Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole Drugs

13 Pages·2010·1.03 MB·English

Requirement for Ergosterol in V-ATPase Function
Underlies Antifungal Activity of Azole Drugs
Yong-Qiang Zhang1, Soledad Gamarra2, Guillermo Garcia-Effron2, Steven Park2, David S. Perlin2, Rajini
Rao1*
1TheJohnsHopkinsUniversitySchoolofMedicineofBaltimore,Maryland,UnitedStatesofAmerica,2PublicHealthResearchInstitute,NewJerseyMedicalSchool-
UMDNJ,Newark,NewJersey,UnitedStatesofAmerica
Abstract
Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular
basisfortheirantifungalactivityisnotunderstood.Weusedmultipleapproachestodemonstrateacriticalrequirementfor
ergosterol in vacuolar H+-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis
mutantsofS.cerevisiaefailedtoacidifythevacuoleandexhibitedmultiplevma2phenotypes.Extractionofergosterolfrom
vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S.
cerevisiaeandthefungalpathogenC.albicans,fluconazoleimpairedvacuolaracidification,whereasconcomitantergosterol
feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges
triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These
findingsprovideamechanisticbasisforthesynergybetweenazolesandamiodaroneobservedinvitro.Moreover,weshow
the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In
summary,wedemonstrateanewregulatorycomponentinfungalV-ATPasefunction,anovelroleforergosterolinvacuolar
ionhomeostasis,aplausiblecellularmechanismforazoletoxicityinfungi,andpreliminaryinvivoevidenceforsynergism
between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic
regimens forpatientpopulations afflictedwith systemic fungalinfections.
Citation:ZhangY-Q,GamarraS,Garcia-EffronG,ParkS,PerlinDS,etal.(2010)RequirementforErgosterolinV-ATPaseFunctionUnderliesAntifungalActivityof
AzoleDrugs.PLoSPathog6(6):e1000939.doi:10.1371/journal.ppat.1000939
Editor:LeahE.Cowen,UniversityofToronto,Canada
ReceivedFebruary18,2010;AcceptedMay5,2010;PublishedJune3,2010
Copyright: (cid:2) 2010 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbyNIHgrantsAI065983toRRandAI069397toDSP.Thefundershadnoroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction organization and function [7,8]. Ergosterol is most abundant in
theplasmamembraneandhasbeenimplicatedinseveralcellular
Pathogenic fungal species, including Aspergillus, Candida, Histo- processesincludingsporulation,pheromonesignalingandplasma
plasma and Cryptococcus among others, cause infections ranging membranefusionduringmatingandendocytosis[9,10].Discern-
frommucocutaneousdisorderstolife-threateninginvasivediseases ableamountsofergosterolhavealsobeenfoundinmembranesof
that can involve any organ. In the past two decades, expanding intracellular organelles including peroxisomes, mitochondria,
populationsofimmunocompromisedpatientsandincreaseduseof vacuoles and ER [11]. Some studies have ascribed a regulatory
invasive devices and implants have led to an increase in the role at these intracellular compartments, including homotypic
incidence of fungal infections [1,2]. Currently, four major vacuolefusion[12],mitochondrialbiogenesisandinheritance,and
categoriesofantifungaltherapeuticsareavailabletotreatinvasive proteinsortingalongexocytosisandendocytosispathways[13,14].
fungal infections: polyenes, azoles, echinocandins and flucytosine The absenceof ergosterol inmammals andsuppression offungal
[3]. Azole drugs are the most widely deployed in clinics, and proliferation by a battery of ergosterol biosynthesis inhibitors
inhibitthebiosynthesisofergosterol,thefungal-specificsterol.The emphasize theimportance andutility ofergosterol asan effective
primary molecular target of azole drugs is Erg11p (Entrez targetinantifungalchemotherapy.Yet,despitenearlytwodecades
GeneID: 856398), a P450 cytochrome that catalyzes 14a- ofuseandthegeneralrecognitionoftheimportanceofergosterol
demethylationoflanosterolintheergosterolbiosynthesispathway tofungalcells ourunderstanding ofthespecific cellular processes
[4]. Besides azoles, a number of other drugs such as allylamines disrupted by ergosterol deprivation following azole therapy
and morpholines used in medicine and agriculture also inhibit remains minimal.
ergosterol biosynthesis [5,6]. The limited categories of antifungal agents and emergence of
Ergosterol is an important constituent of membrane lipids, resistance to existing antimycotics have prompted a search for
similar to vertebrate cholesterol, and modulates the fluidity, compoundswithalternativemodesofaction.Theanti-arrhythmia
permeability and thickness of the membrane. These sterols drug,amiodarone,wasrecentlydocumentedtoexhibitfungicidal
preferentially associate with sphingolipids in microdomains that activity[15,16].Thiscationicamphipathiccompoundinsertsinto
have been postulated to have important roles in membrane thelipidbilayerwhereitelicitsmembranehyperpolarization,and
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855003] and erg24D [Entrez GeneID: 855441]) revealed multiple
Author Summary
vma2 phenotypes, with erg24D displaying the most severe defects.
