The metabolic pathways and transporters of the plastid organelle in Apicomplexa

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Reference

The metabolic pathways and transporters of the plastid organelle in Apicomplexa

KLOEHN, Joachim, LACOUR, Clément, SOLDATI-FAVRE, Dominique

KLOEHN, Joachim, LACOUR, Clément, SOLDATI-FAVRE, Dominique. The metabolic pathways and transporters of the plastid organelle in Apicomplexa. Current Opinion in Microbiology , 2021, vol. 63, p. 250-258

DOI : 10.1016/j.mib.2021.07.016 PMID : 34455306

Available at:

http://archive-ouverte.unige.ch/unige:154385

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The metabolic pathways and transporters of the plastid organelle in Apicomplexa

Joachim Kloehn, Cle´ment EM Lacour and Dominique Soldati-Favre

Theapicoplastistherelictofaplastidorganellefoundinseveral disease-causingapicomplexanparasitessuchasPlasmodium spp.andToxoplasmagondii.Intheseorganisms,theorganelle haslostitsphotosyntheticcapabilitybutharboursseveral fitness-conferringoressentialmetabolicpathways.Although maintainingtheapicoplastandfuellingthemetabolicpathways withinrequiresthechallengingconstantimportandexportof numerousmetabolitesacrossitsfourmembranes,onlyfew apicoplasttransportershavebeenidentifiedtodate,mostof whichareorphantransporters.Herewereviewtherolesof metabolicpathwayswithintheapicoplastandwhatiscurrently knownaboutthefewidentifiedapicoplastmetabolite transporters.Wediscusswhatmetabolitesmustgetinandout oftheapicoplast,themanytransportersthatareyettobe discovered,andwhatrolethesemightplayinparasite metabolismandasputativedrugtargets.

Address

DepartmentofMicrobiologyandMolecularMedicine,Universityof Geneva,CMU,RueMichel-Servet1,1211Geneva,Switzerland Correspondingauthors:Kloehn,Joachim(Joachim.Kloehn@unige.ch), Soldati-Favre,Dominique(Dominique.Soldati-Favre@unige.ch)

CurrentOpinioninMicrobiology2021,64:250–258

ThisreviewcomesfromathemedissueonHost-microbeinterac- tions:parasites

EditedbyBarbaraBurleighandMalcolmMcConville

https://doi.org/10.1016/j.mib.2021.07.016

1369-5274/ã2021TheAuthor(s).PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons.

org/licenses/by/4.0/).

Introduction

Apicomplexansformalargephylumthatcomprisesthou- sands of single-celled eukaryotic organisms, including Plasmodium spp. and Toxoplasma gondii, the causative agentsofmalariaandtoxoplasmosis,respectively.These obligateintracellularparasitesresideinsidethehostcell cytosol surrounded by a parasitophorousvacuole membrane(PVM) thatisformedthroughinvaginationofthe hostcellmembraneduringinvasion [1].Tosurviveand replicate, these parasites must acquire various metabo- litesfromthehost cell, whichis facilitatedthroughthe recruitment of host cell organelles to the PV [2]. The

PVMfunctionsasamolecularsieveandisheavilymodi- fied bythe parasite, enabling theexchange of proteins andmetabolites[3–6].Metabolitessynthesisedortaken upbytheparasitemustthenbedistributedbetweenthe metaboliccompartmentsincludingthecytosol,endoplasmicreticulum(ER),Golgiapparatus,andmitochondrion.

Most of theapicomplexans possess anadditional meta- bolically active compartmenttermed apicoplast (plastid oftheApicomplexa)[7].Theapicoplastisarelicplastid organelle derived from secondary endosymbiosis that fulfills critical metabolic functions. It is now widely accepted thattheapicoplastoriginatesfromtheengulf- ment of a photosynthetic red alga, which previously acquiredits photosyntheticcapability fromthe acquisi- tion of a cyanobacterium [7,8]. While secondary endosymbiosis events occurred independently in several eukaryoticlineages[9],theacquisitionofaredalgalikely gave rise to the common ancestor of dinoflagellates, heterokonts and apicomplexans [8]. Several apicoplast features, including its four membranes and its small circulargenome,arecleartracesindicativeofits(secondary) endosymbiotic origin [10]. A non-photosynthetic vestigial plastid canbe found in several apicomplexans including important pathogens of animal stock such as Eimeriatenella,Babesiabovis,andTheileriaannulataaswell as the human pathogensPlasmodium spp. and T.gondii [11]. Strikingly, the functions of the organelle have diminishedthroughoutevolution,highlightedbytheloss ofphotosynthesis,thereductionofitsgenomeandlossof the organelle altogether in some alveolates including gregarines,colpodellidsandCryptosporidiumspp.[12].

