relaysystemincludesYpd1 andSsk1, MAPKKK Ste11 specificallyinresponsetoheatorosmoticstress. signalling viaCdc42,Ste50,Ste20andCla4,throughthe (reviewed bySmithetal.,2010).TheSho1branchactivatesHog1 MAPK modulerespondstotwowell-definedupstreambranches the upstreamregulatorsofthisstresspathway. and thereappearstohavebeensignificantevolutionaryrewiringof it isnotyetclearhowosmoticstresssignalsaretransducedtoHog1, branch isnotessentialforosmoticstresssignallingeither. Therefore, Hog1 inC.albicans sho1 not transduceosmoticstresssignalstoHog1.Furthermore,a Kruppa andCalderone,2006),suggestingthattheSln1branchdoes osmotic stress-inducedactivationofHog1(Chauhanetal.,2003; albicans Chk1, Nik1)(KruppaandCalderone,2006).However, in + Hog1 isacomponentofhighlyconservedmitogen-activated The inactivationofHog1attenuatesthevirulence fluxes followingphagocytosisbyhostimmunecells(DaSilva- double mutationdoesnotblockosmoticstresssignallingto none ofthesehistidinekinasesorSsk1isessentialforthe − The JournalofExperimentalBiology(2014)doi:10.1242/jeb.088930 1 in thekidneyandbehighurine(Ohnoetal., C. albicansinvitro has orthologuesformanyoftheseproteins S. cerevisiae (Román etal.,2005),indicatingthattheSho1 C. albicans S. cerevisiae C. albicans.InS.cerevisiae and in vitro (Wiederhold etal.,2005;Eisman C. albicans S. pombe cells toosmotic/cationic has highlightedtheroleof . Hence,Hog1-mediated cells areexposedto ( C. albicans C. albicans,this C. albicans , this Sln1, ypd1 C.