Systemic fungal infections impose a significant threat to Inadditiontohypersensitivitytoamiodarone(Fig.1A),erg24Dwas
public health and therapeutic options to treat these unable to grow at alkaline pH (Fig. 1B), a defining phenotype of
diseases remain limited. Azoles represent the largest vma mutants indicative of the inability to acidify vacuoles.
category of anti-fungal drugs and repress fungal growth Furthermore, erg24D exhibited hypersensitivity to Zn2+ toxicity
by inhibiting biosynthesis of ergosterol, an important andtothecalcineurininhibitorFK506,consistentwithbroadion
constituentoffungalmembranes.Despitethewideuseof homeostasis defects characteristic of vma mutants (Fig. 1C–D).
azoles in the clinic for decades, the cellular basis for their
Yeast strains defective in trafficking of chitin synthase, including
antifungal mechanism remains elusive. In this study, we
vmamutants, are moresensitivetotoxicity fromcalcofluor white,
usearangeofgenetic,cellularandbiochemicalapproach-
an antimicrobial agent that binds to cell wall chitin. We showed
es to reveal a requirement for ergosterol in vacuolar H+-
that erg24D shared calcofluor white hypersensitivity with vma2D
ATPase function. V-ATPase plays essential roles in diverse
(Fig. 1E). Poor growth of vma mutants on high concentrations of
cellular processes, and is required for fungal virulence.
non-fermentable carbon sources has been ascribed to oxidative
Concomitant ergosterol feeding restores vacuolar acidifi-
stressfromrespiration.Althougherg24Dwasabletogrowonnon-
cationandgrowthincellstreatedwithfluconazole.These
fermentable carbon sources, growth was significantly impaired
resultssuggestthatthecriticalrequirementforergosterol
in V-ATPase function may underlie the antifungal activity (Fig.1F).Overall,thenovelobservationthatergosterolbiogenesis
of azoles. Moreover, we show in a mouse Candidiasis mutants largely phenocopy vma mutants suggests that cellular
modelthatcombininganionhomeostasis-disruptivedrug ergosterol content may be important for the function of V-
with azole is an effective approach to treat fungal ATPase.
infections.
Ergosterol is critical for V-ATPase function
V-ATPase hydrolyzes ATP and acidifies vacuolar compart-
influxofH+andCa2+intothecytoplasm[15,17].Withinminutes, ments. To assess a possible requirement for ergosterol in V-
amiodaronealsoelicitsatranscriptionalresponsetostarvationand ATPasefunction,wefirstmeasuredthevacuolarpHinergmutants
blocks cell cycle progression [18]. A screen of the yeast haploid using the pH-sensitive fluorescent dye BCECF. The acetoxy
deletionlibraryforamiodaronehypersensitivityrevealedmultiple methylesterofBCECFistakenupbycellsandde-esterifiedinthe
vma genes encoding subunits of the vacuolar membrane H+- vacuolewhereitaccumulates[21].WhilethevacuolarpHofwild-
ATPase [15]. The V-ATPase is critical for generation of a pH type cells was 6.0, vacuoles of vma2D (Entrez GeneID: 852424)
gradientthatdrivessecondarytransporterstomaintaincellularion cells were significantly more alkaline, around pH 7, as would be
homeostasis. Since the fungicidal activity of amiodarone appears expectedforlossofprotonpumpcapacity(Fig.2A).VacuolarpH
to be tightly coupled to ion stress [19], hypersensitivity of vma ofallviableergmutantscloselyresembledthatofthevmamutant,
mutants was ascribed to defects in ion homeostasis. Notably, as shown for erg24D (Fig. 2A). Next, we purified intact vacuolar
deletionmutantsofseveralerggenesintheergosterolbiosynthesis vesicles from wild type, erg24D and vma2D strains, and compared
pathway were also identified in the screen [15], although the V-ATPase function, including rates of proton pumping andATP
underlying mechanism for their amiodarone hypersensitive hydrolysis. There was no V-ATPase activity detectable in vma2D
phenotypewasunclear.Meanwhile,ascreenoftheyeasthaploid vacuoles as expected, whereas in vitro ATPase and H+ pumping
deletionmutantlibraryforstrainswithalterationsinvacuolarpH activitywerebothdiminishedtoabout40%ofwildtypelevelsin
revealed that erg mutants, like vma mutants, had severely alkaline erg24D(Fig.2B).Examinationofsterolprofilesinpurifiedvacuoles
vacuoles(Brett,C.L.,Rao.R.etal.,unpublisheddata).Aseparate confirmedthepresenceofergosterolinwildtypevacuolesandits
study showed that sphingolipid, the other major membrane lipid absence in erg24D (not shown). Taken together, these results
componentfoundassociatedwithergosterolindetergent-resistant provideevidence fora roleforergosterol inV-ATPase activity.
microdomains, was required for the structural integrity of V- The P-type H+-ATPase Pma1 (Entrez GeneID: 852876) has
ATPase domains [20]. In light of these observations, we been documented to associate with ergosterol enriched domains
investigated a potential link between ergosterol and V-ATPase [22,23].Uponglucoseactivation,itpumpsprotonsoutofcellsto
function, which led to a mechanistic basis for the antifungal acidifytheextracellularmedium.Toassesstheeffectofergosterol
activityofazoledrugs.Asafirststepinexploitingourobservations depletion on Pma1function, weexamined extracellular acidifica-
forimprovedmanagementofinvasivefungaldiseases,weassessed tionuponglucoseactivationinerg24Dcells.AsshowninFig.2C,
the efficacy of combining fluconazole with ion homeostasis- the kinetics of medium acidification was substantially similar
disruptive agent amiodaroneina murine Candidiasis model. betweenthewildtypeanderg24D,whileapreviouslycharacterized
PMA1 mutant, pma1-105, reported to have a 65% reduction in
Results activity,exhibitedsloweracidificationrateasexpected[24].These
datasuggestthatergosterolisnotrequiredforPma1functionand
Mutants defective in ergosterol biogenesis exhibit support thespecificity oftheergosterol effect on V-ATPase.