Sinceitsdiscoveryabout25yearsago[13,14],theapicoplast has received considerable attention as a putative drugtargetto battlethedevastating diseases causedby Plasmodium spp. and T. gondii with measurable success [15–17]. Intense research efforts have significantly advancedourunderstanding of theorganelle: itsevolu- tionaryoriginhas beendeterminedand itsgenomehas beensequenced[8,14].Themetabolicpathwayswithinit havebeenlargelydefinedandenzymesofeachpathway havebeentargetedtoprobetheirimportanceinT.gondii aswellasPlasmodiumspp.(reviewedinRefs.[10,18,19]).

Wehave gained insights into apicoplast protein import (reviewedinRef.[20])and,crucially,severaldrugshave beenidentifiedto targetprocesseswithintheapicoplast [15,17,21].Someofthesedrugs,suchasclindamycinand doxycycline, play a vital role in today’s treatment and prophylaxisofmalariaandtoxoplasmosis.Drugstargeting

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the apicoplast typically kill the parasites in the second cyclefollowing treatment,aphenomenonreferredtoas

‘delayed death’(reviewed inRef. [16]).Whilethe slow killingmodeisanundesirablefeatureanddoesnotmeet the criteria for new antimalarials, recent studies have highlighted that known apicoplast drugs can exhibit fast-killing modes, presumablydue to off-target effects athigherconcentrationsorfollowingchemicalmodifica- tion [22,23]. Furthermore, inhibition of the apicoplast- resident non-mevalonate methylerythritol phosphate (MEP)pathwaythroughfosmidomycin[17],andinhibi- tionof proteinimportcauserapiddeathoftheparasites [24],rekindlinginterestintheapicoplastasadrugtarget.

Revisiting therole ofmetabolicpathways harboured within theapicoplast

Theapicoplasthousesseveralmetabolicpathways,whose importance differs between the apicomplexan species T. gondii and Plasmodium spp. as well as between life cyclestagesoftheseparasites.Thepartialhemesynthesis pathwaywithintheapicoplast,thetypeIIfattyacid(FA) synthesis pathway,lysophosphatidicacid(LPA) synthesis,andtheMEPpathwayforthesynthesisofisopentenyl pyrophosphate(IPP) synthesispathwaysprovidecrucial metabolites which are needed throughout the cell, primarily serving the mitochondrion, cytosol, and ER.

Intriguingly,theapicoplastisfoundinverycloseassocia- tion with the mitochondrion and the ER, potentially facilitating the diffusion of lipids between these organelles andenablingdirectexchangeof metabolites(see Figure 1) [25,26]. FA and LPA synthesis inside the apicoplastprovidethesubstratesforbulklipidsynthesis attheER,whileshortchainisoprenoidsproducedinside theapicoplastserveasprecursorsforubiquinonesynthe- sis, atwo-electroncarrierinthemitochondrion,for dolichol synthesis, required for N-glycosylation at theER, andforproteinprenylationandphytoenesynthesisinthe cytosol. Phytoenes are precursors of carotenoids which function as antioxidants and have been detected in extracts of Plasmodium infected red blood cells [27].

Whether carotenoids are also synthesised in T. gondii remains unclear, but T. gondii does exhibit sensitivity to carotenoid biosynthesis inhibitors and was shownto synthesise abscisic acid,aplant hormone, derived from b-carotene[28].

Itisnowclearlyestablishedthatproductionoftheshort isoprenoid IPP isthesole essentialfunction ofthe api- coplastinintraerythrocyticPlasmodiumspp.,allowingthe survivalandreplicationofparasiteswithoutanapicoplast in thepresence ofsufficient exogeneousIPP[29].This ground-breaking findingrevealed that theapicoplast in principleisnotrequiredifoneorseveralcriticalmetabo- lite(s)producedwithinaremadeavailabletotheparasite.

