Lipid Biosynthesis as an Antifungal Target

13 Pages·2017·3.83 MB·English

Journal of
Fungi
Review
Lipid Biosynthesis as an Antifungal Target
JiaoPan1,2,*,CuitingHu1andJae-HyukYu3,* ID
1 DepartmentofMicrobiology,CollegeofLifeSciences,NankaiUniversity,Tianjin300071,China;
[email protected]
2 KeyLaboratoryofMolecularMicrobiologyandTechnology,MinistryofEducation,Tianjin300071,China
3 DepartmentofBacteriology,UniversityofWisconsin-Madison,Madison,WI53706,USA
* Correspondence:[email protected](J.P.);[email protected](J.-H.Y.);
Tel.:[phone](J.P.);[phone](J.-H.Y.)
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:23March2018;Accepted:18April2018;Published:20April2018
Abstract: Lipids, commonly including phospholipids, sphingolipids, fatty acids, sterols, and
triacylglycerols (TAGs), are important biomolecules for the viability of all cells. Phospholipids,
sphingolipids,andsterolsareimportantconstituentsofbiologicalmembranes. Manylipidsplay
important roles in the regulation of cell metabolism by acting as signaling molecules. Neutral
lipids,includingTAGsandsterolesters(STEs),areimportantstoragelipidsincells. Inviewofthe
importanceoflipidmolecules,thisreviewbrieflysummarizesthemetabolicpathwaysforsterols,
phospholipids,sphingolipids,fattyacids,andneutrallipidsinfungiandillustratesthedifferences
betweenfungalandhuman(orothermammalian)cells,especiallyinrelationtolipidbiosynthetic
pathways. Thesedifferencesmightprovidevaluablecluesforustofindtargetproteinsfornovel
antifungaldrugs. Inaddition,thedevelopmentoflipidomicstechnologyinrecentyearshassupplied
uswithashortcutforfindingnewantifungaldrugtargets;thisabilityisimportantforguidingour
researchonpathogenicfungi.
Keywords: lipidmetabolism;pathogenicfungi;antifungaldrugs;targetprotein;lipidomics
1. Introduction
The increasing number of immunocompromised patients has led to the emergence of many
formsoffungalinfectionsinrecentyears. Thecausativefungiaremostcommonlydermatophytic
(Epidermophyton,Microsporum,orTrichophytonspecies)orsaprophyticspecies(Aspergillus,Blastomyces,
Candida,Coccidioides,Paracoccidioides,Cryptococcus,Histoplasma,orSporothrixspecies). Thesaprophytes
are also capable of causing systemic infections, which pose a great threat to human health under
certain conditions [1]. Furthermore, a restricted arsenal of antifungals is available, and the
developmentofresistancetoantifungaldrugsisincreasing,whichmakesitimperativetodiscover
newantifungaltargets.
Theexistenceofthecellwallinfungiisthemostsignificantstructuraldifferencebetweenfungi
and mammalian cells. The main components of the fungal cell wall are β-1,3-glucan, chitin, and
variousglycoproteins, whichareessentialfortheintegrityofthecellwallandthemaintenanceof
intracellularosmoticpressure.Therefore,disruptingthebiosynthesisofthefungalcellwallhasbeenan
importantstrategyforthedevelopmentofantifungaldrugs. Forexample,theechinocandins,clinically
importantfungalinhibitors,areabletoinhibitthesynthesisoffungalβ-1,3-glucanandthusabolish
theintegrityofthecellwall. Althoughthecellwallisimportantforfungi,fromalipidperspective,
theplasmamembraneisimportantasitcontainstheenzymesandproteinsresponsibleforthecell
wallbiosynthesis[2,3]. Inthisreview,wefocusonthefungalmetabolicpathwaysforlipids,including
phospholipids,sphingolipids,fattyacids,andneutrallipids,andhowthebiosyntheticportionsof
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thesepathwaysdifferfromthoseofmammaliancells.Specificenzymesorproteinsnecessaryforfungal
lipidmetabolismmighthavegreatpotentialtobeantifungaltargets. Asimportantconstituentsof
cellularmembranes,lipidsalsohavebeenproventobetargetsofantifungals,whichwillbediscussed
here. Furthermore,someimportantlipidsignalingmoleculescanregulatecellproliferationand/or
celldeath,whichistightlylinkedtothevirulenceandpathogenicityofthefungus. Thus,interference
withtheseregulatorymechanismsmaybeaneffectivemeansofcopingwithfungalinfections.