multiple vma2 phenotypes To investigate the mechanism underlying the requirement of
A genome-wide screen of the S. cerevisiae haploid deletion ergosterol for optimal V-ATPase function, we first asked if V-
collection for hypersensitivity tothe antifungal agent amiodarone ATPase localization was altered in the erg24D mutant. The V-
revealed multiple vma and erg mutants [15]. Given our previous ATPase is made up of 14 subunits organized into the V sector,
o
observationthatamiodaronetriggeredCa2+andH+influxleading integral to the membrane, and the cytoplasmic V sector that
1
to fungal death fromion stress [15,19] andthe importance of V- reversibly dissociates from the membrane [25]. Figure 3A & 3B
ATPase in ion homeostasis, we considered the possibility that showthatrepresentativesubunitsfromtheV sector(Vph1-GFP)
o
ergosterolmaybeimportantforV-ATPasefunction.Asystematic and the V sector (Vma5-GFP) colocalized with the vacuolar
1
examination of viable erg null mutants (erg2D [Entrez GeneID: membrane stain (FM4-64) in erg24D cells, similar to the isogenic
855242],erg3D[EntrezGeneID:850745],erg6D[EntrezGeneID: wild type. It was possible that the ergosterol biogenesis defect
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Figure1.erg24Dexhibitsmultiplevma2phenotypes.GrowthofWT,vma2D,erg24Dstrainsunderdifferentconditions.Valueswerenormalized
togrowthofeachstrainundercontrolcondition(AandC),togrowthofeachstrainatpH4.3(B),togrowthofeachstrainatpH6withoutFK506(D),
ortogrowthofWTunderthetwoconditionswithnon-fermentablecarbonsource(F).(E)GrowthofthestrainsinYPDorYPDsupplementedwith
Calcofluorwhite.Allmeasurementswereintriplicate,andmeansandstandarddeviationsareplotted.pvaluesoftwo-tailedt-testareshown.
doi:10.1371/journal.ppat.1000939.g001
significantly decreased V-ATPase expression or caused the of ergosterol depletion following azole treatment, we used the S.
dissociation of V from V domain. However, analyses of cerevisiaestrainWPY361withagain-of-functionmutationinUPC2
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representative V-ATPase subunits in vacuolar vesicles purified (Entrez GeneID: 851799), upc2-1, that allows overexpression of
from the wild type and erg24D showed similar expression levels ATP-binding cassette transporters required for uptake of exoge-
and V /V ratios that were identical between the two strains nously added sterol under aerobic condition [28,29]. Table 1
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(Fig.3C &3D). shows that growth inhibition caused by fluconazole treatment in
The oligosaccharide Methyl-b-Cyclodextrin (MbCD) extracts WPY361 can be reversed by exogenous supply of ergosterol. To
sterols from cellular membranes. We observed a dose dependent examine whether ergosterol feeding represses endogenous sterol
lossofV-ATPaseactivityfollowingtreatmentofpurifiedvacuolar metabolism and reduces accumulation of intermediates and
vesicles with MbCD, which could be blocked by preloading derivatives, we analyzed sterol profiles in upc2-1 cells after six
cholesterol into MbCD (Fig. 4A). Both ATP hydrolysis rates and hours of exposure to fluconazole and exogenous ergosterol. As
H+ pumping declined at similar rates, suggesting an inhibition of expected,fluconazolealonecausedreductionofergosterolcontent
the intact V1Vo complex. This was verified by immunoblot (nine-fold)andaccumulationoflanosterolandamajorderivative,
analysis of vacuolar membranes collected by centrifugation after likely to be 14-methyl 3,6-diol (Fig. S1). Compared with cells
MbCDtreatment(Fig.4B):wedidnotobservealossofeitherV1 treated with fluconazole alone, cells treated with fluconazole and
(Vma2p) or Vo (Vph1p) subunits, suggesting that the two sectors ergosterolhadthree-foldhigherergosterolcontent,yetthelevelof
remain associated after ergosterol extraction. In contrast, a lanosterol and its derivatives remained the same (Fig. S1). These
previous study pointed to a role for sphingolipids in maintaining dataindicatethatergosterolfeedingdidnotreduceaccumulation
structural integrity of the V-ATPase enzyme complex [20]. We of intermediates and derivatives. Thus, depletion of ergosterol,
conclude that ergosterol constitutes a critical component in the rather than the toxicity of intermediates and derivatives, is a
lipid membrane environmentfor V-ATPasefunction. plausible mechanism fortheantifungal activityof fluconazole.
Basedonourevidencethatergosterolwasrequiredforoptimal
The antifungal drug fluconazole disrupts V-ATPase V-ATPase function, we predicted that azole treatment would
function similarly impair activity of the V-ATPase. Indeed, we show that
Azoledrugsexerttheirfungistaticeffectbyinhibitingergosterol fluconazole treatment resulted in a dose-dependent alkalinization
biosynthesis,specificallytargetinglanosteroldemethylase(Erg11p), of vacuolar pH, consistent with depletion of ergosterol from the
which is the enzymatic step immediately upstream from Erg24p. vacuolar membrane (Fig. 5A). Furthermore, vacuolar membrane
Despitethewidespreaduseofazoleantifungals,ourunderstand- vesicles purified from fluconazole treated cells showed significant
ingofthespecificcellularpathwaysdisruptedbyazolesislimited. reductionsinbothH+pumpingratesandATPaseactivity(Fig.5B).