Lack of IPP was shown to be detrimental by causing defects in protein prenylation, causing disruption of intracellulartrafficking[30].Incontrastto bloodstage

parasites, mosquito and liver stage Plasmodium also criticallydependontheFAandhemesynthesispathways [31–34].

Contrastingly, T. gondii tachyzoites, the fast-replicating parasitesresponsiblefortheacutestageoftoxoplasmosis, areconsideredtobereliantonseveralapicoplast-resident pathways. However recent studies have highlighted a remarkable capacity to circumvent apicoplast-resident pathways throughincreased salvage. Defectsin the FA synthesis pathway and the synthesis of LPA can be overcomethroughincreasedsalvageofexogenouslysup- plemented FAs and LPA, respectively [35,36,37].

Likewise, disruption of the cytosolic synthesis of long chain isoprenoidssuchasfarnesylpyrophosphate(FPP) and geranylgeranyl pyrophosphate (GGPP), which are formed from the apicoplast-generated precursor IPP, canbecompensatedforthroughsalvageoftheseisopre- noids from the host [38]. Finally, defects in the heme synthesis pathway upstreamof the mitochondrial ferro- chelatase can potentially be partially compensated for throughsalvageoftheprecursorprotoporphyrinIXfrom thehostandrewiringofenergymetabolism[36].Thus, all fourknownpathwayswhich generatecriticalmetab- olites within the apicoplast to supply the cell can be compensated for through salvage of supplemented exogenous metabolites, allowing T. gondii to function theoretically withoutanapicoplast.However,cultureof apicoplast-deficient T. gondii has not been reported to date.

Incontrastto thesepathways,thepartialcentralcarbon metabolism within the apicoplast, thelipoate synthesis pathway andthesulfur mobilization (SUF)pathwayfor iron–sulphur cluster ([Fe-S]) generation presumably functionsolelytosustaintheapicoplastitself.Theobser- vationthatthemitochondrial [Fe-S](ISC)pathwayand thecytosolic[Fe-S]assemblymachinery(CIA)pathways areeachessentialinT.gondii,providesevidencethat[Fe- S]clustersarenotexportedfromtheapicoplasttosupply othercompartments[39,40].Similarly,disruptionofthe ISCandCIApathways,impactonlytherespectivecom- partment,indicatingfurtherthatthesepathwaysfunction independently [39,40]. The only known substrate of lipoylationwithintheapicoplastisthepyruvatedehydro- genase(PDH)functioninginFAsynthesis.Basedonthe role of FASII in T. gondii tachyzoites and blood stage Plasmodium, lipoate synthesis within the apicoplast is expected to be fitness-conferring and dispensable in these parasites, respectively. Indeed, a recent study which expressed a lipoamidaseremoving lipoate inside theapicoplastofP.falciparumcausednodefectalthough, arguably the de-lipoylation was potentially incomplete [41]. The need for lipoate inside the mitochondrion is instead met by salvage of hostlipoate in T. gondii and Plasmodium spp.[42].Finally,thepartial centralcarbon metabolism in the apicoplast, including glycolytic and

MetabolitetransportersoftheapicoplastKloehn,LacourandSoldati-Favre 251

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tricarboxylic acid (TCA) cycle enzymes, presumably serves to provide reducing equivalents and nucleotides requiredtosupplyotherpathwaysandapicoplasthouse- keeping[43,44].

Few old andsome new apicoplast transporters

Incontrastto this relativelydetailedcharacterization of themetabolicpathwayswithintheapicoplast,amajorgap inourunderstandingofapicoplastbiologyistheidentifi- cation and characterisation of transporters that shuttle metabolitesacrossitsfourmembranes.Thesemustsup- ply the metabolic pathways, as well as fuel apicoplast housekeeping(replication,transcription, translation,ion homeostasis,redox metabolism)and exportmetabolites produced within the apicoplast. To date, only 7 Plasmodiumand2 T.gondiiputativeapicoplast transporters have been identified [45,46,47,48]. Out of these, only4havebeenassignedasubstrate.Amongstthese,the beststudiedapicoplasttransportersaretheTPTs,which wereidentifiedshortlyaftertheP.falciparumgenomewas sequencedbasedontheirhomologytotheircounterparts inplantchloroplasts[48].TPTstraffictriosephosphates, 3-phosphoglycerate and phosphoenolpyruvate (PEP), providing carbons for the synthesis of FAs, LPA and IPP.Plasmodium spp.encodetwoTPTs, whichlocalise to the inner (PfiTPT) and outer apicoplast membrane (PfoTPT), whereas T. gondii encodes a single TPT (TgAPT),whichpresumably localisesto multiple mem- branesof theorganelle [43].