2. ClassificationandFunctionofLipids
2.1. ClassificationofLipids
There are many kinds of naturally occurring lipid molecules with great differences in their
chemical structures and physical properties. In 2005, to advance the development of lipidomics,
theInternationalLipidClassificationandNomenclatureCommittee(ILCNC)classifiedlipidsinto
eight categories: fatty acyls (FAs), glycerolipids (GLs), glycerophospholipids (GPs), sphingolipids
(SLs),sterollipids(STs),saccharolipids(SLs),prenollipids(PRs),andpolyketides(PKs)[4]. Among
these categories, fatty acids, phospholipids, sphingolipids, glycerolipids, and sterols are common
lipidmoleculesincells(thebasicstructuresofthemainlipidgroupsareshowninFigure1),while
saccharolipids,prenollipids,andpolyketidesaremainlyfoundinbacteria,fungi,andplants[5].
Figure1.Representativestructureof(a)fattyacids,(b)phospholipids,(c)sphingolipids,(d)glycerolipids,
and(e)sterollipids.ThisfigureisadaptedandmodifiedfromRef.[4].
Lipidsareorganicmoleculesthatareinsolubleinwaterbutsolubleinnonpolarorganicsolvents
andhavehydrophobicoramphiphilicproperties; thephysicalpropertiesoflipidsdeterminetheir
physiologicalfunctionincells. Asimportantstoragelipidsincells,triacylglycerols(TAGs)andsterol
esters(STEs)areesterifiedformsoffreefattyacidsandsterols,respectively. Theselipidsareuncharged
andlesstoxictocells. Sphingolipidsandphospholipidsareamphiphiliclipidswithfattyacylchains
thatformhydrophobiccoresandwiththehydrophilicheadsexposedtotheenvironment;thesetypes
oflipidsconstitutethemainbackboneofcellmembranesand,therefore,areimportantstructurallipids
incells.
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2.2. FunctionofLipids
Of the total number of genes in cells, 5% are related to the metabolism of lipid molecules [6],
not only reflecting the complex varieties of lipid molecules but also suggesting their important
biologicalfunctionsincells. Therefore,itisconceivablethatselectiveinterferencewiththemetabolism
oflipidmoleculescaneffectivelyinhibitthesurvivalofpathogenicfungiinhostcells.
First, lipid molecules are important for energy storage in cells. The β-oxidation of fatty acid
chainsprovidesalargeamountofATP,whichallowsotherbiologicalactivitiesofcellstoproceed
properly. Fattyacidsarethesimplestlipidmoleculesincellsandareimportantbuildingblocksof
complexlipidssuchasphospholipidsandsphingolipids. Surplusintracellularfreefattyacidsare
storedinlipiddroplets(LDs)[7]intheformofTAGsandSTEs. LDsarenotonlyanimportantenergy
reservoirforcellsbutarealsoanorganellecloselyrelatedtolipidmetabolism. Alargenumberof
hydrolasesinvolvedinthedegradationofTAGsandSTEsarelocatedonthemembraneandmobilize
thedecompositionofthesetwononpolarlipidswhenneeded[8].
Second, the glycerophospholipid bilayer constitutes the basic skeleton of the cell membrane,
which isolates the cell from the external environment and ensures the independence of
biochemical reactions as well as the formation of internal compartments in which different
biological functions can be completed without interference. Glycerophospholipids can be further
dividedintophosphatidylcholine(PC),phosphatidylethanolamine(PE),phosphatidylinositol(PI),
phosphatidylserine(PS),andsoon,basedontheirheadgroups. Ineukaryoticcells,thecontentand
types of phospholipids vary in the membranes of different organelles and the plasma membrane.
The major phospholipids in total cell extracts from Saccharomyces cerevisiae are PC, PE, PI, and PS
(Table 1) [9]. In addition to glycerophospholipids, sphingolipids and sterols are also important
components of cell membranes. They aggregate dynamically to form lipid rafts, which regulate
downstreamsignalingmoleculesandincreasetheefficiencyofsignaltransduction[10]. Lipidrafts
also play an important role in pathogenicity by containing many of the virulence factors of fungi.
InCryptococcusneoformans,Pma1isaprotonpumpthatmaintainstheintracellularpHoffungalcells
inthephysiologicalrange. Pma1isaraft-localizedproteinanditsassociationwithraftsisrequired
forsurfacedeliveryandproteinstabilityintheplasmamembrane. MutantsofC.neoformansinwhich
Pma1 was not functioning properly showed attenuated virulence, likely due to their inability to
surviveintheacidicenvironmentofthephagolysosome[11]. Moreover,sterolsareabletoregulate
thefluidityandpermeabilityofcellmembranes. Thestructuralfunctionoflipidmoleculesincellsis
irreplaceable. RecentstudiesinHeLacellshaveshownthatthefractionoflipidssuchasPIandPCin
membranesystemschangesduringthecriticalphasesofthecellcycle. Itwasshownthat11kindsof
lipids,forexample,phosphatidicacid(PA)(16:0/16:0),diacylglycerol(DAG)(16:1,12:0,18:1),andPS
(18:0/20:4)accumulateindividingcells. Thecompositionandlocalizationoflipidsareremodulated
duringthecellcycle. Therefore,thelipidcompositionofthecellmembranestructureitselfisvery
importanttothecell[12,13].