Inadditiontoergosteroldepletion,fluconazoletreatmentresultsin Next,weevaluatedtheeffectofergosterolfeedingonvacuolarpH
accumulationoflanosterol(substrateofErg11p)anditsderivative in the upc2-1 mutant. Not only did exogenous ergosterol restore
14-methyl-3,6 diol [26]. Based on analysis of sterol profiles in growth in fluconazole treated cells, vacuolar acidification closely
fluconazolesusceptibleandresistantstrains,itwasconcludedthat resembledthatofuntreatedcells(Fig.5C&5D).Thiscorrelation
14-methyl-3,6 diol toxicity [26,27] was responsible for azole- strengthensthehypothesisthatV-ATPaseinhibitioncontributesto
mediatedgrowtharrest.Tospecificallyassessthefunctionaleffect thecellular mechanism ofazole activity.
Figure2.ERG24deletionimpairsthefunctionofV-ATPasebutnotPma1p.(A)VacuolarpHofWT,erg24Dandvma2Dstrainsmeasured
withpHsensitivefluorophoreBCECF-AM,whichaccumulatesintheyeastvacuole.(B)InitialH+pumpingratewascalculatedfromDA during
490–540
thefirst60safterinitiatingthereaction.ATPaseactivitywascalculatedfromdropofA between3and6minutesafterinitiatingthereaction.
340
Meansandstandarddeviationsareplottedfromdataforatleastthreeindependentvacuolarvesiclepreparations.pvaluesoftwo-tailedt-testare
shown.(C)MediumacidificationbyPma1uponglucoseactivationwasmeasuredasdescribedinMethods.ExtracellularpH(pH )wasrecordedafter
out
glucosewasaddedto2%attime0.
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Figure 3. Expression and localization of V-ATPase in erg24D. Vph1p (A) and Vma5p (B) were tagged with C-terminal GFP at their
chromosomallociinWTanderg24Dstrains.VacuolarmembraneswerestainedwithFM4-64for30mininYPDandchasedforanother30minwith
fresh YPD. (C) Immunoblotting of Vph1p and Vma2p in vacuolar vesicles isolated from WT and erg24D. (D) Vma2p to Vph1p ratio in WT was
designatedasV /V ratioof1.Calculationwasbasedondatafromthreeindependentvacuolarvesiclepreparationsforeachstrain.Meansofthe
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ratiosandstandarddeviationareplotted.
doi:10.1371/journal.ppat.1000939.g003
Figure4.ErgosteroliscriticalforV-ATPasefunction.(A)WTvacuolarvesicleswereincubatedwithMbCDorMbCDpreloadedwithcholesterol
(cholesteroltoMbCDratio1:20byweight)at4uCfor30min.VacuolarvesicleswerespundownandanalyzedforATPasefunction.(B)WTvacuolar
vesiclestreatedwithMbCDfor30minat4uCwerespundownandusedforimmunoblottingtoassesstheabundanceofVph1pandVma2p.
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showthatpretreatmentwithfluconazoleexacerbatesthecytosolic
Table1. Ergosterolfeedingrelieves growth inhibitionby
Ca2+ surge elicited by amiodarone, consistent with defective V-
fluconazole.
ATPase function(Fig.6B).
The proton gradient established by the V-ATPase drives
vacuolar sequestration of excessive cytosolic Ca2+ by H+/Ca2+
Treatment Ab600
exchange mechanisms. The importance of the V-ATPase is
Control 1.0460.01 demonstrated by the inability of purified vacuolar vesicles to
Control+ergosterol 0.9660.06 sequester45Ca2+followingtreatmentwiththeV-ATPaseinhibitor
5mg/mlFLC 1.0060.02 concanamycin A (Figure 6C). As predicted by our hypothesis,
5mg/mlFLC+ergosterol 0.9460.05 vacuolarvesiclespurifiedfromerg24Dorfluconazole-treatedwild-
typeS.cerevisiaebothshowedsimilarimpairmentintheirabilityto
25mg/mlFLC 0.0560.01
sequesterCa2+(Fig.6C).Likewise,vacuolarvesiclespurifiedfrom
25mg/mlFLC+ergosterol 0.9660.01
C. albicans cells treated with fluconazole were also impaired in
100mg/mlFLC 0.0460.02 sequestering Ca2+ (Fig. 6D). Thus, our observations provide a
100mg/mlFLC+ergosterol 0.8860.03 mechanisticbasisforpreviousreportsofsynergismbetweenazoles
andamiodarone againstpathogenic fungi invitro[15].
