Ergosterol biosynthesis in Aspergillus fumigatus: its relevance as an antifungal target and role in antifungal drug resistance.

0.54 MB·English

by  Alcazar-Fuoli, Laura

MINIREVIEWARTICLE
published:10January2013
doi:10.3389/fmicb.2012.00439
Ergosterol biosynthesis in Aspergillus fumigatus: its
relevance as an antifungal target and role in antifungal
drug resistance
LauraAlcazar-Fuoli*andEmiliaMellado
MycologyReferenceLaboratory,CentroNacionaldeMicrobiologia,InstitutodeSaludCarlosIII,Madrid,Spain
Editedby: Ergosterol, themajorsteroloffungalmembranes, isessentialfordevelopmentalgrowth
ElaineM.Bignell,ImperialCollege and the main target of antifungals that are currently used to treat fatal fungal infections.
London,UK
Emergenceofresistancetoexistingantifungalsisacurrentproblemandseveralsecondary
Reviewedby:
resistance mechanisms have been described inAspergillus fumigatus clinical isolates. A
JonY.Takemoto,UtahState
fullunderstandingofergosterolbiosyntheticcontrolthereforeappearstobeessentialfor
University,USA
JeanPaulLatge,InstitutPasteur, improvement of antifungal efficacy and to prevent antifungal resistance. An ergosterol
France biosynthesis pathway in A. fumigatus has been proposed with 14 sterol intermediates
*Correspondence: resultinginergosterolandanothersecondaryfinalcompoundC-24ethylsterol.Transcrip-
LauraAlcazar-Fuoli,Mycology
tomicanalysisoftheA.fumigatusresponsetohost-imposedstressesorantifungalagents
ReferenceLaboratory,Centro
is expanding our understanding of both sterol biosynthesis and the modes of action of
NacionaldeMicrobiologia,Instituto
deSaludCarlosIII,Carretera antifungaldrugs.Ultimately,theidentificationofnewtargetsfornoveldrugdesign,orthe
Majadahonda-PozueloKm2,28220 studyofcombinatorialeffectsoftargetingsterolbiosynthesistogetherwithothermetabolic
Madrid,Spain.
pathways,iswarranted.
e-mail:[email protected]
Keywords:Aspergillus,ergosterolbiosynthesis,antifungaldrugs,resistancemechanisms,transcriptome
ANTIFUNGALSTARGETINGERGOSTEROLANDSTEROL ofallylaminesiscloselyassociatedwiththedevelopmentofhigh
BIOSYNTHESIS intracellularsqualeneconcentrations,whicharebelievedtointer-
Sterolsareneutrallipidsofeukaryoticcells,amongwhichergos- ferewithfungalmembranefunctionandcellwallsynthesis(Ryder,
terolisthemaincomponentof fungalmembranes. Ergosterolis 1992).AllylaminesarenotusedtotreatIA,howeverpotentialuse
involvedinnumerousbiologicalfunctionssuchasmembraneflu- in combination with azoles, polyenes, or echinocandins in the
idity, regulation, activityanddistributionof integralmembrane management of severe drug-resistant or refractory mycoses has
proteins, and control of the cell cycle (Bard etal., 1993; Goo- beenproposed(Krishnan-Natesan,2009).Finally,themaincate-
day, 1995). The essential role of sterols in maintenance of cell goryof antifungalagentsusedagainstAspergillus isazoledrugs,
membranesmakeergosterolanditsbiosyntheticpathwayessen- such as voriconazole (VCZ) and itraconazole, and a new gen-
tial for fungal growth, and a primary target for most, currently erationof azolesthatincludesposaconazole, isavuconazole, and
available, antifungal drugs to treat severe human fungal infec- albaconazole.Azolesrevolutionizedmedicalmycologyduetotheir
tions. Three main classes of antifungal drugs, namely polyenes, broadspectrumandreducedtoxicitycomparedtoAMB.Azoles
allylamines, and azoles, directly target ergosterol itself, or enzy- block the ergosterol biosynthesis pathway via inhibition of 14-α
maticstepsofitsbiosyntheticpathway.Thepolyeneamphotericin steroldemethylase(Cyp51/Erg11),akeyenzymethatremovesthe
B (AMB) has represented, for more than 30 years, the standard methylgroupatpositionC-14ofprecursorsterols.Inhibitionof
antifungaltherapyforinvasiveaspergillosis(IA).TheAMBmode ergosterolsynthesisatthisbiochemicallevelresultsintoxicsterol
of action is still poorly understood. It is widely accepted that accumulationandcelldeath(GhannoumandRice,1999).
AMBkillsyeastprimarilyviachannel-mediatedmembraneper- However, although the use of antifungal drugs has increased
meabilizationleadingtofungalcelldeath(Ermishkinetal.,1976). survivalofpatientswithinvasivefungaldisease,themortalityrate
However, recent research suggests that AMB kills yeast by sim- associatedwiththeseinfectionsremainsextremelyhigh.Thissit-
ply binding ergosterol so that the channel formation represents uationbecomesmorecomplicatedbecauseemergingresistanceto
asecondcomplementarymechanismthatfurtherincreasesdrug theexistingantifungalsisacurrentproblemandsecondaryresis-
potencyandtherateofcelldeath(Grayetal.,2012).Theallylamine tancemechanismshavebeendescribedinclinicalisolates(Howard
group of antimycotics interferes at the early-stage of ergosterol and Arendrup, 2011; Shapiro etal., 2011; van der Linden etal.,
biosynthesisbyinhibitingtheenzymesqualeneepoxidase(Erg1). 2011a). Full understanding of the ergosterol biosynthesis path-
Although squalene epoxidases from various origins have been way in A. fumigatus is essential to new antifungal drug design
investigatedwithrespecttosubstraterequirements,cofactors,and andforimprovingtheactivityofexistingones.Thisreviewsum-
inhibitors, no structural model is available; and the domains marizes current knowledge about the enzymatic sequence and
responsible for enzymatic activity and inhibitor interactions are regulation of the biochemical reactions involved in ergosterol
not well understood (Ruckenstuhletal.,2007). The cidal action biosynthesiswithaviewtounderstandingthemainmechanisms
www.frontiersin.org January2013|Volume3|Article439|1
“fmicb-03-00439” — 2013/1/8 — 22:19 — page 1 — #1
Alcazar-FuoliandMellado Ergosterolbiosynthesisandantifungaldrugresistance
of antifungal resistance to drugs which target ergosterol or its
biosynthesis.