The Journal of Experimental Biology adaptation to stress. Consequently, theinactivation ofCap1 Together, thesefunctionsdetoxify ROS andmediatecellular (e.g. glutathionereductaseand thioredoxin: synthetase: SOD1), glutathionesynthesis(e.g. gamma-glutamylcysteine oxidative stress(e.g.catalaseand superoxidedismutase: 2009). Cap1targets includegenesinvolvedinthedetoxification of promoters (Zhangetal.,2000;Enjalbert2006;Znaidi et al., of itstarget genesviaYap1-responsive elements(YRE)intheir Hog1-independent nuclearaccumulationofCap1andtheactivation that becomeoxidisedfollowingoxidativestress.Thisleadsto the contains redox-sensitivecysteineresiduesnearitscarboxyterminus 1).Cap1 the responseregulatorSkn7(Singhetal.,2004)(Fig. Yap1 andS.pombe transcription factorCap1,whichisanorthologueof Rodaki etal.,2009).ThisresistanceisdependentontheAP-1-like some conditions(Jamiesonetal.,1996;Nikolaou2009; RS,tlrtn vr2 mll mmol (ROS), toleratingover20 Candida albicans of or macrophages(Aranaetal.,2007),butdoesattenuatethevirulence not increasethesensitivityof temperatures (Navarro-Garcíaetal.,1995).Mkc1inactivationdoes of Mkc1conferssensitivitytocellwallstressesandelevated (Paravicini etal.,1996;Alonso-Monge2006).Thedisruption wall stressismediatedthroughproteinkinaseC(Pkc1)signalling MAPK Mkc1(Alonso-Mongeetal.,2006).activationbycell incorporates theMAPKKKBck1,MAPKKMkk1and 2007). pathways areevolutionarilyconservedinotherfungi(Románetal., 1).Both morphogenesis (definedbytheMAPKCek1)(Fig. characterised onthebasisofitsinvolvementinyeast-hypha by theMAPKMkc1)andasecondpathwaythatwasoriginally stress resistanceinC.albicans:thecellintegritypathway(defined et al.,2007).Two additionalMAPKpathwayscontributetocellwall regulates chitinbiosyntheticfunctions(Eismanetal.,2006;Munro The Hog1pathwaycontributestocellwallfunctionalityand response tostressesencounteredduringhost–fungusinteractions. in thewild,aswellcellwallremodellingeventsthatoccur reflect normalcellwallhomeostasisduringgrowthanddevelopment changes thatoccurinresponsetotheseartificialinsultspresumably whereas CalcofluorWhiteperturbschitinassembly. Thecellwall and CongoRedinterferewith et al.,2006;Walker etal.,2008;Leach2012a).Caspofungin REVIEW Oxidative stress module arealsoinvolvedinthe 2002; Alonso-Mongeetal.,2006).ComponentsofthisMAPK MAPKKK Ste11, theMAPKKHst7andMAPKCek1(Brown, 2007). but dodisplayattenuatedvirulence(Csanketal.,1998;Arana mutants arenothypersensitivetomacrophageorneutrophilkilling, et al.,2006;CanteroandErnst,2011). to cellwallstresses(Lebereretal.,1996;Csank1998;Eisman filamentous growthundercertainconditionsandconferssensitivity Inactivation ofcomponentsontheCek1pathwayinhibits wall integrity(Románetal.,2009;CanteroandErnst,2011). response tocellwalldamagingagentsandmutationsthataffect cell The Cek1pathwayisactivatedviathecellsurfacesensorMsb2in et al.,2002),butCek2actsastheMAPKundertheseconditions. The cellintegritypathwayincludesaMAPKKmodulethat The morphogeneticMAPK(Cek1)pathwayincludesthe C. albicans GCS1), redoxhomeostasisandoxidative damagerepair (Diez-Orejas etal.,1997). is relativelyresistanttoreactiveoxygenspecies Pap1 (AlarcoandRaymond,1999),upon C. albicans C. albicans β − 1 -glucan synthesisandassembly, hydrogen peroxide(H to killingbyneutrophils Candida albicanscek1 mating response(Chen GLR1 S. cerevisiae and 2 CAT1 O 2 TRX1). ) under and infections. responses becomelessvitalasthefungusdevelopssystemic stress responsestoprotectitselffromphagocytickilling,but these together, thedatasuggestthat 1999; daSilvaDantasetal.,2010;Cheetham2011). Taken trx1 phagocytes (Fradinetal.,2005;Arana2007),and albicans cap1 following theperturbationofoxidativestressregulators. 2005; Frohneretal.,2009).Similarphenotypesarealsoobserved the fungus(Wysong etal.,1998;Hwang2002;Fradin more sensitivetophagocytickillingandattenuatesthevirulenceof such assuperoxidedismutatesandcatalase,renders al., 2012).TheinactivationofgenesinvolvedinROSdetoxification, stress genes(Enjalbertetal.,2007;AranaMiramón C. albicans expression patternshavebeenconfirmedbysinglecellprofilingof et al.,2007;Walker etal.,2009;Wilson etal.,2009).These are notactivatedtothesameextentduringtissueinfection(Thewes 2005), andduringmucosalinfection(Zakikhanyetal.,2007),but (Rubin-Bejerano etal.,2003;Lorenz2004;Fradin are inducedfollowingexposuretohostmacrophagesandneutrophils profiling experimentshavedemonstratedthatoxidativestressgenes this pathogencarryingsixsuperoxidedismutasegenes.Transcript evolutionary expansionofthe adaptation isessentialforpathogenicity. Therehasbeen to oxidativestressduringinfectionandthat independent fashion(Enjalbertetal.,2006). oxidative stress-inducedtranscriptsareinducedinaHog1- nuclear accumulationofCap1isnotdependentonHog1,andmost oxidative stressresistanceremainanareaofactiveresearch.The downstream molecularmechanismsthatunderlieHog1-mediated Hog1 activationinresponsetoH oxidative stressresistancein al., 2010).Anadditionalresponseregulator(Crr1)contributesto and theperoxiredoxinTsa1 (KruppaandCalderone,2006;Smithet the histidinekinases(Sln1,Chk1,Nik1),responseregulatorSsk1 2010). OxidativestresssignalsappeartobetransducedHog1via and Calderone,2006;daSilvaDantasetal.,2010;Smith Monge etal.,2003;ChauhanSmith2004;Kruppa upstream regulatorsconferoxidativestresssensitivity(Alonso- resistance in (da SilvaDantasetal.,2010). adaptation, ismodulatedbytheredoxregulatorthioredoxin(Trx1) et al.,2006).TheredoxstatusofCap1,andhenceoxidativestress sensitive tooxidativestress(AlarcoandRaymond,1999;Enjalbert attenuates theinductionofthesegenes,rendering static ratherthancidaleffects upon thiol groupsofcysteinesinproteinsandglutathione.RNS exert oxide, causesmoleculardamagesuchastheS-nitrosylationof the Exposure toreactivenitrogenspecies(RNS),forexample nitric 2012). C. albicans: one ofthreegenesencodingflavohaemoglobin-related proteinsin 2005). Inaddition, and NADPHoxidoreductases dehydrogenases(Hromatkaetal., as catalase(Cat1),glutathione-conjugating and-modifyingenzymes, a definedsetofgenesthatincludes oxidativestressfunctionssuch Nitrosative stress Numerous observationsindicatethat The Hog1MAPKpathwayalsocontributestooxidativestress mutants displayattenuatedvirulence(Alonso-Mongeetal., Candida albicans The JournalofExperimentalBiology(2014)doi:10.1242/jeb.088930 cells taggedwithdiagnosticGFP fusionstooxidative C. albicans YHB1, and YHB1 hog1 YHB4 responds tonitrosativestressby activating mutants arekilledmoreeffectively by expression isstronglyinduced. Fg 1).InactivationofHog1andkey (Fig. SOD and C. albicans C. albicans,butisnotrequiredfor YHB5 gene familyin 2 O C. albicans 2 C. albicans (Bruce etal.,2011). The exploits robustoxidative (Ullmann etal., 2004; C. albicans,with cells areexposed C. albicans (Kaloriti etal., C. albicans hog1 Candida YHB1 cells 147 and is

