Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability
2022; Elsevier BV; Volume: 298; Issue: 8 Linguagem: Inglês
10.1016/j.jbc.2022.102243
ISSN1083-351X
AutoresEléa A. Renaud, Sarah Pamukcu, Aude Cerutti, Laurence Berry, Catherine Lemaire-Vieille, Yoshiki Yamaryo‐Botté, Cyrille Y. Botté, Sébastien Besteiro,
Tópico(s)Autophagy in Disease and Therapy
ResumoLike many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets. Like many other apicomplexan parasites, Toxoplasma gondii contains a plastid harboring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are crucial for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway, deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on the parasites. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis, and suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets. Apicomplexan parasites are some of the most prevalent and morbidity-causing pathogens worldwide. Noticeably, they comprise Plasmodium species that can naturally infect humans and cause the deadly malaria in tropical and subtropical areas of the world (1White N.J. Pukrittayakamee S. Hien T.T. Faiz M.A. Mokuolu O.A. Dondorp A.M. Malaria.Lancet. 2014; 383: 723-735Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar). Although less lethal, another apicomplexan parasite called Toxoplasma gondii can cause serious illness in animals, including humans, and has a widespread host range and geographical distribution (2Sanchez S.G. Besteiro S. The pathogenicity and virulence of Toxoplasma gondii.Virulence. 2021; 12: 3095-3114Crossref PubMed Scopus (7) Google Scholar). These protists are obligate intracellular parasites that rely to a large extent on their host cells for nutrient acquisition and for protection from the immune system. Through their evolutionary history, Plasmodium and Toxoplasma have inherited a plastid from a secondary endosymbiosis event involving the engulfment of a red alga whose photosynthetic capability previously originated from the acquisition of a cyanobacterium (3Janouskovec J. Horak A. Obornik M. Lukes J. Keeling P.J. A common red algal origin of the apicomplexan, dinoflagellate, and heterokont plastids.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 10949-10954Crossref PubMed Scopus (340) Google Scholar). Although the ability to perform photosynthesis has been lost during evolution when the ancestors of Apicomplexa became parasitic (4van Dooren G.G. Striepen B. The algal past and parasite present of the apicoplast.Annu. Rev. Microbiol. 2013; 67: 271-289Crossref PubMed Scopus (107) Google Scholar), the plastid has retained critical metabolic functions. For instance, it hosts pathways for the synthesis of heme (together with the mitochondrion), fatty acids (FAs, via a prokaryotic FASII (type II fatty acid synthase) pathway), isoprenoid precursors (through the so-called non-mevalonate or 1-deoxy-D-xylulose 5-phosphate pathway), and iron-sulfur (Fe-S) clusters (5van Dooren G.G. Hapuarachchi S.V. The dark side of the chloroplast: biogenesis, metabolism and membrane biology of the apicoplast.Adv. Bot. Res. 2017; 84: 145-185Crossref Scopus (9) Google Scholar, 6Seeber F. Soldati-Favre D. Metabolic pathways in the apicoplast of apicomplexa.Int. Rev. Cell Mol. Biol. 2010; 281: 161-228Crossref PubMed Scopus (117) Google Scholar). Because of its origin and its metabolic importance, the apicoplast is particularly attractive to look for potential drug targets (7Biddau M. Sheiner L. Targeting the apicoplast in malaria.Biochem. Soc. Trans. 2019; 47: 973-983Crossref PubMed Scopus (16) Google Scholar). For example, drugs inhibiting translation in prokaryotic-like systems like clindamycin or doxycycline, which are used for prophylaxis or treatment of Apicomplexa-caused diseases, primarily target the apicoplast (8Kennedy K. Crisafulli E.M. Ralph S.A. Delayed death by plastid inhibition in apicomplexan parasites.Trends Parasitol. 2019; 35: 747-759Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 9Uddin T. McFadden G.I. Goodman C.D. Validation of putative apicoplast-targeting drugs using a chemical supplementation assay in cultured human malaria parasites.Antimicrob. Agents Chemother. 