AspergillusfumigatusERGOSTEROLBIOSYNTHESIS
PATHWAY
The biosynthesis of ergosterol involves about 20 enzymes and
includesthesynthesisofsqualenefrommevalonate(Ferreiraetal.,
2005;Alcazar-Fuolietal.,2008).ThisrouteiswellknowninSac-
charomyces cerevisiae which provides a model for studying this
pathway in other eukaryotes (Fryberg etal.,1973). Depletion of
ergosterol biosynthetic gene functions is lethal in S. cerevisiae,
due to complete prevention of ergosterol production. Deviating
from the prototypical pathway described in S. cerevisiae, other
morecomplexbiosyntheticpathwayshavebeenproposedinsome
fungiandalsoinplants,suggestingthattheenzymaticpathways
ofergosterolbiosynthesisarespecifictofungaltaxa(Frybergetal.,
1973;Nesetal.,1989).InA.fumigatus,thestudyofthispathway
atagenomiclevel,demonstratedtheexistenceofmultiplegenes
encodingkeyenzymessuchastwodistinct14-αsteroldemethy-
lases (Cyp51A and Cyp51B; Mellado etal.,2001) and three C-5
sterol desaturases (Erg3; Alcazar-Fuoli etal., 2006). The classi-
calergosterolbiosynthesisroutewasreconsideredinA.fumigatus
basedontheexistenceofmultiplegeneswhichputativelyencode
enzymes with the same or duplicated function in the pathway
(Figure 1). Genetic and biochemical analysis of azole suscepti-
blewild-typestrainsandapanelofclinicalazoleresistantstrains
identified a total of 14 sterols showing a similar sterol pattern
composition, with ergosterol as the main sterol for all of them
FIGURE1|Ergosterolbiosyntheticpathway.SterolsofA.fumigatus
(Table1).Eachoftheazoleresistantstrainshadadifferentantifun- wild-typestrain(CM-237),andΔcyp51A,Δcyp51B(markedingreen),
galsusceptibilitypatternthatcorrelatedwithdifferentmutations Δerg3A,Δerg3B,andΔerg3C(markedinred)derivedmutantstrains.
Sterolsareclusteredandrepresentedincakeplotsasthepercentageof
inthecyp51Agene.Thelackofdifferencesbetweensterolshigh-
totalsterols:Δ5sterols(1,2,3,4,6,8ingray),C-4/C-14methylsterols(11,
lightsthefactthattheresistancemechanisminthosestrainswas 12,13,14ingreen),Δ7sterols(5,7,9inred),andethylsterols(10in
target-dependent and not sterol-dependent, as will be discussed purple)accordingtothesterolidentificationinA.fumigatus(Table1).
later. Instead, the sterol composition of single enzyme defec-
tiveA.fumigatus mutantswasratherdifferentdependingonthe
lacking enzyme (Alcazar-Fuoli etal., 2008). Mutants deleted in deletions,at14-αsteroldemethylaseorC-5steroldesaturaselevel,
14-αsteroldemethylases(cyp51Aandcyp51B)hadlessergosterol was lethal for A. fumigatus or influenced its virulence (Mellado
thantheparentalstrainandaccumulatedC-4, andC-14methyl etal.,2005;Alcazar-Fuolietal.,2006).Thissuggeststhatalthough
sterols; mainly eburicol (Figure 1). Accumulation of eburicol, A.fumigatusmightuseoneoftheenzymesCyp51orErg3fornor-
togetherwithadecreaseinergosterol,wasmuchmoreprominent malgrowth,thisfungalpathogenisable,includingduringinfec-
whenthecyp51BgenewasdeletedcomparedtoΔcyp51Astrain. tion,toadaptandcompensatethatlackofanenzymefunction.
Downstreaminthepathway,thesynthesisof ergosterolinvolves
thetransformationoffecosterol,consistingofdouble-bondrear- ANTIFUNGALRESISTANCEMECHANISMSRELATEDTO
rangementsinthesteroidnucleusandinthesidechain,isomeriza- ERGOSTEROLORITSBIOSYNTHETICPATHWAY
tionofthedoubleconnectionintheC-8totheC-7followedbythe Threeofthemostwidelyusedantifungaldrugs,triazoles,polyenes,
desaturationatC-5andC-22,andthereductionoftheC-24.The and allylamines, are aimed at ergosterol, and they are either
enzymaticsequenceofthesethreelaststepscoulddifferbetween fungicidal but toxic to the host (polyenes) or fungi static and
fungaltaxaandgrowthconditions(Nesetal.,1989).Singlegene morevulnerabletoresistance(triazoles). Triazolesareinhibitors
deletionofeachoftheC-5steroldesaturases(Erg3A,Erg3B,and thattargettheergosterolbiosyntheticpathwaybybindingtothe
Erg3C)revealedadifferentsterolprofileintermsoftotalamount Cyp51 family of cytochrome P450s (14-α sterol demethylases)
ofergosterolandsterolcomposition.Againthisphenotypewasdif- causingthedepletionofergosterolbiosynthesisandtheaccumu-
ferentdependingonthedeletedenzyme.Whiletheabsenceofthe lation of lanosterol or eburicol (Kelly etal.,1995). A. fumigatus
geneserg3Aanderg3Cdidnotsignificantlyaffecttotalergosterol contains two different, but related Cyp51 proteins, encoded by
biosynthesis, the deletion of erg3B caused a dramatic ergos- cyp51Aandcyp51B(Melladoetal.,2001)thatarefunctional14-
terol decrease (70%) and a vast accumulation of the Δ7 sterols αsteroldemethylases(Marteletal.,2010; Warrilowetal.,2010).