The Journal of Experimental Biology 148 REVIEW prior exposure to stress 1 does not activate a relevant molecular memory and does not confer protection against stress 3 (lower panel). prior exposuretostress 1doesnotactivatearelevant molecularmemoryanddoesnotconfer protectionagainststress3(lower against stress2(upper panel).However, stress(stress3;green), then ifthiscoretranscriptional responsedoesnotincludegenes thatprotectagainstathird memory that confersprotection genes thatprotectagainstanotherstress (stress2;red).Inthiscase,priorexposuretostress1oftenactivates amolecular response(purple)thatincludes time intervalsbetweenstresses(lower panel).(B)Insomeyeasts,stresses(stress1;blue)activatea coretranscriptional However, willbe lostduringprotracted themoleculesthatrepresentthismemoryhavebiologicalhalf-lives. Therefore,thismolecularmemoryistransient,and tosequentialstresses’). exposure tothatstress(acquired tolerance)(upperpanel).Thisindicatestheexistenceofamolecular memory(see‘Adaptation Acquiredstresstoleranceandcross-protection inyeasts. 2. Fig. repaired, allowing redox homeostasisisrestoredandS-nitrosylatedadductsare al., 2004;Hromatkaet2005).FollowingRNSdetoxification, dioxygenase responsiblefornitricoxidedetoxification(Ullmannet to nitricoxide,andthisgeneencodesthemajoroxide Tillmann etal.,2011). Ofthese,only and Second, incomparisonwiththebenignmodelyeasts MAPK modules,andthetranscriptionfactorsHsf1Cap1. fungal evolution.ExamplesincludetheHog1,Mkc1andCek1 pathways includeregulatorsthathavebeenhighlyconservedduring key stressresponsesinC.albicans.First,thesestress-signalling host immunedefences. during theearlystagesofinfectionwhenfungusisbattlingwith Therefore, thenitrosativestressresponseseemstobemostimportant systemic candidiasis(Hromatkaetal.,2005;Chiranand2008). causes aslightreductioninvirulencethemousemodelof Walker etal.,2009),andtheinactivationofYhb1orCta4only strongly activatedduringsystemicinfection(Thewesetal.,2007; infections (Zakikhanyetal.,2007).However, theresponseisnot Nitrosative stressgenesarealsoupregulatedduringmucosal phagocytic attack(Fradinetal.,2005;Zakikhany2007). surprisingly, therefore,nitrosative stressgenesareinducedfollowing 1995; Vázquez-Torres andBalish,1997;Brown,2011). Not includes RNSthatcontributetofungalkilling(Rementeríaetal., 1). et al.,2004;Chiranand2008)(Fig. either response toRNS(Chiranandetal.,2008),andtheinactivationof factor Cta4isresponsibleforactivating matter. However, ithasbeenshownthatthezincfingertranscription nitrosative stressresponseinC.albicans,orotheryeastsforthat unpublished). Several commonthemesareapparentfromthisbriefoverviewof In additiontoROS,themoleculararmouryofphagocyticcells Little isknownaboutthesignallingpathwaysthatmediate S. pombe,thesestressresponseshavebeenevolutionarilytuned CTA4 or YHB1 C. albicans confers nitrosativestresssensitivity(Ullmann Survival Memory Response Survival Memory Response to resumegrowth(A.T. andA.J.P.B., Stress Stress A YHB1 Stress is inducedinresponse YHB1 expression in Stress S. cerevisiae Time (A) Priorexposuretoastresscanprotect in thehost.Intheseniches above, thispathogeninhabitsdiverse,complexanddynamicniches individual stressesfollowinggrowthonglucose.Yet, asdescribed adaptation in Almost withoutexception,alloftheabovestudiesonstress 2007; Brownetal.,2012a;Román2007). intimately linkedtothevirulenceofthispathogen(Brownetal., during itsinteractionswiththehost.Third,thesestressresponsesare to thetypesandintensitiesofstressesthat molecular memoryistransient(Leachetal.,2012c),withthelength a subsequentstress,leadingtoincreasedsurvival.However, this represent a‘molecularmemory’ thatcanthenprotectthecellagainst mediate adaptationtothatstress.Theseproteinsandmetabolites induction andaccumulationofkeyproteinsormetabolitesthat of amolecularresponsetotheinitialstress,whichrepresents al., 1996).Acquiredstresstoleranceisdependentupontheactivation also beenobservedforoxidativestressin et al.,1990;Piper, 1993;Argüelles, 1997).Acquiredtolerancehas Adaptation tosequentialstresses cerevisiae For example,acquiredthermotolerancehasbeendescribedin 2A). yeast cellsagainstasubsequentdoseofthatsamestress(Fig. time thatpriorexposuretoanon-lethaldoseofstresscanprotect adaptation. section discussestheimpactofcombinatorialinputsuponstress section addressesadaptationtosequentialstresses,andthefollowing factors significantlyinfluencestressadaptationin alternative carbonsources.Recentdatahaverevealedthatthese albicans glucose iseitherlimitingorabsentfrommanyhostniches, the fungusisexposedto‘combinatorialstress’.Furthermore,as other times,multiplestressesareimposedsimultaneouslysuchthat stresses. Attimesthesestressesmaybeimposedsequentially. At With regardtosequentialstresses,ithasbeenknownforsome Survival Memory Response Survival Memory Response , cells mustadapttothesestresseswhilstexploiting S. pombe The JournalofExperimentalBiology(2014)doi:10.1242/jeb.088930 C. albicans B Stress 1 Stress 1 and morerecentlyin Stress 3 Stress 2 have examinedtheresponsesofcellsto C. albicans C. albicans Time will beexposedtomultiple cells againstsubsequent C. albicans C. albicans C. albicans C. albicans.This (Jamieson et (De Virgilio encounters C. S.