2018; 62e01161-17Crossref PubMed Scopus (35) Google Scholar). As some of the earliest catalytic cofactors on earth (10Imlay J.A. Iron-sulphur clusters and the problem with oxygen.Mol. Microbiol. 2006; 59: 1073-1082Crossref PubMed Scopus (445) Google Scholar), Fe-S clusters are found in all kingdoms of life, associated with proteins involved in a number of key cellular functions like the synthesis of metabolites, the replication and repair of DNA, the biogenesis of ribosomes, and the modification of tRNAs (11Lill R. Function and biogenesis of iron–sulphur proteins.Nature. 2009; 460: 831-838Crossref PubMed Scopus (827) Google Scholar). The biosynthesis of Fe-S clusters necessitates a complex machinery for assembling ferrous (Fe2+) or ferric (Fe3+) iron and sulfide (S2−) ions, and delivering the resulting Fe-S cluster to target client proteins (12Braymer J.J. Freibert S.A. Rakwalska-Bange M. Lill R. Mechanistic concepts of iron-sulfur protein biogenesis in Biology.Biochim. Biophys. Acta (Bba) - Mol. Cell Res. 2021; 1868118863Crossref PubMed Scopus (56) Google Scholar). In eukaryotes, Fe-S proteins are present in various subcellular compartments like the cytosol and the nucleus, but also in organelles of endosymbiotic origin like mitochondria or plastids, and thus require compartment-specific biogenesis systems. The three main eukaryotic Fe-S synthesis pathways comprise the iron-sulfur cluster (ISC) machinery, hosted by the mitochondrion, the cytosolic iron-sulfur cluster assembly machinery, important not only for the generation of cytosolic but also of nuclear Fe-S proteins, and the SUF (sulfur utilization factor) pathway that is found in plastids (11Lill R. Function and biogenesis of iron–sulphur proteins.Nature. 2009; 460: 831-838Crossref PubMed Scopus (827) Google Scholar). Like in plants and algae, apicoplast-containing apicomplexan parasites seem to express the machinery corresponding to the three eukaryotic pathways. For instance, recent investigations in T. gondii have shown that the cytosolic iron-sulfur cluster assembly, ISC, and SUF pathways are all essential for parasite fitness (13Aw Y.T.V. Seidi A. Hayward J.A. Lee J. Victor Makota F. Rug M. et al.A key cytosolic iron-sulfur cluster synthesis protein localises to the mitochondrion of Toxoplasma gondii.Mol. Microbiol. 2020; 115: 968-985Crossref PubMed Scopus (6) Google Scholar, 14Pamukcu S. Cerutti A. Bordat Y. Hem S. Rofidal V. Besteiro S. Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.PLoS Pathog. 2021; 17e1010096Crossref PubMed Scopus (3) Google Scholar). From a biochemical point of view, mitochondrial and plastidic Fe-S cluster biosynthesis pathways follow a similar general pattern: cysteine desulfurases produce sulfur from L-cysteine, then scaffold proteins provide a molecular platform allowing assembly of iron and sulfur into a cluster, and finally carrier proteins deliver the cluster to target apoproteins. Importantly, targeting the T. gondii mitochondrial ISC pathway through disruption of scaffold protein ISU1 was shown to lead to a reversible growth arrest and to trigger differentiation into a stress-resistant form; while on the other hand, targeting the plastidic SUF pathway by inactivating NFS2 function led to an irreversible lethal phenotype (14Pamukcu S. Cerutti A. Bordat Y. Hem S. Rofidal V. Besteiro S. Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.PLoS Pathog. 2021; 17e1010096Crossref PubMed Scopus (3) Google Scholar). Like for many apicoplast-hosted pathways, enzymes belonging to the SUF machinery are essentially absent from the mammalian host and as such, they may be seen as good potential drug targets. Noticeably, while most apicoplast proteins are encoded by the parasite nucleus (after gene transfer from the endosymbiont), SUFB is one of the very few proteins still encoded by the apicoplast genome, perhaps illustrating the importance of the pathway for the parasite. This has sparked considerable interest for the SUF pathway in Plasmodium, which has been shown to be important for the viability of several developmental stages of the parasite (15Gisselberg J.E. Dellibovi-Ragheb T.A. Matthews K.A. Bosch G. Prigge S.T. The Suf iron-sulfur cluster synthesis pathway is required for apicoplast maintenance in malaria parasites.PLoS Pathog. 