(Figure1).Althoughthesterolcompositionrevealeddifferences The lethality caused by double gene deletion has been shown,
accordingtotheenzymethatwasdeleted,noneofthesinglegene althoughneithergeneisitself essential(Melladoetal.,2005; Hu
FrontiersinMicrobiology|FungiandTheirInteractions January2013|Volume3|Article439|2
“fmicb-03-00439” — 2013/1/8 — 22:19 — page 2 — #2
Alcazar-FuoliandMellado Ergosterolbiosynthesisandantifungaldrugresistance
Table1|Relativeamountofsterols(%oftotalsterols)indifferentA.fumigatuswild-type(CM-237),cyp51deletedstrains(Δcyp51AΔcyp51B),
andazoleresistancemutantsstrainswithdifferentcyp51Apointmutations(CM-796:G54V;CM-3269:TRL98H;CM-2159:M220K).
Sterolnames Strains
CM-237* Δcyp51A Δcyp51B CM-796* CM-3269* CM-2159*
1)24-methylcholesta-5,7,9(11),22-tetraen-3β-ol 1.09 3.69 2.74 0.85 1.18 0.92
2)24-methylcholesta-5,8,22-trien-3β-ol 0.91 0.87 0.77 0.90 0.80 0.85
3)24-methylcholesta-5,7,9,22-tetraen-3β-ol 0.71 5.69 4.57 0.00 0.00 0.00
4)24-methylcholesta-5,7,22-trien-3β-ol(Ergosterol) 78.12 59.48 48.82 84.08 83.70 88.44
5)24-methylcholesta-7,22(28)-dien-3β-ol 2.62 1.84 1.18 1.51 1.67 1.09
6)24-methylcholesta-5,7,22,24(28)-tetraen-3β-ol 0.48 0.41 0.25 0.32 0.21 0.28
7)24-methylcholesta-7,22,24(28)-trien-3β-ol 0.66 1.44 0.71 0.82 0.51 0.20
8)24-methylcholesta-5,7,24(28)-trien-3β-ol 0.91 0.14 0.34 0.08 0.19 0.16
9)24-methylcholesta-7,24(28)-dien-3β-ol(Episterol) 1.51 2.55 2.01 3.59 1.84 1.08
10)24-Ethylcholesta-5,7,22-trien-3β-ol 7.48 5.48 9.26 2.43 6.46 5.10
11)4,4,14-trimethylcholesta-8,24-dien-3β-ol(Lanosterol) 1.06 1.39 2.87 3.63 0.29 0.18
12)4α,24-dimethylcholesta-8,24(28)-dien-3β-ol 0.99 2.03 0.98 0.73 0.00 0.82
13)4,4,14,24-tetramethylcholesta-8,24(28)-dien-3β-ol(Eburicol) 1.14 9.54 21.95 0.41 1.30 0.23
14)4,4,24-trimethylcholesta-8,24(28)-dien-3β-ol 2.04 5.44 3.56 0.65 0.62 0.65
*GeometricmeanofMICstoantifungalagentsofA.fumigatusstrains:CM-237:ITC(0.25μg/ml),VRC(0.48μg/ml),POS(0.06μg/ml),FLC(1094.55μg/ml),AMB
(0.26μg/ml),TRB(5.4μg/ml);CM-796:ITC(16μg/ml),VRC(1.19μg/ml),POS(1.68μg/ml),FLC(1094.55μg/ml),AMB(0.15μg/ml),TRB(2.38μg/ml;CM-2159:ITC
(16μg/ml),VRC(1.19μg/ml),POS(2μg/ml),FLC(1094.55μg/ml),AMB(0.3μg/ml),TRB(3.17μg/ml);CM-3269:ITC(16μg/ml),VRC(4μg/ml),POS(0.5μg/ml),FLC
(1094.55μg/ml),AMB(0.2μg/ml),TRB(2μg/ml).
etal., 2007). Moreover, the importance of A. fumigatus Cyp51 appears thus far to be limited to mutations which modify the
proteinsinsterolbiosynthesisandtheirsignificanceinazolesus- Cyp51Atargetsite.ThesterolcontentofazoleresistantA.fumi-
ceptibilityhavebeenextensivelystudied(Alcazar-Fuolietal.,2008, gatus strainsbearingdifferentCyp51Apointmutationssuggests
2011; Martel etal., 2010). Also, the in vitro and in vivo corre- thattheirazoleresistantphenotypesarenottheresultofpertur-
lation of azole resistance has been widely documented; with a bationsoftheergosterolbiosyntheticpathway(Table1).Itismore
clearassociationofresistantA.fumigatusstrainisolationandlack likelythatazoleresistanceinthesevariantsresultsfromareduced
of patient response to therapy (van der Linden etal.,2011a). A. affinityofdrugbindingtotheCyp51Aenzyme.Inthissense,3D
fumigatus azole resistance can arise either from modification of proteinmodelsofCyp51Aincombinationwithazoleshavepro-
theCyp51Atarget,orbyitsoverexpression. Resistanceacquired vided the basis to address how point mutations can affect azole
throughexposuretoazolesinthepatientiscorrelatedwithcyp51A drugresistance(Alcazar-Fuolietal.,2011;Fraczeketal.,2011).
single mutations (Diaz-Guerra etal., 2003; Mann etal., 2003; Regardingthepolyeneantifungals,theassociationbetweenA.