The Journal of Experimental Biology driven bytheSty1SAPK.Thecore transcriptional responsehasdivergedsignificantlyin and C.glabrata core transcriptionalresponsestostress inwhichrelativelylargenumbersofgenesarecommonlyinduced responsetodifferent stresses.InS.cerevisiae Three oftheseyeastsdisplay evolutionarily separatedbymanymillions ofyearsandoccupycontrastingniches:green,environmentalniches; red,pathogens. genes (see‘Adaptation tosequentialstresses’). i.3 Corestressresponsesinyeasts. 3. Fig. cerevisiae osmotic orthermalstresspromotesfreeze–thawtolerancein Lewis etal.,1995).Similarly, pre-treatmentwithanoxidative, cells againstasubsequentoxidativestress(Wieser etal.,1991; 2B).ForexampleinS.cerevisiae, amildheatshockprotects (Fig. different typeofstress–aphenomenoncalledcross-protection stress canalsoprotectyeastcellsagainstasubsequentdoseof themselves (Jamiesonetal.,1996). accumulation ofantioxidantenzymesratherthanantioxidants acquired tolerancetooxidativestressisprobablymediatedbythe the osmoticstressisremoved(Klippetal.,2005).Byanalogy, 2012), becauseglycerolisrapidlyextrudedfromyeastcellswhen biosynthetic enzymesratherthantheosmolyteglycerol(You etal., the molecularmemoryisthoughttobemediatedbyglycerol levels declinerapidlyonce trehalose (Argüelles, 1997;Leachetal.,2012c),becausetrehalose trehalose biosyntheticenzymesratherthanthestressprotectant the molecularmemoryin 2A).Forexample,inthecaseofthermotolerance, metabolites (Fig. of thememorydependingupondecayratestheseproteinsand stress isdisplayedbyCandidaglabrata,whichinevolutionaryterms signalling pathway(Görneretal.,1998;Garreau2000). downregulated byglucoseviathecAMP-proteinkinaseA (PKA) 2001). Msn2anditsstress-inducedtranscriptionalactivationare (Mager andDeKruijff, 1995;Gaschetal.,2000;Causton in thepromotersoftheirtarget genestomediatetheiractivation activators Msn2andMsn4,whichbindtostressresponseelements is largely dependentonthefunctionallyredundanttranscriptional (Gasch etal.,2000;Causton2001).Thiscorestressresponse roles instressadaptation,centralmetabolismandenergy generation osmotic, oxidativeorpHstressactivatesseveralhundredgeneswith 3).Forexample,in stress (Fig. activates genesinvolvedinadaptiveresponsestomanytypesof whereby exposuretoanyoneofseveraldifferent typesofstress phenomenon liesinthecoretranscriptionalresponsetostress REVIEW temperatures (Argüelles, 1997).Forosmotolerancein In somecases,priorexposuretoanon-lethaldoseofonetype An analogousMsn2-dependentcoretranscriptionalresponseto (Park etal.,1997).Themolecularbasisforthis the zinc finger transcription factors Msn2 and Msn4 contribute significantly to the core stress response, whereas this response inS.pombe the zincfingertranscriptionfactorsMsn2 andMsn4contributesignificantlytothecorestressresponse,whereas thisresponse Oxidative Osmotic Heat S. cerevisiae C. albicans ~220 genes C. albicans CSR S. cerevisiae Msn2/4 pH The yeastsC.albicans probably representsHSPsand

~60 MYA cells arereturnedtolower Heat Oxidative exposure tothermal, C. glabrata ~400 genes CSR C. albicans Osmotic ~150 MYA Msn2/4 , Saccharomyces cerevisiae

starvation Glucose S. , Oxidative Osmotic response, aswelloftheresponseitself. significant rewiringofthecircuitrythatregulatescorestress Hog1 andCap1(Enjalbertetal.,2006).Clearlytherehasbeen Ramsdale etal.,2008),andthecorestressresponseiscoordinatedby transcription factorshavediverged significantly(Nichollsetal.,2004; (Mitchell etal.,2009).Thisadaptivepredictionisasymmetric,as exhausted andcellsswitchtorespiratoryoxidativemetabolism oxidative stressgenesthatwillberequiredonceglucoseis temperatures associatedwithvigorousfermentationtoinduce prediction isdisplayedby conferring anevolutionaryadvantage.Thistypeofadaptive circuitry thatallowsthemtopredictthesecondstimulus,thereby stimulus. Insuchcasesorganisms mayhaveevolvedaregulatory environmental changeisoftenfollowedbyasecondtypeof relatively predictableenvironments,inwhichonetypeof Mitchell andco-workersargue thatsomemicroorganisms inhabit 4A). microbial adaptiveprediction(Mitchelletal.,2009)(Fig. stress (Enjalbertetal.,2003;Leach2012a). stress doesnotprotect cross-protection isasymmetric,asaninitialtreatmentwithoxidative shock (Nichollsetal.,2009;Leach2012a).However, this correlates withtheinductionofsomeCap1target genesduringheat et al.,2012a).Thiscross-protectionisdependentonCap1and subsequent osmoticorcellwallstress(Enjalbertetal.,2003;Leach albicans during exposuretosequentialstresses.Thermalstressprotects genes (Enjalbertetal.,2006).In response, butonethatcomprisesamuchsmallersubsetofroughly25 Subsequent workrevealedthatthisyeastdoesdisplayacorestress core transcriptionalresponsetostress(Enjalbertetal.,2003). et al.,2009).Incontrast, Rezre l,20)(i.3). (Roetzer etal.,2008)(Fig. lies muchcloserto Hog1 SAPKin driven bySty1(Chenetal.,2003),whichistheorthologueof displays acorestressresponse.However, inthiscasetheresponseis C. albicans This hassignificantimplicationsforthebehaviourof These observationsarereminiscentofthephenomenon ~20 genes CSR C. albicans against asubsequentoxidativestress,butnot >400 MYA ,

metal Heavy The JournalofExperimentalBiology(2014)doi:10.1242/jeb.088930

Candida glabrata

, inwhichthereisarelativelysmallnumber ofcorestress S. cerevisiae S. cerevisiae C. albicans Heat Oxidative C. albicans S. cerevisiae ~140 genes and Schizosaccharomycespombe S. pombe , C. glabrata CSR C. albicans Schizosaccharomyces pombe cells againstasubsequentthermal than to Sty1 Osmotic

was initiallythoughttolacka metal Heavy , whichexploitstheelevated and S. pombe the rolesofMsn2/4-like C. albicans or C. albicans C. albicans (Nikolaou are also 149 is C.