WhilethefunctionoftheTPTsisrelativelywellunder- stoodandtheirsubstrateshavebeenbiochemically validated [46], these transporters still hold some secrets.

Surprisingly,depletionof TgAPTwasreported tocause rapiddeathin T.gondiianddidnotresultinloss ofthe organelle[46].Thisindicatesthattheparasites’deathis notprimarilydue totheinhibition ofFA synthesis,but rather due to disruption of the isoprenoid precursor synthesispathway.Indeed,geneticdisruptionofisopren- oidsynthesisas wellas pharmacologicalinhibitionof 1- deoxy-D-xylulose-5-phosphate reductoisomerase (DOX- PRI)through fosmidomycin havebeen shown to cause rapiddeathandbothdonotresultinlossoftheapicoplast [17,49].DefectsinFAandLPAsynthesisincontrast,are associatedwithlossoftheorganelleanddelayeddeathin T.gondii[35,36,37].Althoughwell-establishedthrough theessentialityofDOXPRI[49],theindispensabilityof theIPPpathwayisnotnecessarilyexpectedgiventhatT.

gondii can efficiently salvage medium and long chain isoprenoids[38].TheIPPisomerdimethylallylpyrophos- phate (DMAPP) is required within the apicoplast to produce methylthio-dimethylallyl tRNA. The known pathwaysin thecytosol, however,suchas dolichol synthesis, protein prenylation and ubiquinone synthesis depend primarily onFPP and GGPP [16], which have beenshowntobesuccessfullysalvaged[38].Presumably,

T.gondii isunable to salvageshorter intermediates and dependscriticallyontheseortheparasitemayinfactbe able to overcome the loss of TgAPT when cultured in freshhostcells,aprerequisiteforisoprenoidsalvage[38], andprovidedwithsufficientlevelsofexogenousFAsand LPA[36,37].

Besides the TPTs, a folate transporter was recently identified in the Plasmodium berghei apicoplast [47].

Twofolatetransportershavepreviouslybeen described insidethechloroplastmembranesofplants[50,51].Folate isacrucialcofactorrequiredforone-carbonmetabolism.

Plasmodiumaswellas T.gondiiencode2 putativefolate transporters.InPlasmodium,thesetransporterswerepre- viouslylocalisedto theplasma membraneusingantibo- dies[52].Subsequentstudies,however,revealedthatthe folateprecursor4-aminobenzoicacid (pABA)canfreely traverse the plasma membrane [53] and tagging of the endogenousfolatetransporter2 revealeditslocalization to the apicoplast [47]. However, the folate-transport activityofthismajorfacilitatorsuperfamily(MFS)trans- porterwasnotformallytestedandtheauthorsobservea secondarylocalisationattheapicaltipofmerozoitesand sporozoites,indicatingapossiblelocalisationatthemem- braneofapicalorganellesortheapicalplasmamembrane [47]. Thus,furtherbiochemical studiesare needed to elucidate the transporter’s function. Whether a folate transportersimilarly localises to the T.gondii apicoplast isunclear but appears unlikely:early biochemicalanal- yses suggested localization of the transporters to the plasmamembraneandmitochondrion[54].

Tworecentseminalstudieshavefuelledthediscoveryof novel putative apicoplast transporters: Barylyuk et al.

assignedatentativesubcellularlocalizationtomorethan 2500proteinsofT.gondiibasedonhyperplexedlocaliza- tionoforganelle proteinsbyisotopetagging(hyperLO- PIT)[55].However,ofthe130proteinsassignedtothe apicoplast,only8featuredmorethanonetransmembrane domain(TMD),includinganMFSproteinwith8TMDs and the previously characterised TgAPT [46], with 6 TMDs(Table 1). By hyperLOPIT,one of theafore- mentionedfolatetransporterswaslocalisedtotheGolgi apparatus,whilenodatawasobtainedfortheother[55].