Table1.PhospholipidcompositioninSaccharomycescerevisiae.
Mol%ofTotalPhospholipids
CellFraction
PC PE PI PS CL PA Others
Homogenate 51.0 25.0 11.4 5.1 3.7 1.1 2.7
Plasmamembrane 11.3 24.6 27.2 32.2 nd 3.3 1.4
Endoplasmicreticulum 38.9 18.6 22.4 6.4 0.3 6.1 10.0
Mitochondria 33.4 22.7 20.6 3.3 7.2 1.7 10.1
Peroxisomes 39.8 17.4 22.0 2.5 2.7 6.1 10.5
This table is adapted and modified from Ref. [9]. PC, phosphatidylcholine; PE, phosphatidylethanolamine;
PI,phosphatidylinositol;PS,phoaphatidylserine;CL,cardiolipin;PA,phosphatidicacid;nd,notdiscovered.
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Finally,lipidssuchasfattyacids[14,15],sterols[16],phospholipids[17],andsphingolipids[18]
are important signaling molecules in cells and participate in intracellular signal transduction.
Inositol1,4,5-triphosphateanddi-acyl-glycerol(DAG),whichoriginatefromphosphatidylinositol
4,5-diphosphate,areveryimportantsignalingmolecules. IP -Ca2+-DAG-dependentproteinkinaseC
3
(PKC)phosphorylatesnumerousdownstreamproteins;thedownstreammitogen-activatedprotein
kinase(MAPK)cascadeshaveimportantregulatoryfunctionsforcellwallintegrityandoxidativestress
responses[19,20]. DAGisalsoakeyintermediatelipidmetaboliteandisinvolvedintheregulation
of lipid metabolism [16]. In response to the catalytic activity of different kinds of phospholipases,
PCcanbecleavedintovarioussecondmessengermoleculessuchaslysophosphatidylcholine(LPC),
phosphatidic acid (PA), and arachidonic acid (AA). These signaling molecules are responsible for
the regulation of many biological processes within the cell [18]. Furthermore, ceramide (Cer),
sphingosine-1-phosphate (S1P), glycosphingolipids (GSLs), and other sphingolipid molecules are
involvedinvitalactivitiessuchascellproliferation,differentiationandapoptosis,proteinsynthesis,
glucosemetabolism,andstressresponse[21].
Polyketides, lipid molecules that are not present in all eukaryotic cells, might be unfamiliar
because they are not commonly thought of as lipids but rather as secondary metabolites in
microorganisms or plants. However, polyketides are of great significance for the virulence of
pathogenicfungi. Dihydroxynaphthaleneisamulti-polyketidecompoundinAspergillusfumigatus;
the gene encoding the responsible polyketide synthase (PKS) is essential for the synthesis of
dihydroxynaphthalene,andtheknockoutofPKSresultedinapigmentationdeficiencyintheasexual
spores.Thesepigment-freeconidiaaremoresusceptibletoengulfmentbyimmunecells[22].Therefore,
melaninisanimportantvirulencefactorinA.fumigatus[23]. Thetargetingofmelaninproductionisan
effectiveantifungalmeansforweakeningthevirulenceandpathogenicityofpathogenicfungi.
Theenergystorage,signaling,andstructuralrolesdescribedabovearethreecommonfunctions
oflipidmoleculesinalmostallorganisms. Thedisruptionoflipidmetabolismleadstothecollapse
oftheentirecellularsystem. However,whenweexploretheantifungaltargetsinpathogenicfungi,
boththeinhibitoryeffectonfungiandthedamagecausedtothehostneedtobeconsidered. Therefore,
wesummarizespecificmetabolicpathwayswithinfungithatdifferfromthoseinmammaliancells
andthushavethepotentialtobeantifungaldrugtargets. Thestudyoflipidfunctionineukaryotic
cellsinstructsusintheexplorationofeffectivemechanismsforfightingpathogenicfungi.
3. SpecificLipidMetabolisminFungi
3.1. MetabolismofErgosterolinFungi
The most important sterols in fungal and mammalian cells are ergosterol and cholesterol,
respectively. Inhibitingthesynthesisofergosterolinfunginotonlyresultsinthestructuraldisruption
of the fungal cell membrane but also interferes with the functional organization of lipid rafts.