The Journal of Experimental Biology drive oxidative stress adaptation(Brownetal., 2012a). more todowiththeneedformetabolic intermediatesandenergy to et al.,2006),mayhavelessto do withanticipatorypredictionand (Gasch etal.,2000;Causton 2001;Chenetal.,2003;Enjalbert induced metabolicactivation), whichisconservedinotheryeasts et al.,2006).However, thisparticularresponse(oxidativestress- activation ofgenesinvolvedin central carbonmetabolism(Enjalbert exposing 1995; Görneretal.,1998;Garreau2000). which hasevolvedinenvironmentalniches(MagerandDeKruijff, cerevisiae unpublished). Indeed,theoppositephenotypeisobserved in relatively recently. Thisappearstobethecase(I.B.andA.J.P.B., glucose-enhanced oxidativestressresistancemusthaveevolved attack fromphagocyticcells.Ifthisweretrue,thephenomenon of glucose, andthismayhelptoprotectthemagainsttheimpending Candida albicans in thebloodstream,islikelytoberelevantdiseasecontext. response, whichistriggeredbytheglucoseconcentrationspresent pathways (I.B.andA.J.P.B., unpublished).Thisanticipatory to beregulatedbyevolutionarilyconservedglucosesignalling 2009). Instead,glucose-enhancedoxidativestressresistanceappears This phenomenondoesnotdependonHog1orCap1(Rodakietal., 4B). resistance toacuteoxidativestress(Rodakietal.,2009)(Fig. following exposuretoglucose,therebyconferringelevated albicans. Inthisfungus,oxidativestressgenesareactivated 150 REVIEW inflammatory responses. attack byphagocyticcellsinresponsetofeversassociatedwith evolved in is consistentwiththeideathatadaptivepredictionmighthave 4B).This induced byanoxidativestress(Enjalbertetal.,2003)(Fig. induced inresponsetoheatshock,butshockgenesarenot observed inC.albicans:general,oxidativestressfunctionsare relationship betweenoxidativeandheatshockgeneregulationis cerevisiae oxidative stressdoesnotinduceheatshockgeneexpressionin glucose exposureandbyactivatingcarbohydratemetabolisminresponsetooxidativestress(see‘Adaptationsequentialstresses’). stressgenesinresponseto response (Mitchelletal.,2009).Thispathogenalsodisplayssymmetricanticipatoryadaptivepredictionbyactivatingoxidative prediction byactivatingoxidativestressgenesinresponsetoelevatedtemperatures. environmental niche.Anticipatoryresponsescanbeasymmetricorsymmetric.(B) inputinitsnormal authors arguethatthisprovidesanevolutionaryadvantagetothemicrobebecausefirstinputisoftenfollowedbysecond (Mitchelletal.,2009).The whereby exposuretooneenvironmentalinputcanleadtheanticipatoryinductionofresponseasecond Anticipatory predictionin 4. Fig. This anticipatoryresponseappearstobesymmetricbecause A secondexampleofadaptiveprediction hasbeendescribedin : glucosereducesstressresistanceinthisbenignyeast, C. albicans C. albicans (Mitchell etal.,2009).Ananalogousasymmetric cells thatenterthebloodstreamareexposedto such thatthepathogenanticipatesoxidative cells tohydrogenperoxideleadsthe B A temperature C. albicans Elevated genes HSP S. cerevisiae Response 1

Stress 1 and S.cerevisiae adaptive prediction Asymmetric stress genes Oxidative Oxidative stress Response 2 Stress 2 . (A)AsdescribedbyMitchellandco-workers,microbesoftendisplayadaptiveprediction, C. S. S. temperature Elevated genes examples ofthishaveemerged recently. between therelevantsignallingpathwaysmightexist.Several (Kaloriti etal.,2012).Ourrationaleisthatunexpectedcross-talk the sumofresponsestocorrespondingindividualstresses responses tosuchcombinatorialstressesmightnotbeequivalent combinatorial stresses?We havepredictedthattheadaptive salt concentrations).Howdo must dealwithendogenousROSwhileadaptingtorelativelyhigh cellular waterbalance)andkidneyinfection(whererespiringcells invasion (whereoxidativestressesareencounteredwhileadjusting be relevantinmanyotherhostniches,suchasduringmucosal 2011; Nüsse,2011). However, combinatorialstressesarelikelyto (Rementería etal.,1995;Vázquez-Torres andBalish,1997;Brown, fungus isbombardedwithROS,RNSandcationicfluxes following phagocytosisbyneutrophilsormacrophages,whenthe inhabit. Possiblythebestexampleofcombinatorialstressoccurs exposed tomultiplestresseswithinthecomplexhostnichesthey As mentionedabove, effective killing of to behighlyrelevanthost–fungus interactionsbecausethe signalling (Phillipsetal.,2003; Phillipsetal.,2006).Thisappears to stimulateapoptoticcelldeath in A.J.P.B., unpublished).Indeed,hydrogenperoxide hasbeenshown levels increase,leadingtocell death(D.K.,M.D.J.,A.T. and and oxidativestressgenesare not induced,andintracellularROS combination ofcationicandoxidativestress.Asaresult, whereby bothCap1andHog1signallingarecompromisedby the The basisforthisappearstobe‘stresspathwayinterference’, (Kaloriti etal.,2012).Thesestresseskill are observedforcombinatorialcationicplusoxidativestresses (A.T. andA.J.P.B., unpublished). Significantly, non-additiveeffects these conditions,indicatingthatCta4signallingiscompromised et al.,2012).However, exert additiveeffects uponthe growthof combinatorial cationic(NaCl)plusnitrosativestressesappear to (dipropylenetriamine-NONOate, DPTA-NONOate) and Adaptation tocombinatorial stresses HSP Saccharomyces cerevisiae Combinatorial oxidative(H Candida albicans Response 1