WPY361(upc2-1mutant)wasgrowninYPDcontainingfluconazole(FLC),
To investigate the potential clinical application of this
ergosterol,oracombinationfor30hoursina96-wellplateat30uCin
quadruplicate.AverageofAbsorbancevaluesat600nm(Ab600)andstandard synergism, we studied the effect of combining fluconazole and
deviationsareshown.Ergosterolstock(5mMinTween:ethanol[1:1])was amiodaroneinamurinecandidiasismodel.Themicrobialburden
addedforafinalconcentrationof50mM.EqualvolumeofTween:ethanolwas of Candida albicans in kidneys was assessed 3 days following
addedtosampleswithoutergosterolascontrol.
intravenousinfectionofBalb/Cmice.AMDtreatmentdoseswere
doi:10.1371/journal.ppat.1000939.t001
presented at 5.0 and 25mg/kg, while FLC doses were 0.5 and
1 mg/kgwithtreatmentsgivenoncedailyforthreedaysafterthe
To extend these observations in the human pathogen Candida
first dose. In the absence of amiodarone, there was a significant
albicans, we monitored vacuolar uptake of the fluorescent weak
dose-dependent effect of FLC on C. albicans (ratio of geometric
base quinacrine. Previous studies have demonstrated pH-depen-
meansofthecfucountper1 mg/kgofFLCwas0.5914,p=0.01).
dentvacuolaraccumulationofquinacrine,whichwasabolishedin
In the absence of FLC, amiodarone did not confer a significant
the homozygous vma72/2 mutant [30]. We observed robust
antifungal activityat theconcentrationstested(ratio ofgeometric
quinacrine fluorescence in C. albicans vacuoles, colocalizing with
means of the cfu count per 1mg/kg of amiodarone was 0.9996,
FM4-64 staining of vacuolar membranes. Fluconazole treatment
p=0.95). Yet in combination with fluconazole, the two doses of
drastically reduced vacuolar accumulation of quinacrine in most
amiodarone significantly reduced C. albicans infection above and
cells, indicative of impaired vacuolar acidification (Fig. 5E).
beyond the dose-dependent effect of fluconazole (ratio of
Additionally, trafficking of FM4-64 to the vacuolar membrane
geometric means of CFU with amiodarone versus without, for
was impaired, consistent with endocytosis defects seen in vma
the same dose FLC=0.4969, p,0.001) (Fig. 6E). These data
mutants[30].Wenotethatfollowing6hoffluconazoletreatment,
provide proof of principle that combining azole drugs with other
cellsfailedtodividebutcontinuetoincreaseinsize,aspreviously
antifungalcompoundsthatdisruptintracellularcationhomeostasis
reported[9].Thesedatasuggestthattherequirementofergosterol
couldbeapromisingtherapeuticstrategytotreatsystemicfungal
for V-ATPase function is conserved in fungi. Given the
infections.
importance of V-ATPase function and vacuolar acidification in
diverse cellular processes, we conclude that disruption of V-
Discussion
ATPasefunctionplaysacriticalroleinantifungalactivityofazole
drugs.Consistentwiththisconclusion,bothvma72/2anderg242/2
In fungal cells, V-ATPase acidifies intracellular compartments
mutantsofC.albicansexhibitdefectivevirulenceinmurinemodelsof
includingthevacuole,endosomes,andlate-Golgi.Mutantslacking
Candidiasis[30,31].
V-ATPase exhibit characteristic phenotypes of growth sensitivity
toalkalinepH,calcofluorwhite,Ca2+andmetalionstress,andare
Impairment of V-ATPase function by azoles underlies unabletogrowonhighconcentrationsofnon-fermentablecarbon
synergism with amiodarone sources [25]. We show that mutants defective in ergosterol
WeshowedpreviouslythatamiodaronetriggeredacytosolicH+ biosynthesisexhibitmostofthesecharacteristicvma2phenotypes.
andCa2+surgeinthebaker’syeastandthatmutantsdefectivein Furthermore, we showed a reduction of vacuolar acidification by
ion homeostasis were hypersensitive to amiodarone toxicity [15]. ergosterol depletion, restoration of vacuolar acidity by ergosterol
Given the pivotal role of the V-ATPase in maintaining feeding,andusedbiochemicalassaysofH+pumpingwithpurified
intracellular cation homeostasis, we predicted that ergosterol vacuolar vesicles to collectively demonstrate the requirement of
depletion by azole treatment would impair V-ATPase function ergosterol for optimal V-ATPase function. This functional link
and exacerbatedisruption ofcation homeostasisby amiodarone. explainssimultaneous identification ofmultiple ergosterol biosyn-
WefirsttestedthishypothesisintheS.cerevisiaemodel.Weused thesisgenes(erg)andV-ATPasesubunitgenes(vma)inanumberof
pHsensitiveGFP(pHluorin)tomonitorchangesincytosolicpHin genome-wide screens, including sensitivity to low Ca2+, alkaline
wildtype,vma2Danderg24Dstrainsuponexposuretoamiodarone stress, and acidstress [33–35]. V-ATPase mutants and ergosterol
[32]. Cytosolic acidification was most pronounced in the vma2D biosynthesis mutants also share defects in endocytosis [9,36].
mutantconsistentwithalossintheabilitytotransportH+fromthe Additionally, in pathogenic fungal species, both vma and erg
cytosoltothevacuole.Depletionofergosterolinerg24Dmutantor mutants are avirulent [30,31,37]. Disruption of V-ATPase
by fluconazole treatment also exacerbated cytosolic acidification, function by ergosterol deprivation provides a mechanistic basis
relative to wild type, upon amiodarone addition (Fig. 6A). We forthese similarities.