Nascimentoetal.,2003;Melladoetal.,2004;Howardetal.,2006; fumigatus susceptibility toAMB in vitro and clinical outcome is
Albarrag etal., 2011), while resistance caused by cyp51A over- unclear. SecondaryresistancetoAMBisgenerallynotobserved,
expression is specifically linked to the combination of a single even in patients whose therapy has failed (Moosa etal., 2002),
amino acid substitution (a leucine to histidine change, L98H), yet resistant mutants have been spontaneously induced in the
together with the presence of two tandem copies of a 34-bp laboratory (Manavathu etal., 1998) and resistance to AMB is
sequence in the promoter of the cyp51A gene. This latter type welldocumentedformanyotherAspergillusspecies(Blumetal.,
of resistance mechanism may have evolved in the environment 2008;vanderLindenetal.,2011b;Hadrichetal.,2012).Acquired
through the exposure of the fungus to azole fungicides used in resistancetoAMBhasbeenmostextensivelyevaluatedinyeasts
agriculture(Sneldersetal.,2008). Inisolationthetandem34-bp and is associated with mutations in the ERG3 gene, which is
promoterinsertionscannotexplaintheobservedazolecrossresis- linked to qualitative and quantitative alterations of membrane
tant phenotype (Mellado etal.,2007), however recent work has lipids and an absence of ergosterol (Kelly etal., 1997; Morio
demonstratedtheessentialityofthis34-bppromotersequencein etal., 2012). However, the deletion of three independent erg3-
maintaining the wild-type expression of cyp51A, and suggested like genes in A. fumigatus did not affect susceptibility to AMB,
the presence of other regulatory elements upstream of the tan- despite marked alterations of sterol composition and a decrease
dem insertions (Paul etal.,2012). Further mechanisms of azole intotalergosterol(Alcazar-Fuolietal.,2006).Otherstudieshave
resistance include overexpression of drug transporters of the determinedthatneitherergosterolcontent,cellwallcomposition,
ABC- and/or MFS-type (Tobin etal., 1997; Slaven etal., 2002; or lipid peroxidation levels correlate with heightened A. terreus
Nascimentoetal.,2003), however clinically observed resistance AMBresistance,onlythatahigherlevelofcatalaseproductionin
www.frontiersin.org January2013|Volume3|Article439|3
“fmicb-03-00439” — 2013/1/8 — 22:19 — page 3 — #3
Alcazar-FuoliandMellado Ergosterolbiosynthesisandantifungaldrugresistance
A.terreus mightcontributetoAMBresistancesinceitpromotes In a similar manner, analysis of sterol composition in
oxidativedamageoffungalcellmembranesthroughgenerationof iron-depleted and replete cultures revealed decreased ergosterol
reactiveoxygenspecies(Blumetal.,2008). biosynthesisduringironstarvationandtheaccumulationoftwo
Terbinafinebelongstotheallylamineclassof antifungalsthat types of sterol intermediates C-4 and Δ7. The genome-wide
inhibit squalene epoxidase (Erg1). Erg1 catalyzes the first oxy- expressionresponseofA.fumigatusinashiftfromiron-depleted
genation step in sterol biosynthesis and is suggested to be one toiron-repleteconditions(10,30,60,120,and240mincompared
of the rate-limiting enzymes in this pathway. Alterations in withthatfrom0min)showedthatErg24andErg25functionswere
A. fumigatus erg1 gene dosage can promote terbinafine resis- down-regulatedforallof thetimepoints,andincontrastErg11
tance (Liu etal., 2004), however, the first terbinafine resis- was up-regulated at 30 and 60 min of iron exposure (Schrettl
tance mechanism was described in A. nidulans and associated etal.,2008). In conditions of iron limitation, A. fumigatus uses
with the activity of salicylate 1-monooxygenase (salA), a well- siderophore-assistedironuptake,whichinturnusesmevalonate
characterized naphthalene-degrading enzyme, suggesting that asaprecursor.Theresultsdescribedabove,togetherwiththechar-
resistance could follow degradation of the naphthalene ring acterizationoffunctionalsiderophoredeletedmutants,probedthe
contained in terbinafine (Graminha etal., 2004). Also, a point linkbetweenfungalergosterolandsiderophorebiosynthesisinA.
mutation,F391L,inthesqualeneepoxidaseenzymewasfoundto fumigatusviathecompoundmevalonate.Thelatterwouldappear
conferastrongterbinafineresistancephenotype. Theequivalent to feed both biochemical pathways (Yasmin etal., 2012). Also,
mutationwasintroducedintothehomologousgeneof A.fumi- in hypoxic conditions SrbA was found to activate siderophore-
gatusresultinginterbinafineresistance(Rochaetal.,2006).Since mediatedironuptakeinresponsetohypoxiaandironstarvationin
terbinafine is not currently used in the management of IA, the partbytranscriptionalactivationofanothertranscriptionfactor,
appearance of terbinafine-resistant strains in the clinical setting HapX(Blatzeretal.,2011).
isunlikely, however, itspotentialuseincombinationwithother Transcriptional regulation of ergosterol biosynthesis in A.
antifungaldrugsshouldbeconsidered. fumigatushasalsobeenstudiedunderantifungalexposureinlabo-
ratorycultures.Microarrayanalysisindicateddown-regulationof
ALTERNATIVESTOIMPROVEANTIFUNGALEFFICACYAND theerg6geneunderbothAMBandVCZexposure(daSilvaFerreira
TOMINIMIZEDRUGRESISTANCE etal.,2006;Gautametal.,2008). Erg6hasaroleinthesynthesis
Globalgeneexpressionstudiesidentifiedthatergosterolbiosyn- ofsecondarysterols(ethylsterols),anditsdown-regulationsug-
thesis is likely to be highly sensitive to environmental perturba- geststhatA.fumigatusaltersthesynthesisofsecondarysterolsin
tions. Among them, adaptation to the host environment when responsetoAMBorVCZ.However,differentresultswerefoundfor
A.fumigatusinitiatesdiseaseisparticularlyinteresting.Thetran- theA.fumigatusCyp51-encodinggenes,whichwerebothdown-
scriptomeof A.fumigatus duringearlystagesof infectioninthe regulated under VCZ treatment and up-regulated with AMB.