Stress 1 adaptive prediction stress genes The JournalofExperimentalBiology(2014)doi:10.1242/jeb.088930 C. albicans Symmetric Oxidative Oxidative stress displays ananalogousasymmetricanticipatoryadaptive C. albicans Response 2 Stress 2 C. albicans displays asymmetricanticipatoryadaptive YHB1 Metabolic exposure Glucose genes gene inductionisattenuatedunder C. albicans cells byhuman neutrophils appears 2 O cells areoftensimultaneously 2 ) plusnitrosativestresses C. albicans C. albicans C. albicans cells respondtosuch via Ras-cAMP synergistically. cells (Kaloriti

The Journal of Experimental Biology toxic compoundsthatcandamagelipids,proteinsandnucleicacids activation ofdifferent subsetsofstressgenes. chlorine species(RCS) canbegeneratedspontaneously andbyenzymaticcatalysis(Brown etal.,2009;Brown2011), leading tothe presumably unpublished). (D)Thereiscross-talk atthechemicallevel,wherebydifferent species(RNS)andreactive reactiveoxygenspecies(ROS),nitrogen stress pathwayinterference,whereby Hog1andCap1signallingareaffected M.D.J.,A.T. byoxidativeandcationicstress,respectively(D.K., andA.J.P.B., oxidativestressleadsto Hsf1 andtheMAPKsinresponseto thermalfluctuations(Leachetal.,2012a;Leach2012b).(C)Combinatorial cationicplus growth pathway:Ste11, MAPKKK;Hst7,MAPKK;Cek1,MAPK.(B)Hsp90 actsasabiologicaltransistor, thetranscription factor modulating theactivitiesof Mating/invasive signalling pathway:Ssk2,MAPKKK; Pbs2,MAPKK;Hog1,MAPK/SAPK.Cellintegritypathway:Bck1,MAPKKK; Mkk1,MAPKK;Mkc1,MAPK. albicans Mechanismsunderlyingcombinatorialstress effectsin 5. Fig. Combinations ofH et al.,2005).Cross-talkalsoexistsatthechemicallevel. phosphorylation andtheinhibitionofMkc1(Arana fluctuations havebeenshowntoinfluence activation ismodulatedbyHsp90(Leachetal.,2012a).Temperature in thesepathwaysareallclientproteinsofHsp90,andtheir Cek1 pathwaysmodulatecellwallfunctionality. TheMAP kinases (Leach etal.,2012a).Asdescribedabove,theHog1,Mkc1and 2012a). ThisstressinteractionappearstobemediatedviaHsp90 White), buthavelittleeffect uponosmo-sensitivity(Leachetal., sensitivity of other stresses.Forexample,elevatedtemperaturesdecreasethe oxidative stresses(D.K.,M.D.J.,A.T. andA.J.P.B., unpublished). to dependontheextremepotencyofcombinatorialcationicplus REVIEW combine toformnitrylchloride(NO form peroxynitrite(ONOO hypochlorous acid(HOCl),andnitricoxidesuperoxidereactto whereby thedeletionof stress signallingpathways.Thisissuggestedbymutationalanalyses appears tobesignallingcross-talkbetweentheMAPKsincritical 5).First,there by whichcombinatorialcross-talkcanoccur(Fig. available datahaverevealedseveraldistinctmolecularmechanisms 1).The pathways thatmediateresponsestospecificstresses(Fig. stresses exertunexpectedeffects upontheclassicalregulatory ambient temperatureofthecell(Leachetal.,2012b). environmental responses,includingcellwallremodelling,tothe been proposedtoactasabiologicaltransistorthattunes (Leach etal.,2012a;Heilmann2013).Therefore,Hsp90has cell wallproteome,andHsp90depletionaltersarchitecture Leach etal.,2012c).Furthermore,ambienttemperatureaffects the (Nicholls etal.,2009;Diezmann2012;Leach2012a; as wellthebindingofHsp90toitsclientproteinsin Combinatorial effects arealsoobservedbetweenthermaland Therefore, aspredicted(Kaloritietal.,2012),combinatorial (see ‘Adaptation to combinatorial stresses’). (A) Classical cross-talk occurs between the MAPK signalling pathways (Alonso Monge etal.,2006).Hog1 (see ‘Adaptationtocombinatorialstresses’). (A)Classicalcross-talkoccursbetweentheMAPKsignallingpathways (AlonsoMonge C. albicans 2 O 2 A C HOG1 and NaClleadtotheformationof B cells toacellwallstress(Calcofluor – ), andnitritehypochlorousacid stress genes Ste11 Cek1 Hst7 leads tothederepressionofCek1 Cationic Cationic stress 2 Cl), generatingcocktailsof Pbs2 Ssk2 Hog1 plus HSP90 stress genes Oxidative Oxidative stress expression levels Bck1 Mck1 Mkk1 C. albicans C. albicans. D hypochlorous HOCl acid Hsp90 Several distinctmechanismscontributetocombinatorialstresseffects in adding tothedynamismofhostniches.Theregulatorycircuitrythat modify thepHofitsmicroenvironment(Vylkova etal.,2011) Miramón etal.,2012).Themetabolicactivityof cells (Lorenzetal.,2004;Fradin2005;Barelle2006; utilisation (Barelleetal.,2006),asdophagocytosed infecting thekidneyactivatepathwaysforalternativecarbon (Ueno etal.,2011), andasignificantproportionof assimilation isessentialfor al., 2006;RamírezandLorenz,2007).Furthermore,lactate virulence (LorenzandFink,2001;Barelleetal.,2006;Piekarska gluconeogenesis andtheglyoxylatecycle,arerequiredforfull assimilation ofthesealternativecarbonsources,suchas Not surprisingly, metabolicpathwaysthatareessentialforthe alternative carbonsourcesifitistogrowandcolonisetheseniches. fatty acids.Consequently, sources suchasaminoacids,carboxylicacidslactate,and Instead, thesenichescontaincomplexmixturesofalternativecarbon sugars suchasglucoseorcontainatlowconcentrations. C. albicans Metabolic changeswithinhostnichesalsoaffect stressadaptationin stress adaptationishighlyrelevanttonaturalenvironments. that underliecombinatorialstressadaptation.Yet, combinatorial reasonably wellunderstood,littleisknownaboutthemechanisms While theresponsesoffungalcellstoindividualstressesarenow pathways involvedincellularadaptation(Leachetal.,2012b). this case,Hsp90coordinatestheactivitiesofmultiplesignalling combinatorial effects canbemediatedbyabiologicaltransistor. In and A.J.P.B., unpublished).Inaddition,wehaveshownthat pathway interferenceandultimatelycelldeath(D.K.,M.D.J.,A.T. stresses leadstothebuildupofintracellularROS,causingstress In thiscasetheinhibitionofkeydetoxificationfunctionsbycationic combinatorial effects canalsobetriggeredatthebiochemicallevel. (reviewed byBrownetal.,2009).We havenowshownthat Impact of dynamichost niches adaptation uponstress Impact of