havepreviouslydemonstrateddefectiveclearanceofcytosolicCa2+ To investigate the underlying basis for the requirement of
in vma mutants following exposure to amiodarone [15]. We now ergosterolinV-ATPasefunction,wefirstcheckedthelocalization
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Figure5.FluconazoletreatmentdisruptsV-ATPasefunction.(A)VacuolarpHofWT(BY4742)culturestreatedwithfluconazoleatspecified
concentrationsfor6hoursinYPD.(B)ProtonpumpingrateandATPaseactivityofWT(BY4742)culturestreatedwithorwithoutfluconazolefor
6hours in YPD. Three independent batches of vacuolar vesicles were isolated and used to assay V-ATPase function. Cultures of upc2-1 mutant,
WYP361, were treated with fluconazole (100mg/ml), ergosterol (50mM) or their combination at OD 0.1. Growth (C, representative of three
independentexperiments)wasassessedat8hourpost-treatment,andvacuolarpH(D)wasassessedat6hourpost-treatment.(E)WTC.albicanscells
(SC5314)weregrowninYPDwithorwithoutfluconazolefor5hours.FM4-64wasaddedtotheculturestostainvacuolesfor30min.Cellswere
chasedwithfreshYPDwithorwithoutfluconazolefor20minfollowedbyquinacrineadditionforanother5min.Fluorescencemicroscopicimagesof
thecellsweretakenimmediatelyafterwashing.Meansandstandarddeviationsareplotted.pvaluesoftwo-tailedt-testareshown.
doi:10.1371/journal.ppat.1000939.g005
and abundance of V-ATPase in erg24D. Fluorescent microscopy While the molecular target of azole drugs, Erg11p, has been
and immunoblot analysis ruled out possible mislocalization and extensively characterized, not much is known about the cellular
reduced abundance of V-ATPase in erg24D vacuolar membrane. basis of fungal growth inhibition. Inhibition of Erg11p by
In yeast, the V-ATPase complex undergoes rapid reversible fluconazoleresultsinaccumulationoflanosterolanditsderivative
dissociation into non-functional V and V sectors in response to 14-methyl-3,6 diol. In azole resistant erg3 mutants, 14-methyl
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glucosewithdrawal[38].AstudywithBabyHamsterKidneycells fecosterol accumulates upon treatment with fluconazole [26,27].
suggestedthatincreasingratioofIntactV /V alongtheendocytic Thishasledtothenotionthattoxicityofsterolderivativessuchas
1 o
pathway effectively increased acidity along the compartments in 14-methyl-3,6diolmediatestheactionoffluconazole[45,46].We
the pathway [39]. Analyses of immunoblot results in this study exploited the ability of a recently described upc2-1 mutation that
showthatV andV domainsarestillassociated,andV /V ratio allowsuptakeofergosterolunderaerobicconditionstodistinguish
1 o 1 o
remains unchanged upon ergosterol deprivation by treating cells between the effects of byproduct accumulation and ergosterol
withfluconazoleandtreatingvacuolarvesicleswithMbCD.These depletion on cell growth. The ability of exogenous ergosterol to
data rule out dissociation of V from V domain as the cause of reverse growth inhibition by fluconazole supports a plausible
1 o
reduced V-ATPase function, and indicate that ergosterol directly alternative hypothesis that antifungal activity of azoles is due to
modulatestheactivityofV-ATPase.Sphingolipid,anothermajor ergosterol depletion. Although we ruled out a corresponding
decrease in lanosterol and other derivatives in the ergosterol fed
componentoflipidraft,wasthoughttoaffectV-ATPasefunction
cells, we cannot exclude the possibility that a specific ratio of
by maintaining the structural integrity of V-ATPase because V
1
ergosteroltoothersterolsmaycounterpotentialtoxiceffects.This
subunits(Vma1p,Vma2pandVma5p)dissociatefromV domain
o
possibility could be tested by varying ratios of sterols in upc2-1
duringFicollgradientprocedureinthesphingolipidmutantsur4D.
feedingexperiments;however,14-methyl-3,6diolisnotcommer-
In contrast, Vma2p remained associated with Vph1p after Ficoll
ciallyavailableanditspotentialtoxicitycannotbedirectlyassessed
gradient procedure in erg24D mutant and after ergosterol
at this time. Furthermore, our results warrant more careful
extraction by MbCD in the wild type. Thus, these two key
examination of the role of 14-methyl fecosterol in the potential
membranelipidcomponentsplaydistinctrolesinmaintainingV-
compensationof ergosterol functionin membranes.
ATPase function.
SomereportssuggestaroleforROSinthetoxicityofazolesto
The precise molecular basis of V-ATPase regulation by
fungalcells[47].ItisworthnotingthatcellularROSlevelinthese
ergosterol remains to be determined. In mammalian cells, V-
studies was measured with the fluorescent dye DCFH-DA (29,79-
ATPase has been shown to associate with cholesterol-rich
dichlorofluorescin-diacetate) which has also been documented to
microdomains, with loss of vesicular acidification reported upon
respondtopHalterations[48].Giventheeffectofazoledrugson
treatment with b-methylcyclodextrin [40]. A number of studies
pH homeostasis, the role of ROS in azole-induced growth
showed that inhibitors could interact with lipid bilayer and affect
inhibition mayneedtobere-evaluated.
V-ATPase function by restricting its structural flexibility [41,42].