Anotherstudyusingproximitybiotinylation-basedprote- omicsandcomputationalanalysesofPlasmodiumspp.was more successful in identifying novel proteins of this organelle, revealing 346 putative apicoplast proteins, 60of whichcontaintwo ormore TMDs[56].Ofthese, 6 belong to transporter families, including 3 putative ATP-binding cassette (ABC) transporters, a putative monocarboxylate transporter as well as the two known TPTs[48](Table1).Thelocalizationofseveralofthese transporters was further validated and mutants characterised in a groundbreaking study bySayers et al. [57].

Searchesfortransportershomologoustothoseidentified inplantchloroplastshavenotyieldedsignificantmatches

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besidesidentificationoftheTPTs.Thislimitednumber of identified putative apicoplast transporters stands in sharpcontrasttothemanymetabolitesthatareexpected to beimportedintoandexportedfromtheapicoplast.

The manyunknownimporters tosustain the apicoplast andfuel itsmetabolicpathways Most apicoplasttransportersare expectedtofunctionin housekeepingbasedonthemanymetabolitesrequiredto sustainreplication,transcriptionandtranslation,including amino acids (AAs), nucleotides as well as cofactors and vitamins.Whilenucleotidetransportershavebeenidenti- fiedinthechloroplastofplants[58],nosimilartransporters have been identified in apicomplexans to date. PEP is imported into the apicoplast through the PfiTPT, the PfiTPTandtheTgAPTandisconvertedtopyruvateby thepyruvatekinase(PKII)insidetheapicoplast,generat- ing nucleotide triphosphates (NTPs) from nucleotide diphosphates (NDPs). Surprisingly, PKII was shown to be dispensable in T. gondii tachyzoites but essential for P. falciparum blood stageparasites [59,60]. Swiftet al., elegantlydemonstratedthattheessentialfunctionofPKII inbloodstagePlasmodiumisnotthegenerationofpyruvate, but rather the generation of deoxy-(d)NTPs [59]. (d)

NTPs are required as energy equivalents and building blocksfor transcriptionandreplicationwithintheorgan- elle.T.gondii,butnotPlasmodium,alsopossessesaphos- phoglyceratekinase(PGKII)insidetheapicoplast,which can potentiallycompensate for the loss of PKII. These findings haveseveralimplicationsfor transportersinside theapicoplastmembranesoftheseparasites.Presumably, the Plasmodium apicoplast harbours transporters for (d) NDPs,butnotfor(d)NTPs.Pyruvateappearstobeunable to traverse the apicoplast membranes efficiently in Plasmodium.Instead,T.gondii,potentiallyexpressesapi- coplasttransporterscapableoftransporting(d)NTPsand, inadditiontoTgAPT,maypossessapyruvatetransporter [60].BesidesthecriticalfunctionofPKIIinP.falciparum, theTIMbarrelapicoplastproteinApicoplastMinusRes- cue1(AMR1),hasbeenproposedtobeinvolvedinmain- tainingNTP balanceinsidetheorganelleand providing cytosine triphosphate (CTP) for the synthesis of IPP.

DisruptionofAMR1leadstoanincreaseofearlyisoprenoid precursors,butadecreaseofthedownstreamintermedi- ates,whichrequireCTPasacofactor[61].

As for nucleobases, (d)NDPs or (d)NTPs, it is unclear how AAs for apicoplast-resident protein synthesis are

Table1

Apicoplast transporters inPlasmodium berghei,Plasmodium falciparum andToxoplasmagondii.Two proteomicandbioinformatic studies havecontributed totheidentificationofnew putativeapicoplasttransporters inadditionto thepreviouslyidentifiedtriose phosphatetransporters(TPTs)[55,56].SeveraloftheP.bergheitransporterswerecharacterisedinaseminalstudybySayersetal.[57].