RCS Hsf1 hydrogen peroxide H 2 Thermotolerance O Temperature 2 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.088930 Cek1 Stress genes Fg 6).Inparticular, manyhostnicheseitherlack (Fig. superoxide ROS O 2 Hog1 – oxide nitric NO Mck1

C. glabrata C. albicans peroxynitrite RNS ONOO – to colonisetheintestine must assimilatethese C. albicans C. albicans C. albicans C. cells 151 can

The Journal of Experimental Biology 152 REVIEW (Barelle etal., 2006). Thisisbecausethemicroenvironments of populations displayheterogeneous behavioursinhostniches molecular behaviourofthefungal populationasawhole,andfungal Wilson et al., 2009).However, microarraystudies averagethe (Fradin etal.,2005;Thewes al.,2007;Zakikhanyet Bernie Hube’s groupgosomeofthewaytoaddressingthisquestion disease progression?Theelegant microarraystudiesperformedby stress responsesregulatedtemporallyduringhostcolonisation and stress adaptationremainslargely unexplored.Inparticular, howare dynamic, andtheimpactofthiscomplexitydynamism upon fungal pathogenofhumans.However, host nichesarecomplexand because stressadaptationcontributestothevirulenceofthismajor elaboration ofspecificstresssignallingpathways.Thisisimportant adaptation in Significant advanceshavebeenmadeinourunderstandingof stress cells intheseniches(Eneetal.,2012a). efficacy ofantifungaldrugtreatments againstindividual and diseaseprogression.Significantly, thisisalsolikelytoaffect the dynamic microenvironmentsitoccupiesduringhostcolonisation environmental adaptationofthisfunguswithinthecomplexand albicans, andthisfurthercomplicatesourunderstandingof 2013). Clearly, metabolicadaptationaffects stressresponsesin and mucosalmodelsofinfection(Eneetal.,2012a;Ene cells andinfluencethevirulenceofthispathogeninbothsystemic alterations atthecellsurfaceaffect hostrecognitionof 6).These of thecellwall(Eneetal.,2012a;Ene2012b)(Fig. of thecellwall,whichinturnimpactuponbiophysicalproperties alternative carbonsourcesontheproteomiccontentandarchitecture on Hog1orMkc1signalling.Instead,itrelatestotheeffects of antifungal drugs.Thisincreasedstressresistanceisnotdependent more resistanttoosmoticstress,cellwallstressesandsome adaptation in alternative carbonsources,suchaslactateoroleicacid,affects stress Recently, weshowedthatgrowthonphysiologicallyrelevant performed oncellsgrowninmediacontaining2%glucose. niches, moststudiesofstressadaptationinC.albicans adaptation (discussedabove). Lavoie etal.,2009;Sandai2012),justasisthecaseforstress evolutionary rewiring(Ihmelsetal.,2005;Martchenko2007; regulates carbonassimilationin Impact ofcarbonsourceonC.albicans. 6. Fig. Outlook micrographs ofcellwallsfrom wall. Thisaffects Transmission stressadaptation,immunerecognitionandvirulence(Eneetal.,2012a;Ene2012b;2013). electron Despite thefactthatglucoseislimitingorabsentinmanyhost C. albicans C. albicans,andprogressisbeingmadetowardsthe (Ene etal.,2012a).Lactate-growncellsare C. albicans adaptation Stress Carbon source cells grownonglucoseorlactateareshowntheright. C. albicans Virulence Cell wall Changes incarbonsourceaffect theproteome,architectureandbiophysicalpropertiesof recognition Immune has undergone C. albicans C. albicans have been properties Biophysical Proteome Architecture C. therapeutically. cationic plusoxidativestresscould,inprinciple,beexploited such astheacutesensitivityof laboratories (DolginandMotluk,2011). Therefore,observations potential ofHsp90inhibitorsisbeingpursuedbyanumber of development ofnovelantifungaltherapies.Indeed,thetherapeutic provide targets fortranslational researchdirectedtowardsthe are revealingpointsoffragilityin driven therecentevolutionofthispathogeninitshost. Such studieswillprovideimportantinsightsintotheforcesthat have the developmentofatrueunderstandingtheseadaptiveprocesses. predictive mathematicalmodellingseemsespeciallyimportantfor their complexity, acombinationofexperimentalapproachesand many oftheseresponsesaredynamicanddosedependent.Given transcriptional, biochemicalandchemicalcross-talk.Furthermore additive behavioursthatreflectunexpectedsignalling, exploration oftheseissues,itisalreadyclearthattheyinvolvenon- influence stressresistancewithinhostniches?Despitethelimited and howaretheyregulated?Third,doesmetabolicadaptation stress responsesin controlled atthemolecularlevel?Second,whichcombinatorial disease progression,andhowaretheseanticipatoryresponses anticipatory responsesinC.albicans direct relevance A.J.P.B., unpublished). are beingpursuedbytheAberdeenFungalGroup(J.P., S.S.and example byexploitinglasercapturemicroscopy. Theseapproaches sequencing technologiesandincreasingtheirspatialresolution,for Miramón etal.,2012),orbyincreasingthesensitivityofRNA profiling methods(Barelleetal.,2006;Enjalbert2007; individual cells infection. Thismusteitherbedonebyexaminingtheresponsesof spatial regulationofstressadaptationmustalsobeexaminedduring individual cellsvaryevenwithinspecifichostniches.Therefore,the special mention. Netea, FransKlis,Leah Cowen,StephanieDiezmannand JoeHeitmandeserve Brown, JanetQuinn,KenHaynes,Christophe d’Enfert,BernardHube,Mihai discussions andhelpfuladvice.NeilGow, FrankOdds,CarolMunro, Gordon Consortium, theFINSysBNetworkand theCowenlaboratoryforstimulating We thankourfriendsandcolleaguesintheAberdeenFungalGroup, theCRISP Acknowledgements In closing,itisworthemphasisingthatstudiesofstressadaptation In addition,atleastthreeaspectsofstressadaptationthatare Glucose Lactate The JournalofExperimentalBiology(2014)doi:10.1242/jeb.088930 in vivo in vivo C. albicans , forexampleusingGFP-basedsingle-cell need furtherdissection influence host–fungusinteractions, C. albicans C. albicans influence hostcolonisationand towards combinatorial that couldpotentially in vitro C. albicans . First,which cell