The far-reaching effect of V-ATPase is illustrated by diverse
Mechanisms proposed to explain the regulation of Ca2+-ATPase
phenotypes exhibited by V-ATPase mutants. By disrupting the
andNa+,K+-ATPasebymembranelipidsincludeloweringoffree
functionofV-ATPasethroughergosteroldeprivation,azoledrugs
energy of activation and proper packing at protein-protein
can affect a wide range of cellular processes, including cation
interfaces[43,44].Alteredsterolcompositionsareknowntoaffect
homeostasis, protein sorting, processing and degradation. Impor-
membrane packing and rigidity: fluorescence anisotropy probes
tantly, V-ATPase function and vacuolar processing of virulence
haverevealedincreasedmembranefluidityandpermeabilityupon
factorsarerequiredforpathogenesis[30,37].Althoughwecannot
fluconazoletreatment[45],consistentwithalterationsinactivityof preclude additional cellular targetsof azole toxicity,disruption of
membrane-localized pumps and transporters, and a critical role V-ATPase function is sufficient to repress growth and attenuate
for ion homeostasis mechanisms in drug treated cells. It is also fungalvirulenceandislikelytobeacriticalmechanismunderlying
possible that ergosterol may affect V-ATPase function indirectly antifungal activity ofazole drugs.
by modulating regulatory interactions with other proteins. The Amiodaroneexhibitsantimicrobialactivityagainstawiderange
complex multi-subunit structure of V-ATPase and its intimate of fungi and protozoa through disruption of H+ and Ca2+
associationwithsterolsandspingolipidsindicatethatsophisticated homeostasis[15,16,49].Additionally,invitrostudiesshowedazoles
regulatory mechanisms must be in place to ensure proper weresynergisticwithamiodaroneagainstfungalpathogens,e.g.C.
assembly, configuration, and communication among these com- albicans andC.neoformans [15].Interestingly, amiodaroneinteracts
ponents. Ergosterol depletion may affect other membrane with fluconazole synergistically against fluconazole-resistant clin-
functions besides vacuolar acidification. The plasma membrane ical isolates of A. fumigatus and C. albicans [50,51]. Moreover, the
H+-ATPase,Pma1,isamajoreffluxmechanismforprotons,andis synergy ofamiodarone andposaconazole hasbeen shownon the
also found associated with ergosterol-rich microdomains [22,23]. protozoan T. cruzi both in vitro and in vivo [49]. Uncovering the
However,wefoundthatPma1-mediatedprotonpumpingfunction mechanismunderlyingthissynergismmayprovideinsightguiding
wasnotalteredinerg24D,incontrasttothepronouncedeffectseen thedesign ofmore potentantifungal therapy.
on the V-ATPase. This suggests that membrane proteins have Data in this study show that ergosterol is required for the
specific lipid requirementfor theirfunctions. optimalfunctionofV-ATPase,acentralplayerinmaintainingH+
PLoSPathogens | www.plospathogens.org 8 June2010 | Volume 6 | Issue 6 | e1000939
AzolesDisruptFungalV-ATPaseFunction
PLoSPathogens | www.plospathogens.org 9 June2010 | Volume 6 | Issue 6 | e1000939
AzolesDisruptFungalV-ATPaseFunction
Figure6.Synergybetweenfluconazoleandamiodarone.(A)EarlylogphasecellsexpressingpHluorinweregrownfor6hourstomidlog
phase(OD,1)inSCminusleucinemediumwithorwithoutfluconazole.Amiodarone(10mM)wasinjectedatthearrow.Measurementwasdonein
triplicate,andthemeansandstandarddeviationsareplotted[32].(B)EarlylogphaseWTcellsexpressingaequorinweregrownwithorwithout
fluconazole for 4hours. Amiodarone (10mM) was injected at the arrow. Ca2+-dependent Aequorin luminescence was measured in triplicate as
previouslydescribed[15].For45Cauptake,earlylogphaseWTcellsofS.cerevisiae(C)andC.albicans(D)weregrownwithorwithoutfluconazole
(20mg/mlforS.cerevisiae,1mg/mlforC.albicans)for6hours.VacuolarvesiclesisolatedfromthesecultureswereassayedforMgATP-dependent
45CaCl uptakeasdescribedinMaterialsandMethods[33].ConcanamycinAwasaddedtoassessV-ATPasedependenceofCaCl uptake.pvaluesof
2 2
two-tailed t-test were shown. (E) Mice infected with C. albicans (ATCC 36082) were treated with vehicle, fluconazole, amiodarone, and their
combinationintraperitoneally.Onday4post-infection,mousekidneys(5pergroup)wereremoved,weighed,homogenized,seriallydilutedandthen
platedontoYPDagarplatescontainingchloramphenicolandampicillin.CFUwerecountedafter48hours.MeansofCFUpergramofkidneyunder
eachtreatmentandstandarderrorsareplotted.
doi:10.1371/journal.ppat.1000939.g006
andCa2+homeostasis.Therefore,depletionofergosterolwouldbe sequencewasamplifiedfromBY4742genomicDNAwithprimers
expectedtoexacerbatethedisruptionofH+andCa2+homeostasis XbaITEF1andBamHITEF1,whichincorporaterestrictionsitesof
upon amiodarone treatment. Indeed, upon ergosterol depletion XbaI and BamHI. CYC1 terminator sequence was amplified with
either by erg mutations or by azole treatment, 45Ca uptake by primers BamHICYC1 and EcoRICYC1, which incorporate
purifiedvacuolarvesicleswasreducedwhilecytosolicH+andCa2+ restriction sites of EcoRI and BamHI. pHluorin gene sequence
surges increased upon exposure to amiodarone. Thus, we was amplified from pCB190YpHc plasmid with primers Bam-
conclude that disruption of V-ATPase function in maintaining HIPhluoandBamHIPhluoR,whichincorporateBamHIrestriction
cation homeostasis by azoles contributes to the synergy between site. The amplicons were digested with corresponding restriction
azoles andamiodarone. enzymes and ligated sequentially with the backbone of pYE-
Recently, we demonstrated that a combination of amiodarone plac181,resultinginthegeneforpHluorinbeingflankedbyTEF1
and fluconazole in C. albicans resulted in dampening of the promoterandCYC1terminator.pZR4.1wastransformedtoyeast
transcriptionalresponsetoeitherdrugalone[52].Thiseffectcould strains to monitor cytosolic pH change upon exposure to
potentiallystunt cellularstressresponsesoccurring downstreamof amiodarone.