neutropenicmurinelungidentifiedreducedexpressionof genes Theseresultsmayindicatetheexistenceof differentialresponses
encodingergosterolpathwayfunctions(Erg11,Erg24,andErg3) for overcoming the distinct stresses imposed by these drugs. In
relativetolaboratorycultures(McDonaghetal.,2008). addition,genesencodingforErg24,Erg25,andErg3werediffer-
Althoughthetargetsiteofazoleactivityiswellstudied,therole entially expressed when A. fumigatus cells were cultured in the
of other proteins in the mode of action of these drugs in fungi presenceofVCZ(daSilvaFerreiraetal.,2006).
ispoorlyunderstood. Recently,acriticalroleforSrbA-mediated Collectively, the above observations suggest that ergosterol
regulation of ergosterol biosynthesis and triazole drug interac- biosynthesis is prone to perturbation by environmental condi-
tions in A. fumigatus has been reported (Blosser and Cramer, tions,andinamannerdependentuponErg6,Erg11,Erg24,Erg25,
2012). SrbA was identified by transcriptional profiling under andErg3functions,therebyhighlightingthesegenefunctionsas
hypoxiaconditionsasaregulatorofergosterolbiosyntheticgenes alternative or synergic inhibitors of the pathway. An important
in response to low oxygen levels. The ΔsrbA mutant strain was findinginA.fumigatuswastheidentificationofC-24ethylsterols.
attenuated in virulence and it was hypersusceptible to flucona- These sterols can also be final metabolites of the sterol pathway
zoleandVCZ(Willgeretal.,2008).Also,severalgenesencoding andtheyarepredominantlyfoundinhigherplants,thoughabsent
enzymes that require high levels of oxygen were found to be in mammalian cells, which cannot alkylate the C-24 of sterols.
transcriptionallyrepressedintheabsenceof SrbA,includingthe InplantsC-24ethylsterolshavemultiplerolestoplayingrowth
enzymes Erg6, Erg11, Erg24, Erg25, and Erg3. The relationship anddevelopment,however,fewreportsexistonthedetectionof
betweenergosterolregulationandhypoxiawasalsodemonstrated 24-ethyl sterols in fungi, and their role in A. fumigatus remains
by the analysis of the sterol profile of the ΔsrbA which showed unknown.C-24alkylationiscatalyzedbyS-adenosyl-methionine-
a decrease in ergosterol levels and accumulation of C-4 methyl sterol-C-methyltransferases(SAMs).InS.cerevisiaethemethyla-
sterols,suggestingablockageof C-4demethylation.Inaddition, tioniscatalyzedbyerg6whichconvertszymosterolintofecosterol,
the apparent control of cyp51 transcript levels by SrbA suggests and at least two methyltransferases are involved in consecutive
an additional target for drug development. A role for SrbA in methylationreactionsleadingto24-ethylsterolsinhigherplants
the development of triazole resistance has also been suggested, (Bouvier-Nave etal., 1998). In A. fumigatus, only one Erg6 has
since alterations in this transcription factor or its DNA-binding been identified that could transform lanosterol to eburicol and
affinity could transcriptionally initiate changes that could alter may also be involved in the production of C-24 ethyl sterols.
target abundance and would lead to triazole resistance (Blosser The sterol profile analysis of A. fumigatus strains showed that
andCramer,2012). thesynthesisof24-Ethylcholesta-5,7,22-trien-3β-olissensitiveto
FrontiersinMicrobiology|FungiandTheirInteractions January2013|Volume3|Article439|4
“fmicb-03-00439” — 2013/1/8 — 22:19 — page 4 — #4
Alcazar-FuoliandMellado Ergosterolbiosynthesisandantifungaldrugresistance
pathwayperturbations.InaΔcyp51Bmutantalackofergosterol is able to synthesize ergosterol, even with suppression of sev-
is compensated by an increase of 24-Ethylcholesta-5,7,22-trien- eralenzymesinthepathway.Biosyntheticcontrolunderdifferent
3β-ol, probably because the activity of the pathway becomes environmental circumstances is observed, demonstrating that
diverted toward the synthesis of alkyl sterols. However, when Aspergillushasalternativestoovercomeseveredrawbacks.There-
erg3Bissuppressed,ergosterollevelsaredecreasedtogetherwith fore,thepreciseknowledgeofthiscomplexbiosyntheticpathway
24-ethylcholesta-5,7,22-trien-3β-ol,confirmingthatthisenzyme couldfacilitatethefuturedevelopmentof novelandmoreselec-
wouldalsobeinvolvedinthedesaturationofC-5,7alkylsterols. tiveantifungaldrugsinordertoimproveefficacyandtominimize
Genomewidestudiesareprovidinguswithaveryusefulplat- drugresistance.
form from which to dissect the linkages between cellular sterol
biosynthesisandothercellfunctionsormetabolicpathways.Such ACKNOWLEDGMENTS
newfindingsmightbefurtherexploredfornoveldrugdevelop- EmiliaMelladowassupportedbytheEuropeanScienceFounda-
mentorforpossiblecombinatorialtherapeuticstrategiestofight tion: Fuminomics 06-RNP-132 and the Research Projects from
invasive fungal diseases. Among them, genes encoding enzymes theSpanishMinistryofScienceandInnovation:SAF2008-04143
involvedincellularstress,cellwallsynthesis,andtransporthave andERA-NETPathogenomics(7thFP),BFU2008-04709-E/BMC.
beenfoundtobedifferentiallyexpressedunderantifungalexpo- LauraAlcazar-FuoliisfundedbySpanishFondodeInvestigación
sure or underhost imposed stresses. To conclude, A. fumigatus SanitariawithaMiguelServetfellowship:MPY991/12.
REFERENCES of erg11 (cytochrome P450 lanos- ionicchannelsinducedinlipidbilay- ergosterol. Proc. Natl. Acad. Sci.
Albarrag, A. M., Anderson, M. J., teroldemethylase)mutationsinSac- ers by polyene antibiotics ampho- U.S.A.109,2234–2239.