The Journal of Experimental Biology led,R . rne . lrct . hws . ae . dad,J.E., Edwards, M., Laue, S., Thewes, A., Albrecht, S., Brunke, R.S., Almeida, rw,A.J.P. Brown, rw,A .P,Hye,K n un,J. Quinn, and K. N.A.R. Haynes, A.J.P., Gow, Brown, and F. C. Odds, A.J.P., Brown, rc,C . mt,D . ogr,D,d iv ats . aClu,D.M., MacCallum, A., daSilvaDantas, D., Rodgers, D.A., Smith, White, and C.R., M.G. Bruce, Netea, S.M., Levitz, N.A.R., Gow, D.W., Denning, G.D., Brown, Trust [grantnumber096072].DepositedinPMCforimmediaterelease. Scholarship andaSirHenryWellcome PostdoctoralFellowshipfromtheWellcome 080088, 097377].M.D.L.wasalsosupportedbyaCarnegie/Caledonian BB/F010826/1; BB/F00513X/1],andbytheWellcome Trust [grantnumbers [grant numbersBBS/B/06679;BB/C510391/1;BB/D009308/1;BB/F000111/1; 2009-AdG-249793], bytheUKBiotechnologyandBiologicalResearchCouncil the EuropeanCommission[FINSysB,PITN-GA-2008-214004;STRIFE,ERC- We aregratefultoourfundingbodiesfortheirsupport.Thisworkwassupportedby by A.J.P.B. andM.D.L. All authorscontributedtothewritingofthisreview, theinitialdraftbeingprepared The authorsdeclarenocompetingfinancialinterests. REVIEW led,R . isn .adHb,B. Hube, and D. Wilson, R.S., Almeida, M. Raymond, and A.M. Alarco, rw,G.D. Brown, A.J.P. Brown, ulr . amse,M . i,M . ats .A . atiua,S,Munro, S., Sakthikumar, M.A.S., Santos, M.F., Lin, M.D., Rasmussen, G., Butler, rw,A .P,Hye,K,Gw .A .adQin J. Quinn, and N.A.R. Gow, K., Haynes, A.J.P., Brown, References Funding Author contributions Competing interests lnoMne . aar-aca . oeo . izOea,R,Gsi,M,Pla, M., Gustin, R., Diez-Orejas, G., Molero, F., Navarro-García, R., Alonso-Monge, lnoMne . aar-aca . oá,E,Ngeo .I,Esa,B., Eisman, A.I., Negredo, E., Román, F., Navarro-García, R., Alonso-Monge, ae,C . ot,L . orls .R n ono,A.D. Johnson, and T. R. Sorrells, L.N., Booth, C.R., Baker, J.C. Argüelles, emn .adSdey P. E. Sudbery, and J. Brown, Berman, and F. C. Odds, N.A., Gow, D.M., Maccallum, C.L., Priest, C.J., Barelle, rn,D . lnoMne . u . adrn,R n l,J. Pla, and R. Calderone, C., Du, R., Alonso-Monge, D.M., Arana, C. Nombela, and J. Pla, D.M., Arana, E., Román, J. R., Pla, and Alonso-Monge, E. Rial, C., Nombela, S., Carvaihlo, R., Alonso-Monge, Brand, rn,D . obl,C,Aos-og,R n l,J. Pla, and R. Alonso-Monge, C., Nombela, D.M., Arana, Candida albicans B. Hube, and S.G. Filler, J. Bacteriol. involved inmultidrugresistanceandoxidativestressresponse Int. 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