The interaction of lipid rafts with proteins in a distinct biophysical environment may change the
raft-localized proteins’ conformation and bioactivity—Pma1 in C. neoforman, for example. It can
therebycausegreatdamagetofungalcellswithminimaleffectsonhumancells. Theinhibitionof
ergosterolsynthesisisthemodeofactionofmanyantifungaldrugs,suchastheazoles,theallylamines,
and the morpholines. As shown in Figure 2, different drugs interfere with the specific process of
ergosterolbiosynthesisinSaccharomycetes[24]. Theazoleshaveaninhibitoryeffectoncytochrome
P450-dependent lanosterol 14α-demethylase, the allylaminesand thiocarbamates inhibitsqualene
epoxidase,andamorolfinandothermorpholinedrugshaveinhibitoryeffectsonsterol∆14-reductase
and sterol ∆8,∆7-isomerase. Distinct from these, the polyene macrolides, such as amphotericin B,
areabletobindergosterolinthefungalcellmembrane,therebyforminghydrophilicchannelsthat
changethepermeabilityofthecellmembraneandresultingintheoutflowofintracellularcontents[25].
Thesedrugsareclinicallyimportantinhibitorsforthetreatmentofinfectioninducedbypathogenic
fungi. However,thedevelopmentanduseoftheseantifungaldrugsareaccompaniedbyseveralissues:
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(i)toxicsideeffectstothehumanbodyareeasilyproduced;forexample,amphotericinBinhibitsfungi
effectivelywithabroad-spectrumantifungaleffectbutismoretoxictotheliverandkidneysinhumans
thanareotherdrugs[26];(ii)pathogenseasilybecometoleranttodrugs,especiallytheazoles,which
justcausetemporarydamagetopathogenicfungibutarenotabletokillthemdirectly,allowingthe
pathogenstoreboundeasilyanddevelopantifungalresistance;and(iii)someofthesedrugscannotbe
takenorally: theuseoftheechinocandinsdoesnotreadilyresultinfungalresistance,andthesedrugs
arelesstoxictopatients,buttheymustbeadministeredintravenously[1]. Theemergenceofliposomal
formulationsofamphotericinBandcombinedantifungaltherapyisdestinedtosolvetheselimitations
andexpandtheclinicalapplicationsofthesedrugs[27–30].
Figure2. Antifungalinterferencewithergosterolbiosynthesis. Thisfigureisadaptedandmodified
fromRef.[24].HMG-CoA,β-Hydroxy-β-methylglutaryl-CoA.
3.2. MetabolismofPhospholipidsinFungi
Phospholipids, which are the most abundant lipids in cells, comprise 40–60% of lipids in
eukaryotic cells [6]. The metabolism of phospholipids in cells is very complicated, and there are
substantialdifferencesinthemetabolicpathwaysinfungalandmammaliancells. Itisnecessaryto
clarifythemetabolicnetworkofphospholipidsinfungalcells(Figure3). ThenumbersinFigure3
representthecatalyticenzymesineachreaction. DetailsoftheenzymesarelistedinTable2.
TheanabolismofphospholipidmoleculesbeginswithPA.ThedenovosynthesisofPAisviathe
two-stepacylationofglycerol3-phosphate(G-3-P)ordihydroxyacetonephosphate(DHAP)((cid:13)1 and
(cid:13)2 ). CDP-DAG(Cytidinediphosphate-diacylglycerol)synthasecatalyzesthereactionbetweenthe
substratesPAandcytidinetriphosphate(CTP)thatsynthesizesCDP-DAG((cid:13)5 ),whichisthedonor
ofthephosphatidylgroup. Undertheactionofthecorrespondingenzymes,inositol(Ins),glycerol
3-phosphate,andserineactasheadgroupsinthegenerationofPI((cid:13)6 ),phosphatidylglycerolphosphate
(PGP)((cid:13)7 ),andPS((cid:13)10),respectively. PGPisdephosphorylatedbytheactionofphosphatasetoform
phosphatidylglycerol(PG),andPGiscondensedwithamoleculeofCDP-DAGtoformcardiolipin
(CL) ((cid:13)9 ). In S. cerevisiae, PS is an important substrate for PC synthesis. PS is converted to PE by
phosphatidylserinedecarboxylase((cid:13)11)andthensynthesizedthroughathree-stepmethylationreaction
thatisdependentonS-adenosylmethionine. Thefirststepinthemethylationreactioniscatalyzed
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byphosphatidylethanolaminemethyltransferase((cid:13)12),encodedbyCHO2,andthelattertwostepsare
catalyzedbytheOPI3-encodedphospholipidmethyltransferase((cid:13)13);thispathwayistheendogenous
pathwayforthesynthesisofPCdrivenbymethylationreactionsandisthemainsourceofPCinS.
cerevisiae. AnalternativepathwayforPCsynthesisistheKennedypathway,inwhichexogenously
obtainedethanolamine(Etn)orcholine(Cho)arephosphorylatedtogeneratephosphoethanolamine
(P-Etn)((cid:13)16)orphosphocholine(P-Cho)((cid:13)19),respectively,undertheactionofthecorrespondingkinases.