2013; 9e1003655Crossref Scopus (69) Google Scholar, 16Haussig J.M. Matuschewski K. Kooij T.W.A. 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Mishra S. et al.[Fe-S] cluster assembly in the apicoplast and its indispensability in mosquito stages of the malaria parasite.FEBS J. 2017; 284: 2629-2648Crossref PubMed Scopus (20) Google Scholar). To better understand the contribution of the SUF pathway to T. gondii viability, we have generated a conditional mutant for the scaffold protein TgSUFC and conducted a thorough phenotypic characterization of this mutant, together with the TgNFS2 mutant we have previously generated (14Pamukcu S. Cerutti A. Bordat Y. Hem S. Rofidal V. Besteiro S. Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.PLoS Pathog. 2021; 17e1010096Crossref PubMed Scopus (3) Google Scholar). Our results confirm that inactivating the plastid-hosted SUF pathway in T. gondii leads to irreversible and marked effects on membrane homeostasis, impacting the division process and parasite viability. We show that these effects are likely due to impairment in the function of several key plastidic Fe-S proteins, which have pleiotropic downstream metabolic consequences for the parasite. Searching for homologs of the plant SUF system in the ToxoDB.org database, we have previously shown an overall good conservation for the plastidic Fe-S cluster synthesis pathway (14Pamukcu S. Cerutti A. Bordat Y. Hem S. Rofidal V. Besteiro S. Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.PLoS Pathog. 2021; 17e1010096Crossref PubMed Scopus (3) Google Scholar). Among the candidates for members of the SUF machinery, we have identified a potential T. gondii homolog of SUFC, member of a Fe-S cluster scaffold complex comprising SUFC, SUFB, and SUFD (Fig. 1A). This complex is also present in prokaryotes, where it was first characterized (20Takahashi Y. Tokumoto U. A third bacterial system for the assembly of iron-sulfur clusters with homologs in archaea and plastids.J. Biol. Chem. 2002; 277: 28380-28383Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar): it was shown that bacterial SufC is an ABC-like ATPase component essential for proper Fe-S cluster assembly (21Hirabayashi K. Yuda E. Tanaka N. Katayama S. Iwasaki K. Matsumoto T. et al.Functional dynamics revealed by the structure of the SufBCD complex, a novel ATP-binding cassette (ABC) protein that serves as a scaffold for Iron-Sulfur cluster biogenesis.J. Biol. Chem. 2015; 290: 29717-29731Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Alignment of the amino acid sequences of the T. gondii SUFC candidate (entry TGGT1_225800 in the ToxoDB.org database (22Harb O.S. Roos D.S. ToxoDB: functional genomics resource for Toxoplasma and related organisms.Met. Mol. Biol. 2020; 2071: 27-47Crossref PubMed Scopus (15) Google Scholar)) and its Escherichia coli counterpart showed a good overall conservation (56% of identity), particularly in the motifs that are characteristic of ABC ATPAses (Fig. 1B). The T. gondii protein presents an N-terminal extension when compared with E. coli SufC, which may contain a transit peptide for targeting to the apicoplast. Accordingly, it was predicted with high probability to be a plastid-localized protein by the Deeploc 1.0 (https://services.healthtech.dtu.dk/service.php?DeepLoc-1.0) algorithm (23Almagro Armenteros J.J. Sønderby C.K. Sønderby S.K. Nielsen H. Winther O. DeepLoc: prediction of protein subcellular localization using deep learning.Bioinformatics. 2017; 33: 3387-3395Crossref PubMed Scopus (524) Google Scholar), although the exact position of the N-terminal transit peptide sequence could not be defined. Data from global mapping of T. gondii proteins' subcellular location by hyperLOPIT spatial proteomics (24Barylyuk K. Koreny L. Ke H. Butterworth S. Crook O.M. Lassadi I. et al.A comprehensive subcellular atlas of the Toxoplasma proteome via hyperLOPIT provides spatial context for protein functions.Cell Host & Microbe. 2020; 28: 752-766.e9https://doi.org/10.1016/j.chom.2020.09.011Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) also suggested an apicoplast localization for TGGT1_225800. To assess whether this protein is a real functional homolog, we first performed complementation assays of an E. coli SufC mutant, for which growth is slowed, especially when limiting iron availability with a specific chelator (25Outten F.W. Djaman O. Storz G. A suf operon requirement for Fe-S cluster assembly during iron starvation in Escherichia coli: suf operon role during iron starvation.Mol. Microbiol. 