Howard,S.J.,Robson,G.D.,Warn, charomyces cerevisiae and Candida tericinBandnystatine. Nature262, Hadrich, I., Makni, F., Neji, S.,
P. A., Sanglard, D., etal. (2011). albicans.Lipids28,963–967. 698–699. Cheikhrouhou, F., Bellaaj, H.,
Interrogationofrelatedclinicalpan- Blatzer, M., Barker, B. M., Willger, Ferreira,M.E.,Colombo,A.L.,Paulsen, Elloumi, M., etal. (2012). Ampho-
azole-resistant Aspergillus fumigatus S. D., Beckmann, N., Blosser, S. I.,Ren,Q.,Wortman,J.,Huang,J., tericinBinvitroresistanceisasso-
strains: G138C,Y431C,andG434C J., Cornish, E. J., etal. (2011). etal. (2005). Theergosterolbiosyn- ciated with fatal Aspergillus flavus
singlenucleotidepolymorphismsin SREBP coordinates iron and ergos- thesis pathway, transporter genes, infection.Med.Mycol.50,829–834.
cyp51A,upregulationofcyp51A,and terolhomeostasistomediatetriazole and azole resistance in Aspergillus Howard, S. J., and Arendrup, M. C.
integrationandactivationof trans- drug and hypoxia responses in the fumigatus. Med. Mycol. 43, S313– (2011). Acquired antifungal drug
posonAtf1inthecyp51Apromoter. human fungal pathogen Aspergillus S319. resistance in Aspergillus fumigatus:
Antimicrob. Agents Chemother. 55, fumigatus. PLoSGenet. 7:e1002374. Fraczek, M. G., Bromley, M., and epidemiology and detection. Med.
5113–5121. doi:10.1371/journal.pgen.1002374 Bowyer, P. (2011). An improved Mycol.49,S90–S95.
Alcazar-Fuoli,L.,Cuesta,I.,Rodriguez- Blosser, S. J., and Cramer, R. A. model of the Aspergillus fumigatus Howard, S. J., Webster, I., Moore, C.
Tudela, J. L., Cuenca-Estrella, M., (2012). SREBP-dependent triazole CYP51Aprotein.Antimicrob.Agents B., Gardiner, R.E., Park, S., Perlin,
Sanglard,D.,andMellado,E.(2011). susceptibilityinAspergillusfumigatus Chemother.55,2483–2486. D.S.,etal.(2006).Multi-azoleresis-
Three-dimensional models of 14α- ismediatedthroughdirecttranscrip- Fryberg, M., Oehlschlager, A.C., and tanceinAspergillusfumigatus.Int.J.
sterol demethylase (Cyp51A) from tionalregulationoferg11A(cyp51A). Unrau,A.M.(1973).Biosynthesisof Antimicrob.Agents28,450–453.
Aspergillus lentulus and Aspergillus Antimicrob. Agents Chemother. 56, ergosterolinyeast.Evidenceformul- Hu,W.,Sillaots,S.,Lemieux,S.,Davi-
fumigatus: an insight into dif- 248–257. tiplepathways.J.Am.Chem.Soc.95, son, J., Kauffman, S., Breton, A.,
ferences in voriconazole interac- Blum, G., Perkhofer, S., Haas, H., 5747–5577. etal. (2007). Essential gene identi-
tion. Int. J. Antimicrob. Agents 38, Schrettl, M., Würzner, R., Dierich, Gautam, P., Shankar, J., Madan, T., fication and drug target prioritiza-
426–434. M. P., etal. (2008). Potential basis Sirdeshmukh, R., Sundaram, C. S., tion in Aspergillus fumigatus. PLoS
Alcazar-Fuoli,L.,Mellado,E.,Cuenca- for amphotericin B resistance in Gade, W. N., etal. (2008). Pro- Pathog. 3:e24. doi: 10.1371/jour-
Estrella,M.,andSanglard,D.(2011). Aspergillusterreus.Antimicrob.Agents teomicandtranscriptomicanalysisof nal.ppat.0030024
Probingtheroleofpointmutations Chemother.52,1553–1555. Aspergillusfumigatusonexposureto Kelly, S. L., Lamb, D. C., Cor-
inthecyp51AgenefromAspergillus Bouvier-Nave, P., Husselstein, T., and amphotericin B. Antimicrob. Agents ran, A. J., Baldwin, B. C., and
fumigatusinthemodelyeastSaccha- Benveniste,P.(1998).Twofamiliesof Chemother.52,4220–4227. Kelly, D.E.(1995). Modeofaction
romyces cerevisiae. Med. Mycol. 49, sterolmethyltransferasesareinvolved Ghannoum, M. A., and Rice, L. B. and resistance to azole antifun-
276–284. inthefirstandthesecondmethyla- (1999). Antifungalagents: modeof gals associated with the formation
Alcazar-Fuoli, L., Mellado, E., Garcia- tionstepsofplantsterolbiosynthesis. action,mechanismsofresistance,and of14alpha-methylergosta-8,24(28)-
Effron, G., Buitrago, M. J., Lopez, Eur.J.Biochem.256,88–96. correlationofthesemechanismswith dien-3 beta,6 alpha-diol. Biochem.
J. F., Grimalt, J. O., etal. (2006). da Silva Ferreira, M. E., Malavazi, I., bacterial resistance Clin. Microbiol. Biophys.Res.Commun.207,910–915.