P-Etn or P-Cho binds to a single molecule of CTP to generate cytidine diphosphate ethanolamine
(CDP-Etn)((cid:13)17)orcytidinediphosphocholine(CDP-Cho)((cid:13)20),respectively;thesereactionsarecatalyzed
byacytidylyltransferase,andCDP-EtnandCDP-ChofinallyreactwithDAGtoformPE((cid:13)18)orPC
((cid:13)21),respectively. Inthereversereaction,PCcanbedecomposedintoDAGandPAundercatalysisby
phospholipaseC(PLC)((cid:13)22)andphospholipaseD(PLD)((cid:13)23),respectively[31].
Figure3. MetabolicnetworkofphospholipidsinS.cerevisiae. G-3-P,Glycerol-3-phosphate;DHAP,
Dihydroxyacetonephosphate;PA,Phosphatidicacid;Lyso-PA,Lyso-Phosphatidicacid;Acyl-DHAP,
Acyl-Dihydroxyacetonephosphate;CDP-DAG,Cytidinediphosphate-diacylglycerol;PI,Phosphatidyl
inositol;Ins,Inositol;PGP,Phosphatidylglycerolphosphate;PG,Phosphatidylglycerol;CL,Cardiolipin;
PS,Phosphatidylserine;PE,Phosphatidylethanolamine;PME,Phosphatidylmonomethylaminoethanol;
PDE, Phosphatidyl dimethylaminoethanol; PC, Phosphatidylcholine; DAG, Diacylglycerol; Etn,
Ethanolamine;P-Etn,Phospho-ethanolamine;CDP-Etn,Cytidinediphosphate-ethanolamine;Cho,
Choline;P-Cho,Phospho-choline;CDP-Cho,Cytidinediphosphate-choline.
PS synthase ((cid:13)10), PS decarboxylase ((cid:13)11), and phospholipid methylase ((cid:13)13), shown with bold
arrowsinFigure3,havebeenreportedintheliteratureaspotentialantifungaltargets[32]. TheCandida
albicansPSsynthasemutant,∆cho1,lackingPS,hasadecreasedPEcontentandisavirulentinamouse
modelofsystemiccandidiasis. The∆cho1mutantexhibitsdefectsincellwallintegrity,mitochondrial
function,andfilamentousgrowth. Inaddition,Cho1doesnothaveahomologinmammaliancells;
therefore,theinhibitionofCho1activityislikelytobecomeanewbasisforantifungaltherapy. Cho1
isconservedinfungi,anditsinhibitorsmayhaveabroad-spectrumantifungaleffect[33–35]. PSisa
substrateforthedenovosynthesisofPE.TheC.albicansPSdecarboxylasecannotbeexpressedatall
whenbothpsd1andpsd2areknockedout.Theresultingphenotypeissimilartothatresultingfromcho1
deletion,includingreducedPEcontentandcellwalldefects,butthedecreaseinvirulenceresulting
from the former mutation is not as significant as that resulting from the latter [34]. In Aspergillus
nidulans,thelossofchoC(opi3inyeast)resultsinseverelyimpairedvegetativegrowth,swellingof
thehyphaltips,andthecompleteblockageofasexualandsexualdevelopment. Thisenzymealsohas
nohomologousproteininthehumanbody. Therefore,ChoChasapotentialtobeadrugtargetin
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thetreatmentofaspergillosis,althoughthealveolarsurfacemayprovideanalternativesubstratefor
thesynthesisofphospholipidsbypathogenicfungi[35]. Astudyintheentomopathogenicfungus
MetarhiziumrobertsiifoundthatthedeficiencyofPEmethylase(12)reducedthePCcontentofcells
andimpairedtheproductionofconidiabuthadnosignificanteffectonvirulence. Thereasonthat
mostenzymesrelatedtothemethylationreactionsinthesynthesisofPCcanbepotentialantifungal
targetsisthattheendogenouspathwayisessentialforthesynthesisofPCinfungalcells,whilePCis
mainlysynthesizedviatheKennedypathwayinthecellsofhighereukaryotes. Inhumans,only30%
ofPCissynthesizedviathemethylationpathwayinlivercells,amuchhigherpercentagethaninother
organs[8,36,37].
Table2.EnzymescatalyzingphospholipidsmetabolisminS.cerevisiae.