drug toxicity and thereby contribute to synergistic effects of the Wildtype(matingtypea)Vph1-GFPandVma5-GFPstrainsin
drugs.Thesedatarevealanadditionalmechanismcontributingto whichGFPwasfusedtotheC-terminusofthetwoproteinswere
the synergism that is distinct from the ion homeostasis defects purchased from Invitrogen. The Vph1-GFP and Vma5-GFP
presented here. We and othershave arguedthat non-overlapping fragmentswereamplifiedfromthesestrainswithprimersVph1L2
butcomplementaryinsightscanbeobtainedfromphenotypeversus +Vph1R1,andVma5L2+vma5R1,respectively.Theamplicons
transcriptional profiling [18]. Thus, genes involved in membrane weretransformedtoerg24DtoreplacetheendogenousVPH1and
integrity and ion homeostasis such as VMA and ERG, determine VMA5genesbyhomologousrecombination.Primersequencesare
phenotypeofgrowthsensitivitytoamiodarone.Thesegenestendto available uponrequest.
be constitutively expressed, have a non redundant function and
represent pathways upstream of the transcriptional response. On
Ergosterol feeding and sterol analysis
theotherhand,genesthataredifferentiallyregulatedinresponseto
Ergosterol (Sigma) was dissolved in Tween 80:ethanol (1:1) as
drugappeartoplayacollective,ratherthanindividual,responseto
5 mM stock. Stocks of ergosterol, fluconazole or their combina-
adaptationtostress.Takentogether,thesemechanismscontribute
tion were added at the same time to WPY361 cells in YPD. For
toamorecompletepictureofthecomplexcellularresponsetodrug
growthassay,stationaryculturesweregrownin96-wellplatesfor
toxicity.Finally,inthisstudy,weevaluatedtheclinicalpotentialof
30 hours at 30uC. For vacuolar pH measurement and sterol
combiningfluconazoleandamiodaroneintreatingfungalinfections
analysis, log phase cells were treated with ergosterol and
inamurineCandidiasismodel.Thesynergydemonstratedinthis
fluconazole for 6 hours. Total sterols were extracted from
experimentisproof-of-principlethatcombiningazoleswithagents
,56107 log-phase cells after washing twice with water and
disruptivetocationhomeostasisisapromisingapproachtobetter
analyzedwithanAgilent6850gaschromatographwithanHP-1
managefungalinfections.
columnandFIDasdescribedpreviously[53].Retentiontimesfor
cholesterol, ergosterol and lanosterol were determined using
Materials and Methods
standards. Cholesterol was added to each sample to normalize
Yeast strains, media, and reagents extraction efficiency.
S. cerevisiae erg2 and vma2 mutant strains are from MATa
deletion library (Invitrogen, Carlsbad, CA). WPY361 (MATa Vacuolar vesicle purification, MbCD treatment and 45Ca
upc2-1ura3-1his3-11,-15leu2-3,-112trp1-1)waskindlyprovided uptake assay
by Dr. Will Prinz (NIDDK). Yeast cells were grown in standard Vacuolarvesicleswerepreparedasdescribedpreviouslyexcept
SC (synthetic complete) medium or YPD (yeast extract, peptone that 10% Ficoll, instead of 8% Ficoll, was used in the second
and dextrose) medium at 30uC with shaking at 250rpm unless ultracentrifugation step to facilitate purification of vesicles from
specified otherwise. Media with non-fermentable carbon source erg24D and fluconazole treated cells [54]. For 45Ca uptake assay,
contains 1%Bacto-yeast extract, 2% Bacto-peptone, 3% glycerol vacuolar vesicles were incubated in reaction buffer containing
(v/v) plus 2% ethanol (v/v), or 2% sodium lactate. Antibodies 5 mM CaCl with tracer quantities of 45CaCl . After 5 min of
2 2
against Vph1p and Vma2p were purchased from Invitrogen incubation, vacuolar vesicles were filtered onto nitrocellulose
(Carlsbad,CA)orprovidedbyDr.PatriciaKane(UpstateMedical membranes. The filters were washed and processed for liquid
University, New York). scintillation counting. Concanamycin A was added to 0.5 mM to
assess the dependence of 45Ca uptake by vacuolar vesicles. For
Plasmid construction and yeast genetic manipulation MbCD treatment, vacuolar vesicles were incubated with MbCD
PlasmidpZR4.1withpHluoringeneunderTEF1promoterwas orMbCDpreloadedwithcholesterol(cholesteroltoMbCDratio
constructedtomeasureyeastcytosolicpH.Briefly,TEF1promoter 1:20 by weight, Sigma, C4951) for 30min at 4uC with gentle
PLoSPathogens | www.plospathogens.org 10 June2010 | Volume 6 | Issue 6 | e1000939