2004; 52: 861-872Crossref PubMed Scopus (339) Google Scholar). We could show that expressing the predicted functional domain of TGGT1_225800 restored bacterial growth (Fig. 1C), even in the presence of the iron chelator, confirming this protein (hereafter named TgSUFC) is functional. In order to detect TgSUFC expression and assess its subcellular localization in the tachyzoite stage (the fast-replicating stage associated with acute toxoplasmosis (2Sanchez S.G. Besteiro S. The pathogenicity and virulence of Toxoplasma gondii.Virulence. 2021; 12: 3095-3114Crossref PubMed Scopus (7) Google Scholar)), we epitope-tagged the native protein. This was performed by inserting a sequence coding for a C-terminal triple hemagglutinin (HA) epitope tag at the endogenous TgSUFC locus by homologous recombination (Fig. S1). It was achieved in the tetracycline-inducible transactivator (TATi) ΔKu80 cell line, favoring homologous recombination and allowing transactivation of a Tet operator-modified promoter that we subsequently used for generating a conditional mutant in this background (26Fox B.A. Ristuccia J.G. Gigley J.P. Bzik D.J. Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining.Eukaryot. Cell. 2009; 8: 520-529Crossref PubMed Scopus (197) Google Scholar, 27Huynh M.-H. Carruthers V.B. Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80.Eukaryot. Cell. 2009; 8: 530-539Crossref PubMed Scopus (335) Google Scholar, 28Sheiner L. Demerly J.L. Poulsen N. Beatty W.L. Lucas O. Behnke M.S. et al.A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor.PLoS Pathog. 2011; 7e1002392Crossref PubMed Scopus (150) Google Scholar). Immunoblot analysis with an anti-HA antibody revealed two products, likely corresponding to the immature and mature forms (resulting from the cleavage of the transit peptide upon import into the organelle) of TgSUFC (Fig. 2A). Immunofluorescence assay (IFA) with the anti-HA antibody and costaining with an apicoplast marker confirmed that TgSUFC localizes to this organelle in T. gondii tachyzoites (Fig. 2B). Next, we generated a conditional TgSUFC mutant cell line in the TgSUFC-HA–expressing TATi ΔKu80 background. Replacement of the endogenous promoter by an inducible-Tet07SAG4 promoter was achieved through a single homologous recombination at the locus of interest, yielding the conditional knockdown (cKD) TgSUFC-HA cell line (Fig. S2). In this cell line, the addition of anhydrotetracycline (ATc) can repress transcription through a Tet-Off system (29Meissner M. Brecht S. Bujard H. Soldati D. Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii.Nucl. Acids Res. 2001; 29: E115Crossref PubMed Google Scholar). Initial phenotypic characterization was performed on two independent clones, which were found to behave similarly and thus only one was analyzed further. It should be noted that the promoter replacement resulted in a slightly higher expression of TgSUFC but did not change the maturation profile of the protein (Fig. 2A). Downregulation of TgSUFC was assessed by growing the parasites in the presence of ATc. Immunoblot and IFA analyses showed a decrease of TgSUFC to almost undetectable levels after as early as 1 day of ATc treatment (Fig. 2, A and B). We also generated a complemented cell line constitutively expressing an additional TY-tagged (30Bastin P. Bagherzadeh A. Matthews K.R. Gull K. A novel epitope tag system to study protein targeting and organelle biogenesis in Trypanosoma brucei.Mol. Biochem. Parasitol. 1996; 77: 235-239Crossref PubMed Scopus (222) Google Scholar) copy of TgSUFC from the uracil phosphoribosyltransferase (UPRT) locus, driven by a tubulin promoter (Fig. S3). This cell line, named cKD TgSUFC-HA comp, was found by immunoblot (Fig. 2C) and IFA (Fig. 2D) to be stably expressing TgSUFC while the HA-tagged copy was efficiently downregulated in the presence of ATc. We first evaluated the consequences of TgSUFC depletion on parasite fitness in vitro by performing a plaque assay, which determines the capacity of the mutant and complemented parasites to produce lysis plaques on a host cells' monolayer in the absence or continuous presence of ATc for 7 days (Fig. 3). Depletion of TgSUFC prevented plaque formation, which was restored in the complemented cell lines (Fig. 3, A and B). Our previous analysis of another SUF pathway mutant (TgNFS2, (14Pamukcu S. Cerutti A. Bordat Y. Hem S. Rofidal V. Besteiro S. Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.PLoS Pathog. 2021; 17e1010096Crossref PubMed Scopus (3) Google Scholar)) suggested that the impact on the pathway leads to irreversible death of the parasites, so we sought to verify this by removing the ATc after 7 days of incubation and monitoring plaque formation. We confirmed that depleting TgSUFC was irreversibly impacting parasite viability, as ATc removal did not lead to the appearance of plaques (Fig. 3C). We next assessed whether this defect in the lytic cycle is due to a replication problem. Mutant and control cell lines were preincubated in the absence or presence of ATc for 48 h and released mechanically, before infecting new host cells and were then grown for an additional 24 h in ATc prior to parasite counting. We noted that the incubation with ATc led to an accumulation of vacuoles with fewer TgSUFC mutant parasites, but that it was not the case in the complemented cell lines (Fig. 3D). Overall, our data show that depleting TgSUFC has an irreversible impact on parasite growth, as previously described for other SUF mutant TgNFS2 (14Pamukcu S. Cerutti A. Bordat Y. Hem S. Rofidal V. Besteiro S. Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.PLoS Pathog. 2021; 17e1010096Crossref PubMed Scopus (3) Google Scholar). T. gondii tachyzoites divide by a process called endodyogeny, whereby two daughter cells will assemble inside a mother cell (31Francia M.E. Striepen B. Cell division in apicomplexan parasites.Nat. Rev. Microbiol. 2014; 12: 125-136Crossref PubMed Scopus (156) Google Scholar). Among the structures which are essential as a scaffold for daughter cell formation is the inner membrane complex (IMC), a system of flattened vesicles underlying the plasma membrane and is supported by a cytoskeletal network. The IMC also supports anchorage for the glideosome, the protein complex–powering parasite motility (32Harding C.R. Meissner M. The inner membrane complex through development of Toxoplasma gondii and Plasmodium.Cell Microbiol. 2014; 16: 632-641Crossref PubMed Scopus (52) Google Scholar). As for several other cellular structures, there is a combination of de novo assembly and recycling of maternal material during IMC formation in daughter cells (33Ouologuem D.T. Roos D.S. Dynamics of the Toxoplasma gondii inner membrane complex.J. Cell Sci. 2014; 127: 3320-3330Crossref PubMed Scopus (37) Google Scholar). To get more precise insights into the impact of the impairment of the SUF pathway on parasite division, we incubated the TgNFS2 and TgSUFC mutant parasites with ATc for up to 2 days and stained them for IMC protein IMC3 to detect growing daughter cells (Fig. 4A). IMC3 is an early marker of daughter cell budding (34Gubbels M.-J. Wieffer M. Striepen B. Fluorescent protein tagging in Toxoplasma gondii: identification of a novel inner membrane complex component conserved among apicomplexa.Mol. Biochem. Parasitol. 2004; 137: 99-110Crossref PubMed Scopus (80) Google Scholar), which is usually synchronized within the same vacuole. However, after 2 days of ATc treatment, an increasing portion of the vacuoles showed a lack of synchronicity for daughter cell budding for both mutant cell lines, although the effect was more pronounced for the TgSUFC mutant (Fig. 4, A and B). Then, we used electron microscopy to get a subcellular view of the consequences of TgNFS2 and TgSUFC depletion on the cell division process. Strikingly, in parasites grown in the continuous presence of ATc for 3 days, we observed cytokinesis completion defects. As budding daughter cells emerge, they normally incorporate plasma membrane material that is partly recycled from the mother, leaving only a basal residual body. Here, in both TgNFS2 and TgSUFC mutant cell lines, daughter cells remained tethered through patches of plasma membrane (Fig. 4C). Hence, this highlighted an early and important defect in plasma membrane biogenesis and/or recycling during daughter cell budding. We previously observed major cell division defects after long term (5 days or more) continuous incubation of cKD TgNFS2-HA parasites with ATc (14Pamukcu S. Cerutti A. Bordat Y. Hem S. Rofidal V. Besteiro S. Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.PLoS Pathog. 2021; 17e1010096Crossref PubMed Scopus (3) Google Scholar). When assessing the cKD TgSUFC-HA parasites in the same conditions, costaining with apicoplast and IMC markers revealed similar defects, including organelle segregation problems and abnormal membranous structures (Fig. 4D). Computational prediction of the Fe-S proteome combined with hyperLOPIT localization data suggests there are a limited number of apicoplast proteins potentially containing Fe-S clusters (14Pamukcu S. Cerutti A. Bordat Y. Hem S. Rofidal V. Besteiro S. Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.PLoS Pathog. 2021; 17e1010096Crossref PubMed Scopus (3) Google Scholar). However, these candidates are supposedly very important for the parasite. They include the following: IspG and IspH, two oxidoreductases involved in isoprenoid synthesis (35Imlay L. Odom A.R. Isoprenoid metabolism in apicomplexan parasites.Curr. Clin. Microbiol. Rep. 2014; 1: 37-50Crossref PubMed Scopus (45) Google Scholar); LipA, a lipoyl synthase important for the function of the pyruvate dehydrogenase (PDH) complex (36Thomsen-Zieger N. Schachtner J. Seeber F. Apicomplexan parasites contain a single lipoic acid synthase located in the plastid.FEBS Lett. 2003; 547: 80-86Crossref PubMed Scopus (65) Google Scholar); MiaB, which is likely a tRNA modification enzyme (37Pierrel F. Douki T. Fontecave M. Atta M. MiaB protein is a bifunctional radical-S-adenosylmethionine enzyme involved in thiolation and methylation of tRNA.J. Biol. Chem. 2004; 279: 47555-47563Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar); as well as proteins that are directly involved in Fe-S synthesis, and the plastidic ferredoxin (Fd) that is an important electron donor that regulates several apicoplast-localized pathways (38Henkel S. Frohnecke N. Maus D. McConville M.J. Laue M. Blume M. et al.Toxoplasma gondii apicoplast-resident ferredoxin is an essential electron transfer protein for the MEP isoprenoid-biosynthetic pathway.J. Biol. Chem. 2022; 298: 101468Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 39Frohnecke N. Klein S. Seeber F. Protein–protein interaction studies provide evidence for electron transfer from ferredoxin to lipoic acid synthase in Toxoplasma gondii.FEBS Lett. 2015; 589: 31-36Crossref PubMed Google Scholar, 40Swift R.P. Rajaram K. Elahi R. Liu H.B. Prigge S.T. Roles of ferredoxin-dependent proteins in the apicoplast of Plasmodium falciparum parasites.mBio. 2022; 13e0302321Crossref Google Scholar). Dataset from a CRISPR-based genome-wide screen suggests that most of these candidates are important for fitness in vitro (41Sidik S.M. Huet D. Ganesan S.M. Huynh M.-H. Wang T. Nasamu A.S. et al.A genome-wide CRISPR screen in Toxoplasma identifies essential apicomplexan genes.Cell. 2016; 166: 1423-1435.e12Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). We first assessed whether depletion of TgSUFC led to a partial apicoplast loss, as was previously shown for TgNFS2 (14Pamukcu S. Cerutti A. Bordat Y. Hem S. Rofidal V. Besteiro S. Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.PLoS Pathog. 2021; 17e1010096Crossref PubMed Scopus (3) Google Scholar). Although slowed down in growth, some parasites eventually egressed during the course of the experiments and were used to reinvade host cells and were kept for a total of 5 days in the presence of ATc (Fig. 5A). This is reminiscent to the so-called "delayed death" effect, observed when inhibiting apicoplast metabolism, that often results in slow-kill kinetics (8Kennedy K. Crisafulli E.M. Ralph S.A. Delayed death by plastid inhibition in apicomplexan parasites.Trends Parasitol. 2019; 35: 747-759Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Quantification of the apicoplast marker TgCPN60 showed a progressive loss of this protein (Fig. 5, A and B). As t
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