Number Enzyme Abbreviation
1 G-3-Pacyltransferase SCT1
2 G-3-Pacyltransferase GPT2
3 1-acyl-DHAPreductase AYR1
4 Lyso-phospholipidacyltransferase SLC1/ALE1
5 CDP-DAGsynthase CDS1
6 PIsynthase PIS1
7 PGPsynthase PGS1
8 PGPphosphatase GEP4
9 CLsynthase CRD1
10 PSsynthase CHO1
11 PSdecarboxylase1/2 PSD1/PSD2
12 PEmethylase CHO2
13 Phospholipidsmethylase OPI3
14 PAphosphatase PAH1
15 DAGkinase DGK1
16 Etnkinase EKI1
17 P-Etncytidylyltransferase ECT1
18 Etnphosphotransferase EPT1
19 Cholinekinase CKI1
20 P-chocytidylyltransferase PCT1
21 Cholinephosphotransferase CPT1
22 PhospholipaseC PLC
23 PhospholipaseD SPO14
3.3. MetabolismofSphingolipidsinFungi
Sphingolipids,whichareimportantcomponentsofcellmembranesandactassignalingmolecules
incells,areacomplexfamilyofcompoundsthatshareacommonstructuralfeature,abackboneof
sphingoidbasesthatissynthesizeddenovofromserineandalong-chainfattyacyl-CoA.Condensation
withfattyacidsviaamidebondsand/orhydroxylationatpositionC1ofthesphingoidbasegives
risetothecomplexityanddiversityofsphingolipidmolecules. Figure4showsthedenovosynthesis
ofsphingolipidsinS.cerevisiae[34]. Thesynthesisofsphingolipidsbeginswithareactionbetween
palmitoyl-CoAandserine;thisreactioniscatalyzedbyserinepalmitoyltransferase(SPT)andgenerates
3-ketodihydrosphingosine,whichisreducedtodihydrosphingosineinanNADPH-dependentmanner.
Dihydrosphingosineisconvertedtophytosphingosinebytheactionofhydroxylaseandceramide
synthase,andthenphytoceramidebindsamoleculeofPItosynthesizeinositolphosphatyl-ceramide
(IPC) through a reaction catalyzed by IPC synthase. IPC can either be further mannosylated or
condensedwithPItogeneratethemorecomplexsphingolipidsMIPCandM(IP) C,respectively.
2
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Figure4. DenovosynthesisofsphingolipidsinS.cerevisiae. Thisfigureisadaptedandmodified
from Ref. [38]. IPC, inositolphosphatyl-ceramide; MIPC, mannose inositol-P-ceramide; M(IP) C,
2
mannose-(inositol-P)2-ceramide.
Mostenzymesrequiredforthedenovosynthesisofsphingolipidshavehomologousproteinsin
mammaliancells;therefore,theseenzymescannotbeusedastargetsforantifungaldrugsbecauseof
theirpoorselectivity. Surprisingly,IPCsynthaseisaspecificcatalyticenzymeinfungi,whichisnot
presentinthehumanbody;therefore,ithaspotentialtobecomeanantifungaltarget[38]. Furthermore,
some antifungals have already been reported to effectively inhibit pathogenic fungi. Rustimicin
48andgalbonolideBhaveaninhibitoryeffectontheIPCsynthaseofCryptococcusneoformans[39].
Khafrefungin 50 has exhibited different inhibitory effects on sphingolipid synthesis in C. albicans,
S.cerevisiae,andC.neoformans. TheseinhibitorsofIPCsynthaseshowastrongantifungaleffectand
excellentselectivity,buttheyarenotactiveagainstA.fumigatus;thus,thestructuralremodulationof
theseinhibitorsmaybroadentheirantifungalspectrum[40].
3.4. MetabolismofFattyAcidsinFungi
Fatty acids, which act as building blocks for complex lipids such as phospholipids and
sphingolipids, as well as energy reservoirs and signaling molecules, are the most fundamental
lipidmoleculesincells. Fattyacidmoleculeshaveacommonbasicstructurewithspecificdiversity
determinedbythechainlengthandthedegreeofunsaturation,andFAsarerapidlymetabolized[4].