PlasmodiumtransportersconservedinT.gondiiandviceversaareshown.Ofthe10putativenewapicoplasttransportersinP.berghei andP.falciparum,onlyasingletransporter,anABCtransporterwithrelativelyweakhomology,wasshowntolocalisetotheapicoplastin T.gondiibyhyperLOPIT[55].Transportersforwhichtheapicoplastlocalisationwasconfirmedbyendogenoustaggingarehighlighted throughgreenshading.TheimportanceofeachtransporterforP.bergheifitnessaregivenandhighlightedinacolourschemefromgreen (dispensable)tored(essential).Similarly,theT.gondiifitnessscreenscoresaremarkedinaheatmapcolourschemewithpositivescores (green)indicatingdispensabilityandhighnegativescores(red)indicatingessentiality.Abbreviations:Acc.No.-accessionnumber;TMDs -transmembranedomains;PM-plasmamembrane;ND-notdetermined;NA-notapplicable.

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imported.PlantsprimarilysynthesiseAAswithinplastids andmustexportthese,aswellasimportthosesynthesised insidethemitochondriaandcytosol.OnlyoneAAtrans- porter has been identified in chloroplasts to date, a glutamate/malate translocator [62]. In contrast to the chloroplast,theapicoplasthasnoAAsynthesiscapacity.

T.gondiireliesontheuptakeofseveralessentialAAsfrom the host and synthesises few within the cytosol and mitochondrion[63].AllAAsmustthenbetraffickedfrom the cytosol into the apicoplast through unknown transporters. Given that no AA transporter has been identified to date, it appears likely that a low number ofpromiscuousAAtransportersfulfilthis task.

Besides housekeeping, a multitude of transporters is expectedtoberequiredtomaintainthemetabolicpath- wayswithintheapicoplast.Todate,only theTPTsare known transporter participating in this task. It is note- worthythat sustaining themetabolic pathways requires many additional metabolites: FA synthesis alone also relies on the availability of adenosine di/-triphosphate (ADP, ATP), guanosine diphosphate (GDP), nicotin- amide adenine dinucleotide(phosphate) (NAD⁺, NADPH),coenzymeA(CoA)/dephospho-CoA,S-adenosylmethionine (SAM), thiamine and biotin [64]. How thesecofactorsandvitamins,whichareequallyimportant astriosephosphatestosustainFAsynthesis,areimported intotheapicoplastremainsunknown.Asdiscussedabove, NADPHcanbegeneratedwithintheapicoplastthrough its partial central carbon metabolism includingthrough glyceraldehyde-3-phosphate dehydrogenase 2 (GAPDH2), PDH or the isocitrate dehydrogenase 1 (ICDH1) [43,44].However, the oxidized forms, NAD⁺ and NADP⁺, must be imported into the apicoplast throughunknownmechanisms.[44].AnNADtransporter wasinitially localisedtoplant chloroplastsbutwas later showntoresideinsidethemitochondrialmembrane[65].

Similarly,itsclosesthomologueinT.gondiilocalisestothe mitochondrionbasedonitsannotation.Thereductionof NADPtogenerateNADPHbyICDH1furtherrelieson theimport ofcitrateintotheapicoplastandpresumably onthe exportof a-ketoglutarate,which possibly enters themitochondrionthroughaknownoxoglutarate/malate translocatortocontributetotheTCAcycle[44].Atrans- porterforthecofactorSAMwaspreviouslyidentified in plantchloroplast[66],butmayalsoexhibitduallocaliza- tion to the mitochondrion and plastid [67]. Its closest homologuein T.gondii isamitochondrial carriersuper- familyproteinwhich,accordingtohyperLOPITanalysis, localises exclusively to the mitochondrion [55].

Whetherothersimilarsolute carriersalsolocalisetothe apicoplast remains unclear. Intriguingly, arecent study localised the P. falciparum dephospho-CoA (deP-CoA) kinase(DPCK), thefinal enzyme in theCoA synthesis pathway, to the apicoplast [68].CoA is critical for FA synthesis inside the apicoplast [64,69] but is equally important inside the mitochondrion for a functional

TCAcycleandinthecytosolforFAelongation,histone modificationand proteinacetylation [70].Incontrast to P.falciparum,T.gondiiDPCKlocalisestothecytoplasm (Lunghi et al., unpublished data). These findings have several implications for apicoplast transporters: in P. falciparum, an importer for deP-CoA and a CoA- exporter are required, while T. gondii is expected to possessaCoAimporterinsidetheapicoplastmembranes.