Aspergillus fumigatus C-5 sterol Savoldi,M.,Brakhage,A.A.,Gold- Rev.12,501–517. Kelly, S. L., Lamb, D. C., Kelly,
desaturases Erg3A and Erg3B: man,M.H.,Kim,H.S.,etal.(2006). Gooday, G. W. (1995). “Cell mem- D. E., Manning, N. J., Loef-
role in sterol biosynthesis and TranscriptomeanalysisofAspergillus brane,”inTheGrowingFungus, eds fler, J., Hebart, H., etal. (1997).
antifungal drug susceptibility. fumigatus exposed to voriconazole. N. A. R. Gow and M. G. Geoffrey Resistancetofluconazoleandcross-
Antimicrob. Agents Chemother. 50, Curr.Genet.50,32–44. (London:Chapman&Hall),62–64. resistancetoamphotericinBinCan-
453–460. Diaz-Guerra, T. M., Mellado, E., Graminha,M.A.,Rocha,E.M.,Prade, dida albicans from AIDS patients
Alcazar-Fuoli, L., Mellado, E., Garcia- Cuenca-Estrella,M.,andRodriguez- R. A., and Martinez-Rossi, N. M. caused by defective sterol delta5,6-
Effron, G., Lopez, J. F., Grimalt, Tudela,J.L.(2003).Apointmutation (2004).Terbinafineresistancemedi- desaturation.FEBSLett.400,80–82.
J. O., Cuenca-Estrella, J. M., etal. in the 14alpha-Sterol demethylase ated by salicylate 1-monooxygenase Krishnan-Natesan, S. (2009). Terbi-
(2008).Ergosterolbiosynthesispath- genecyp51Acontributestoitracona- in Aspergillus nidulans. Antimicrob. nafine: apharmacologicalandclin-
wayinAspergillusfumigatus.Steroids zoleresistanceinAspergillusfumiga- AgentsChemother.48,3530–3535. ical review. Expert Opin. Pharma-
73,339–347. tus. Antimicrob. Agents Chemother. Gray, K.C., Palacios, D.S., Dailey, I., cother.10,2723–2733.
Bard, M., Lees, N.D., Turi, T., Craft, 47,1120–1124. Endo,M.M.,Uno,B.E.,Wilcock,B. Liu, W., May, G. S., Lionakis, M.
D., Cofrin, L., Barbuch, R., etal. Ermishkin, L. N., Kasumov, K. M., C., etal. (2012). Amphotericinpri- S., Lewis, R. E., and Kontoyian-
(1993).Sterolsynthesisandviability andPotzeluyev,V.M.(1976).Single marilykillsyeastbysimplybinding nis, D. P. (2004). Extra copies
www.frontiersin.org January2013|Volume3|Article439|5
“fmicb-03-00439” — 2013/1/8 — 22:19 — page 5 — #5
Alcazar-FuoliandMellado Ergosterolbiosynthesisandantifungaldrugresistance
of the Aspergillus fumigatus squale- of cyp51A alterations. Antimicrob. inAspergillusfumigatus.Antimicrob. Warrilow, A. G., Melo, N., Martel,
neepoxidase gene confer resistance AgentsChemother.51,1897–1904. AgentsChemother.50,2533–2536. C. M., Parker, J. E., Nes, W. D.,
to terbinafine: genetic approach to Mellado,E.,Garcia-Effron,G.,Alcazar- Ruckenstuhl,C.,Lang,S.,Poschenel,A., Kelly,S.L.,etal.(2010).Expression,
studyinggenedose-dependentresis- Fuoli, L., Cuenca-Estrella, M., Eidenberger,A.,Baral,P.K.,Kohút, purification,andcharacterizationof
tancetoantifungalsinA.fumigatus. andRodriguez-Tudela, J.L.(2004). P.,etal. (2007). Characterizationof Aspergillusfumigatussterol14-alpha
Antimicrob. Agents Chemother. 48, Substitutions at methionine 220 squaleneepoxidase of Saccharomyces demethylase(CYP51)isoenzymesA
2490–2496. in the 14alpha-sterol demethylase cerevisiae by applying terbinafine- andB.Antimicrob.AgentsChemother.
Manavathu, E. K., Alangaden, G. J., (Cyp51A)ofAspergillusfumigatusare sensitivevariants.Antimicrob.Agents 54,4225–4234.
andChandrasekar,P.H.(1998).In- responsibleforresistanceinvitroto Chemother.51,275–284. Willger,S.D.,Puttikamonkul,S.,Kim,
vitroisolationandantifungalsuscep- azole antifungal drugs. Antimicrob. Ryder,N.S.(1992).Terbinafine:mode K.H.,Burritt,J.B.,Grahl,N.,Met-
tibility of amphotericin B-resistant AgentsChemother.48,2747–2750. ofactionandpropertiesofthesquale- zler, L. J., etal. (2008). A sterol-
mutants of Aspergillus fumigatus. J. Mellado,E.,Garcia-Effron,G.,Buitrago, neepoxidaseinhibition.Br.J.Derma- regulatory element binding protein
Antimicrob.Chemother.41,615–619. M. J., Alcazar-Fuoli, L., Cuenca- tol.126,S2–S7. isrequiredforcellpolarity,hypoxia
Mann,P.A.,Parmegiani,R.M.,Wei,S. Estrella, M., andRodriguez-Tudela, Schrettl, M., Kim, H. S., Eisendle, adaptation, azole drug resistance,
Q.,Mendrick,C.A.,Li,X.,Loeben- J. L. (2005). Targeted gene disrup- M., Kragl, C., Nierman, W. andvirulenceinAspergillusfumiga-
berg, D., etal. (2003). Mutations tionofthe14-alphasteroldemethy- C., Heinekamp, T., etal. (2008). tus. PLoS Pathog. 4:e1000200. doi:
inAspergillusfumigatus resultingin lase(cyp51A)inAspergillusfumigatus SreA-mediated iron regulation in 10.1371/journal.ppat.1000200
reduced susceptibility to posacona- anditsroleinazoledrugsusceptibil- Aspergillusfumigatus.Mol.Microbiol. Yasmin, S., Alcazar-Fuoli, L.,
zole appear to be restricted to a ity.Antimicrob.AgentsChemother.49, 70,27–43. Gründlinger, M., Puempel, T.,
singleaminoacidinthecytochrome 2536–2538. Shapiro,R.S.,Robbins,N.,andCowen, Cairos,T.,Blatzer,M.,etal.(2012).