Inyeastcells,acetyl-CoA,derivedeitherfromcitricaciddegradationorfromacetate,iscarboxylated
intomalonyl-CoAundertheactionofacetyl-CoAcarboxylase;thisreactionisthefirststepinthede
novosynthesisoffattyacidsandoccursinthecytoplasmorinmitochondria. Theenzymecatalyzing
thecarboxylationreactioninthecytoplasmisencodedbyACC1,whereastheacetyl-CoAcarboxylase
inmitochondriaisencodedbyHFA1. FAsynthesisiscarriedoutinthecytosolbythehexamericfatty
acidsynthase(FAS)complexconsistingofsixFas1pandsixFas2psubunits,eachofwhichexhibit
morethanoneenzymaticfunction. Incontrast,themitochondrialFAsynthasecomplexconsistsof
sixproteins,eachhavingonlyoneenzymaticactivity. BoththecytosolicandthemitochondrialFAS
machineriesaddtwocarbonsfrommalonyl-CoAtoasaturatedacylchain. Whilethemitochondrial
FASsystemprimarilyproduceslipoicacid,achainlengthofupto18carbonscanbeproducedbythe
cytosolicFAsynthase. FurtherelongationtoformverylongchainFA(VLCFA)ofupto26carbonsin
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lengthbythestepwiseadditionoftwocarbonsfrommalonyl-CoAiscarriedoutintheendoplasmic
reticulum(ER)bydifferentelongases. DesaturationandhydroxylationofFAalsooccurintheER[9].
ThefattyacidsynthaseoftheBasidiomyceteyeastC.neoformansiscomposedoftwosubunits
encoded by the FAS1 and FAS2 genes. The suppression of Fas1 and Fas2 expression blocks the
denovosynthesisoffattyacidsandseverelyinhibitsvegetativegrowthofC.neoformans. However,
supplementationwithexogenousfattyacidswasnotabletorescuecelldeathinducedbyFASdeficiency.
ThetreatmentofC.neoformanswithfluconazolewasshowntohaveanincreasedinhibitoryactivity
andtoevenbecomefungicidalwhenexpressionofeitherFAS1orFAS2wassuppressed. Inaddition,
Fas1andFas2aredifferentfromthefattyacidsynthaseinthehumanbodyandthusmaybepotential
antifungaltargetproteins[41].
3.5. MetabolismofNeutralLipidsinFungi
NeutrallipidsinfungiprimarilyincludeTAGsandSTEs,whichareresponsibleforthestorageof
freefattyacids,sterols,andDAG.DAGoriginatingfromthebreakdownofPAservesastheprimary
backboneforthesynthesisofTAG.ThelaststepofdenovoTAGformationinyeastcanbeaccomplished
throughdifferentroutes. Lro1p,ahomologofthehumanlecithin-cholesterolacyltransferase(LCAT),
catalyzes the transfer of an acyl group from the sn-2 position of PE or PC; this activity appears to
be restricted to the ER. In an acyl-CoA-dependent reaction, the activated fatty acid can be linked
to the glycerol backbone of DAG by Dga1p, which is localized to the ER and to LDs [42]. Are1p
andAre2p,locatedintheER,alsohaveaminorroleintheconversionofDAGtoTAG,whichalso
dependsonacetyl-CoA.Thesetwoisomeraseshave16%and17%homology,respectively,withhuman
acetyl-CoA:cholesterolacyltransferase(ACAT).Are1pandAre2paretwoessentialSTEsynthasesin
S.cerevisiae. Lipidanalysisof∆are1and∆are2deletionstrainsrevealedthatAre2pandAre1putilize
sterol substrates invivo with different efficiency; Are2p has a significant preference for ergosterol
asasubstrate, whereasAre1pesterifiessterolprecursors(mainlylanosterol, aswellasergosterol).
Nevertheless,terbinafine,aninhibitorofergosterolbiosynthesis,inhibitsgrowthof∆are1∆are2cells
moreefficientlythangrowthofwild-typecells. Inagrowthcompetitionexperiment,∆are1∆are2cells
grow more slowly than wild-type cells after several rounds of cultivation, suggesting that Are1p
and Are2p or STEs, the product formed by these two enzymes, are more important in the natural
environmentthanunderlaboratoryconditions[43].
Itisnotcertainwhetheranyproteininvolvedinthesynthesisofneutrallipidshaspotentialto
beanantifungaltarget. However,therelationshipbetweenautophagyandTAGhasalreadybeen
reportedinMetarhiziumrobertsii. IthasbeenshownthatnitrogenstarvationisabletoinduceTAG
accumulation in cells and activate autophagic activity but inhibits the internalization of LDs into
vacuolesfordegradation. ProteomicanalysisofLDsidentifiedanarrayofdifferentiallyaccumulated
proteins,includingautophagy-related(ATG)proteins,heatshockproteins,andenzymesinvolvedin
TAGmetabolismandphospholipidbiosynthesis,inresponsetofungalgrowthunderdifferentnutrient
conditions. ThissuggeststhatTAGmetabolismandcellularnitrogenstarvationaretightlyrelated;
however,theresponsiblemechanismisnotyetclear. Furtherresearchinthisfieldmightprovidenew
insightsintotheexplorationofantifungaltargets[44].