Figure1 providesanoverview overthefew knownand many unknown metabolite importers as well as the unknown exporters. The metabolites that must be imported and exported are based on the recently pub- lishedT.gondiimetabolicmodeliTgo[36],theLibrary of Apicomplexan Metabolic Pathways (LAMP) [71] as wellas thecurrentliteraturediscussed here.

Unknownexporters tosupply thecell with metabolitesgenerated insidetheapicoplast The list of predicted exporters of critical metabolites across the apicoplast membranes is short and yet none of these exporters have been identified to date. These includeacoproporphyrinogenIIItransporterrequiredto trafficthishemeintermediateintothecytosol,whereitis convertedtoprotoporphyrinogenIXwhichisthentrans- portedintothemitochondriontocompletehemesynthe- sisintwoadditionalsteps.AputativeFAexporter,which likelytrafficsmyristateandpalmitate(C14:0andC16:0) outoftheapicoplast.FAsandLPAmustbetraffickedto theERforfurtherelongation,desaturation,andsynthesis of various phospholipids [35,69]. FA exporters (FAX) have been identified in plant chloroplasts [72], but no homologues were identified in apicomplexans to date.

Similarly,anLPAexporter isexpectedtobepresent in theapicoplastmembrane.Itremainsunclear,whetherFA andLPAtraffickingoccursthroughfreediffusion across the membrane network, or through active transport of fatty acyl-CoAs [25,26]. As detailedabove, the P. falci- parumDPCKwaslocalisedtotheapicoplast,necessitat- ing a CoA exporter inside the Plasmodium apicoplast membranes. Lastly, apicomplexans must possess an exporterof IPPand DMAPP,theisoprenoidprecursors generated within the apicoplast, which allow for the generation of geranyl-pyrophosphate (GPP), FPP and GGPPaswellaslongerchainisoprenoidsinthecytosol.

Thesemetabolite exporters likelymake excellent drug targets. Despite their localization inside the apicoplast membranes,thesetransporterswouldprimarilyinterfere with metabolic pathways within the cytosol, ER and mitochondrionand likely causerapid death rather than theapicoplastrelated‘delayeddeath’.Potentially,accumulation ofthetransporter’s substrateinside theapicoplast could be toxic and cause a secondary deleterious effect and further enhance the efficacy of a putative inhibitor. However, unless toxic accumulation occurs, exportofmostmetabolites,islikelydispensableinblood stagePlasmodium,exceptforessentialexportofIPPand

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Figure1

Current Opinion in Microbiology

Apicoplastmetaboliteimportandexport.

Theschemeprovidesanoverviewoverthefewknown(blue)andmanyunknownmetaboliteimporters(orange)thatsupplymetabolicpathways withintheapicoplastanditshousekeeping(replication,translation,transcription),aswellastheunknownexportersthatareexpectedin Plasmodiumspp.andToxoplasmagondii(yellow)oronlyinPlasmodium(pink).Metabolitesthataregeneratedbypathwayswithintheapicoplast areshownbyhorizontalgreyarrows.Forexample:TheFASIIsynthesispathwayprovidesoctanoyl-ACPrequiredforlipoatesynthesis.Theend productofthelipoatesynthesispathwayisrequiredintheFASIIpathway(lipoylationofthePDHsubunitE2).FASIIalsoreliesonNADPH,which canbeprovidedthroughactivityofthepartialcarbonmetabolismandactivityofisocitratedehydrogenaseIandpyruvatedehydrogenaseor glyceraldehyde-3-phosphatedehydrogenaseII(notshown).Acyl-ACPfromtheFASIIpathwayisprovidedasasubstrateforLPAsynthesis.The onlyessentialapicoplast-derivedmetaboliteinPlasmodiumspp.blood-stageparasites,IPP,ishighlightedinblue.Essentialmetaboliteswhichare derivedfromapicoplast-residentmetabolicpathwaysandhavebeenshowntobesalvagedbyT.gondiiarehighlightedinred.CoenzymeA(CoA), whichisonlyexpectedtobegeneratedinsideandexportedfromtheapicoplastofPlasmodiumbutnotT.gondii,ishighlightedinpurple.Very closeinteractionsbetweentheapicoplastandtheendoplasmicreticulumandmitochondrionhavebeendescribed,potentiallyfacilitatingthedirect exchangeoflipidsandsolutesthroughunknowntransporters,asindicatedbythedashedarrows.NotethatsomedifferencesbetweenT.gondii andPlasmodiumspp.arenotindicatedinthefigure:AfolatetransporterwasthusfaronlyidentifiedinsidetheP.bergheiapicoplast[47].In additiontotheTPs,theT.gondiiapicoplastlikelyalsoimportspyruvateandNTPscanbegeneratedfromNDPsinsidetheapicoplastof PlasmodiumthroughpyruvatekinaseII,whileT.gondiipotentiallyimportsNTPs[59,60].Potentialion(Cl ,Na⁺,K^+,Ca²⁺,H⁺,Fe²⁺)transporters werenotincludedhere.GraphicswerepartiallytakenandmodifiedfromsmartServierMedicalArt:https://smart.servier.com/.Abbreviations:5- ALA,5-aminolevulinicacid;TPs,triosephosphates;ADP,adenosinetriphosphate;ADP,adenosinediphosphate;GDP,guanosinediphosphate;