P450 14alpha-demethylase. Antimi- Moosa,M.Y.,Alangaden,G.J.,Man- L. E. (2011). Regulatory circuitry Mevalonate governs interdepen-
crob. Agents Chemother. 47, avathu,E.,andChandrasekar,P.H. governingfungaldevelopment,drug dencyofergosterolandsiderophore
577–581. (2002).ResistancetoamphotericinB resistance, and disease. Microbiol. biosynthesesinthefungalpathogen
Martel,C.M.,Parker,J.E.,Warrilow,A. doesnotemergeduringtreatmentfor Mol.Biol.Rev.75,213–267. Aspergillus fumigatus. Proc. Natl.
G.,Rolley,N.J.,Kelly,S.L.,andKelly, invasive Aspergillosis. J. Antimicrob. Snelders, E., vanderLee, H.A., Kui- Acad.Sci.U.S.A.109,E497–E504.
D. E. (2010). Complementation Chemother.49,209–213. jpers,J.,Rijs,A.J.,Varga,J.,Samson,
of a Saccharomyces cerevisiae Morio, F., Pagniez, F., Lacroix, R. A., etal. (2008). Emergence of
ERG11/CYP51(sterol14α-demethy- C., Miegeville, M., and Le Pape, azoleresistanceinAspergillusfumiga-
lase) doxycycline-regulated mutant P. (2012). Amino acid substitu- tusandspreadofasingleresistance Conflict of Interest Statement: The
andscreeningoftheazolesensitivity tionsintheCandidaalbicans sterol mechanism. PLoSMed. 5:e219. doi: authors declare that the research was
of Aspergillus fumigatus isoenzymes {Delta}5,6-desaturase(Erg3p)confer 10.1371/journal.pmed.0050219 conductedintheabsenceofanycom-
CYP51A and CYP51B. Antimi- azole resistance: characterization of Slaven,J.W.,Anderson,M.J.,Sanglard, mercial or financial relationships that
crob. Agents Chemother. 54, 4920– twonovelmutantswithimpairedvir- D.,Dixon,G.K.,Bille,J.,Roberts,I. couldbeconstruedasapotentialcon-
4923. ulence.J.Antimicrob.Chemother.67, S.,etal.(2002).Increasedexpression flictofinterest.
McDonagh, A., Fedorova, N. D., 2131–2138. ofanovelAspergillusfumigatusABC
Crabtree, J., Yu, Y., Kim, S., Nascimento, A. M., Goldman, G. H., transporter gene, atrF, in the pres- Received: 24 July 2012; accepted: 19
Chen,D.,etal.(2008).Sub-telomere Park, S., Marras, S. A., Delmas, enceofitraconazoleinanitracona- December 2012; published online: 10
directed gene expression during G., Oza, U., etal. (2003). Mul- zoleresistantclinicalisolate. Fungal January2013.
initiation of invasive aspergillo- tiple resistance mechanisms among Genet.Biol.36,199–206. Citation: Alcazar-Fuoli L and Mel-
sis. PLoS Pathog. 4:e1000154. doi: Aspergillus fumigatus mutants with Tobin,M.B.,Peery,R.B.,andSkatrud, ladoE(2013)Ergosterolbiosynthesisin
10.1371/journal.ppat.1000154 high-levelresistancetoitraconazole. P. L. (1997). Genes encoding mul- Aspergillusfumigatus:itsrelevanceasan
Mellado, E., Diaz-Guerra, T. M., Antimicrob. Agents Chemother. 47, tipledrugresistance-likeproteinsin antifungaltargetandroleinantifungal
Cuenca-Estrella,M.,andRodriguez- 1719–1726. AspergillusfumigatusandAspergillus drugresistance. Front. Microbio. 3:439.
Tudela, J. L. (2001). Identifica- Nes, W. D., Xu, S. H., and Haddon, flavus.Gene200,11–23. doi:10.3389/fmicb.2012.00439
tionoftwodifferent14-alphasterol W.F.(1989).Evidenceforsimilarities van der Linden, J. W., Snelders, E., ThisarticlewassubmittedtoFrontiersin
demethylase-related genes (cyp51A anddifferencesinthebiosynthesisof Kampinga, G. A., Rijnders, B. J., FungiandTheirInteractions,aspecialty
andcyp51B)inAspergillusfumigatus fungalsterols.Steroids53,533–558. Mattsson,E.,Debets-OssenkoppY.J., ofFrontiersinMicrobiology.
andotherAspergillusspecies.J.Clin. Paul, S., Klutts, J. S., and Moye- etal.(2011a).Clinicalimplicationsof Copyright©2013Alcazar-FuoliandMel-
Microbiol.39,2431–2438. Rowley, W. S. (2012). Analysis azole resistance inAspergillus fumi- lado. Thisisanopen-accessarticledis-
Mellado,E.,Garcia-Effron,G.,Alcazar- of promoter function in Aspergillus gatus, TheNetherlands, 2007–2009. tributedunderthetermsoftheCreative
Fuoli, L., Melchers, W. J., Verweij, fumigatus. Eukaryot. Cell 11, 1167– Emerg.Infect.Dis.17,1846–1854. Commons Attribution License, which
P. E., Cuenca-Estrella, M., etal. 1177. vanderLinden,J.W.,WarrisA.,andVer- permitsuse, distributionandreproduc-
(2007). A new Aspergillus fumi- Rocha,E.M.,Gardiner,R.E.,Park,S., weijP.E.(2011b).Aspergillusspecies tioninotherforums,providedtheorigi-
gatus resistance mechanism confer- Martinez-Rossi,N.M.,andPerlin,D. intrinsically resistant to antifungal nalauthorsandsourcearecreditedand
ringinvitrocross-resistancetoazole S.(2006).APhe389Leusubstitution agents. Med. Mycol. 49(Suppl. 1), subjecttoanycopyrightnoticesconcern-
antifungals involves a combination inergAconfersterbinafineresistance S82–S89. inganythird-partygraphicsetc.
FrontiersinMicrobiology|FungiandTheirInteractions January2013|Volume3|Article439|6
“fmicb-03-00439” — 2013/1/8 — 22:19 — page 6 — #6