4. SearchingforPotentialAntifungalDrugTargets
Thedevelopmentofmodernbiotechnology,high-throughputsequencing,andchromatography
and mass spectrometry for the separation and identification of biomolecules, as well as the
cross-convergenceofmultiomics, haveenablednewinsightsinthesearchforpotentialantifungal
drugtargets.
In2016,Martin,Ketal. combinedthreetechniquesforthediscoveryofanti-A.fumigatustargets:
(i) modeling metabolic networks by elementary mode analysis and flux estimate approximations
incorporating expression data from biochemical databank KEGG (Kyoto Encyclopedia of Genes
and Genomes) and Metacyc [45–47]; (ii) targeting metabolic genes by transcriptome analysis to
J.Fungi2018,4,50 10of13
identifycondition-specifichighlyexpressedenzymes[48–50];and(iii)analyzingenzymestructureand
interconnectedness(“hubs”)andidentifyingpathogen-specificenzymesusingorthologyrelationships.
Sixty-fourtargetcandidateswerefinallyobtained. Thepotentialantifungaltargetproteinsassociated
with lipid metabolism that have been reported in the literature are listed in Table 3. The first five
genes/proteins are responsible for PC synthesis via the methylation pathway and the fatty acid
synthesispathway.
Pho88(AFUA_5G01960)isaninorganicphosphatetransporterthatisinvolvedinphospholipid
synthesis in A. nidulans, A. fumigatus, and other Aspergillus species [51]. The homologous gene of
AFUA_3G12320inyeastisLpl1,whichencodesaphospholipaseB;Lpl1isessentialforthemorphology
of lipid droplets in yeast cells but is dispensable for cell survival because of the presence of other
phospholipaseBenzymes. However,fromaclinicalpointofview,aphospholipaseBblockerwould
beconsideredaninterestingdrug,becausesuchadrugwouldprobablyblockallphospholipaseB
activityingeneral,resultinginlethality[52]. InS.cerevisiae,Vtc4pisrequiredduringthefusionof
inorganicphosphate-containingvesiclestothevacuolarmembraneandtheconsequentaccumulation
ofphosphatestoredaspolyphosphate(polyP)inthevacuole. InthemaizepathogenU.maydis,∆vtc4
mutants were found to have reduced virulence in maize seedlings [53]; however, the loss of Vtc4
inCandidablocksthesynthesisofpolyPanddoesnotleadtoanenhancedsensitivitytooxidative
stress[54]. Therefore,furtherstudiesareneededtodeterminewhetherthisproteincanbeconsidered
anantifungaldrugtarget.
Table3.Antifungaltargetproteinsrelatedtolipidmetabolism.
Gene Annotation FungiTested* Reference
AFUA_4G13680 Phosphatidylserinesynthase C.albicans [33]
AFUA_2G15970 PhosphatidylethanolamineN-methyltransferase S.cersvisiaeetal. [55]
AFUA_1G09050 Methylene-fatty-acyl-phospholipidsynthase A.nidulans [35]
AFUA_3G04210 FattyacidsynthaseαsubunitFasA C.neoformans [41]
C.neoformans
AFUA_3G04220 FattyacidsynthaseβsubunitFasB [41,56]
C.parapsilosis
A.nidulans
AFUA_5G01960 PhosphatetransporterPho88 [51]
A.fumigatus
AFUA_3G12320 Lipase/Serineesterase S.cerevisiae [57]
Ustilagomaydis
AFUA_2G09040 Vacuolartransporterchaperone(Vtc4) [53,54]
Candida
*“Fungitested”meansthatfungilistedherearealreadywelldescribedinliteratureandtheresearchershave
publishedevidencesupportingthemaspromisingdrugtargets.
5. ConclusionsandOutlook
In this review, we summarize fungal lipid metabolic pathways in comparison with those of
humans,anddiscussthespecificitiesonwhichtheantifungalmechanismsofthecurrentlyexisting
andyet-to-be-developedfungalinhibitorsarebased. Theincreasednumberofimmunocompromised
patientsasaresultofHIVinfectionororgantransplantationshasattractedmoreandmoreattention
tostudiesinthisfield,especiallyinrelationtothevirulenceandpathogenicityofpathogenicfungi
includingC.neoformans,C.albicans,andA.fumigatus;suchinvestigationscreateanewvisionforthe
treatmentoffungalinfections. Multiomicdataarebecomingincreasinglyusefulanddefinitewith
theprogressofbiotechnologyandtheaidofcomputertechnologiesincludingtheMetatoolprogram,
YANA software package Borland JBuilder 2005, R statistics software, and so on; these advances
providemoreaccurateandfastertechnicalmethodsfortheexplorationofnewantifungaltargetsand
areexpectedtodriveantifungalresearch.