NAD+,nicotinamideadeninedinucleotide;NADPH,nicotinamidedinucleotidedinucleotidephosphate(reduced);CoA,coenzymeA;deP-CoA, dephospho-CoA;DPCK,dephospho-CoenzymeAkinase;SAM,S-adenosylmethionine;TPP,thiaminepyrophosphate;(d)NTPs,(deoxy)nucleotide triphosphate,(d)NDP,(deoxy)nucleotidediphosphate;AAs,aminoacids;LPA,lysophosphatidicacid;ACP,acylcarrierprotein;FASII,typeIIfatty acidsynthase;SUF,sulfurmobilization;IPP,isopentenylpyrophosphate;MEP,methylerythritolphosphate;DOXP,1-Deoxy-D-xylulose-5-phosphat;

DMAPP,dimethylallylpyrophosphate;aKG,a-ketoglutarate;CPOIII,coproporphyrinogenIII;FA,fattyacid;PPgenIX,protoporphyrinogenIX;PPIX, protoporphyrinIX;TCA,tricarboxylicacid;ER,endoplasmicreticulum;GPP,geranylpyrophosphate;FPP,farnesylpyrophosphate;GGPP,geranyl- geranylpyrophosphate.

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DMAPP.Instead, exportof IPPfrom theT. gondiiapi- coplast may be dispensable based on the salvage of cytosoliclongchainisoprenoids[38].Conversely,allother exporters, trafficking FAs, LPA, and coproporphyrino- gen-III are expected to be highly fitness-conferring in T.gondii.Identificationofexporters couldbefacilitated bythenotionthatlossofthesetransportersmaynotcause lossoftheorganelleincontrasttothemanytransporters expectedtobeinvolvedinimportingmetabolitesessen- tialto maintaintheapicoplast. Inthiscontext, acritical transporter identified by Sayers et al., was proposed to potentiallybeinvolvedin IPP/DMAPP export,but this requiresfurtherinvestigation[57].

Conclusions andperspectives

As detailedabove, several recent studiesrevealed new insights intothe role ofmetabolic pathways withinthe T.gondiiandPlasmodiumapicoplast.StudiesonT.gondii have revealed a remarkable flexibility of the parasite, showing that it can compensate for defects in several apicoplast-residentpathwaysthroughincreaseduptakeof supplemented exogenous metabolites [35,36,37,38].

However,amajorgapinourunderstandingofapicoplast biology is the limited knowledge of metabolite import and export. Three studies have identified and characterised previously unidentified apicoplast transporters [55,56,57],butmanymorearepostulatedtobeneeded tofulfilthediversemetabolicfunctionsoftheorganelle.

The characterisationof apicoplast transporterswill con- tribute to our understanding of the functions of this peculiarorganelle andwill likelyprovide new potential targets for intervention against these deadly parasites.

Especially,exportersofcriticalmetabolitesareexpected tomakeexcellentdrugtargetsthatwouldresultinrapid death.

Conflictofintereststatement Nothingdeclared.

Authorcontribution statement

J.K.,C.E.M.L.andD.S.-F.wrotethemanuscript.

Acknowledgements

ThisworkwassupportedbytheEuropeanResearchCouncil(ERC)under theEuropeanUnion’sHorizon2020researchandinnovationprogramunder Grantagreementno.695596.C.E.M.L.issupportedbyCarigestSA.

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